Diese Arbeit wurde von der National Institute for Mental Health und der National Science Foundation unterstützt. Besonderer Dank geht an Sive lab Mitglieder für viele hilfreiche Diskussionen und konstruktive Kritik, und Olivier Paugois für Experten Fische Tierhaltung.

Ein. Vorbereitung für Mikroinjektion <ol><li> Bereiten Mikroinjektion Nadeln durch Ziehen Kapillarrohre mit Sutter Instrumente Nadel abziehen. </li><li> Last Mikroinjektionsnadel mit fluoreszierenden Farbstoff (FITC-Dextran). </li><li> Montieren Nadel auf Mikromanipulator und Mikroinjektionsvorrichtung. </li><li> Vorsichtig brechen Mikroinjektionsnadel mit einer Pinzette auf ungefähr 2 um in der Breite, wird dies jedoch abhängig von Ihren Mikroinjektor Setup. Für unsere Mikroinjektion Nadeln, entspricht dies dem ersten Bereich der Nadel von der Spitze, die sich nicht verbiegt. </li><li> Messen Tropfengröße in Öl, Einstellen Einspritzzeit und Druck, so daß jeder Injektion 1 nl liefert. Beispiel Einstellungen für Harvard Apparatus Mikroinjektor sind: P balance = 1,4 psi, P out = 1,4 psi, injizieren P = 22,9 psi, P clear = 67,8 psi mit einer Einspritzzeit von 0,4 bis 0,7 sec. Der Durchmesser unserer Nadel mit diesen Einstellungen ist etwa 2 um. Allerdings werden die Einstellungen Mikroinjektor spezifisch sein, und variieren einach Nadel Durchmesser. </li></ol> 2. Vorbereitung der Embryonen <ol><li> Coat 2 Gerichte mit 1% Agarose in Wasser für jede Bedingung, poke Löcher in Agarose mit einer 1-200 ul Pipettenspitze und entfernen Agarose Stecker. Füllen Sie Gerichte mit Embryo Medien. </li><li> Mit einer Pinzette, dechorionate Embryonen 18 hpf oder älter unter einem Stereomikroskop sind. Embryonen werden nach Kimmel et al inszeniert. 11. </li><li> Übertragen dechorionated Embryonen in erster Agarose-beschichteten Gericht. </li><li> Embryonen zu betäuben, fügen Tricain (0,1 mg / ml) in die Schale, bis Embryonen bewegen Anschlag (hergestellt nach Westerfield 12). </li></ol> 3. Einspritzen der Hirnkammern <ol><li> Orient Embryonen, so dass Sie werden mit ihrem dorsalen Seite suchen, indem Sie den Schwanz des Embryos in das Loch. Wenn Ihr Mikromanipulator ist auf der rechten, dann den Embryo, so dass das Vorderhirn auf der linken Seite und Hinterhirn nach rechts. </li><li> Position Nadel widest Punkt Hinterhirn Ventrikels. </li><li> Sorgfältig durchbohren Dachblech Rautenhirn Ventrikel sicher zu sein, um nicht durch die Tiefe des Gehirns in den Dotter (1A) zu gehen. </li><li> Injizieren 1-2 nl der Fluoreszenzfarbstoff in die Ventrikel sicherstellen der Farbstoff erfüllt die ganze Länge der Hirnkammern. </li><li> Übertragen Embryonen in die zweite Agarose-beschichtete Schale mit Embryo Medien befüllt und wieder zu betäuben wie in 2.4 beschrieben. </li><li> SOFORT starten Bildgebung, wie in Abschnitt 4 beschrieben, um einen Zeitpunkt Null Bild zu bekommen. </li></ol> 4. Imaging <ol><li> Orient Embryonen mit ihren Schwanz im Loch wie in 3.1 beschrieben. </li><li> Verwenden Sie ein Binokular mit beiden gesendeten und Fluoreszenzlicht eine Hellfeld dorsalen Bild aufzunehmen. Halten Sie die Vergrößerung konstant zwischen Bildgebung von verschiedenen Embryonen. Dies ermöglicht den direkten Vergleich von Analysen mit Bild J (5,2-6). </li><li> Ohne Bewegung des Embryos, Mikroskop oder Teller, nehmen einentsprechendes Fluoreszenzbild. </li><li> Wiederholen Sie dies für jeden Embryo gewünschten Zeitpunkten. </li></ol> 5. Quantifizierung von Dye Movement <ol><li> Merge Hellfeld und Fluoreszenz-Bilder in Photoshop wie zuvor von Gutzman und Sive 10 beschrieben. </li><li> Messen Sie den Abstand der Farbstoff vor bewegt sich in Image J Software verfügbar <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> . </li><li> Offene zusammengeführte Datei in Image J und verwenden Tool, um eine Zeile aus dem Vorderhirn Scharnier-Punkt nach vorne zu einem 10-20 °-Winkel vom Neuroepithel (Abbildung 1A) färben ziehen. Diese Region wurde ausgewählt, weil es das erste und auffälligste Website des Farbstoffs Auslaufen des Wildtyp Neuroepithel ist. </li><li> Wählen Messwerkzeug zur Länge der Leitung berechnen. </li><li> Wiederholen Sie für jede Zeit-Punkt. </li><li> Berechnen net Abstand der Farbstoff vor der Zeit bewegt durch Subtrahieren Abstand zum Zeitpunkt t = 0 von anderen Zeitpunkten. </li><li> Plot aufgraph. </li></ol> 6. Repräsentative Ergebnisse Ein Beispiel der Ergebnisse in einem Test unter Verwendung neuroepithelialen Permeabilität Wildtyp Embryos gewonnen ist in 1B-D gezeigt. Um genau zu unterscheiden Permeabilität, ist es nützlich, Farbstoffe mit unterschiedlichen molekularen weightsto testen identifizieren eine Größe, die nur geringfügig undicht in Wildtyp oder Kontrolle Embryonen (Abbildung 2). Dies ermöglicht die Identifizierung von genetischen Mutanten oder Umweltbedingungen, dass entweder Anstieg oder Abnahme Permeabilität (Figur 1D, grünen und roten Linien jeweils). Für den 24 hpf Zebrafisch Neuroepithel, 70 kDa FITC Dextran Lecks langsam über 2 Stunden, während 2.000 kDa nicht und 10 kDa fast sofort austreten. Deshalb 70 kDa ist die ideale Molekulargewicht Bedingungen, dass sowohl steigen als auch neuroepithelialen Durchlässigkeit zu verringern identifizieren. Wenn die Nadel verfehlt das ventrikuläre Lumen, Fluoreszenz wKranke außerhalb des Gehirns erscheinen t = 0 (für ein Beispiel siehe Gutzman und Sive 2009 10). Diese Embryonen zu verwerfen, da der injizierte Farbstoff wurde zunächst nicht innerhalb des Gehirns enthalten und keine klare Aussage zur Bewegung des Farbstoffs und Permeabilität des neuropeithelium vorgenommen werden können. Schließlich, wenn Embryonen haben kleine Ventrikeln oder nicht aufgeblasenen Hirnkammern, Voreinspritzung von Ventrikel mit einer Salzlösung kann vor der Injektion des Fluoreszenzfarbstoffs durchgeführt werden. Dieser bläst die Ventrikel Herstellung anschließender Visualisierung der Ventrikel leichter beim Injizieren mit dem Fluoreszenzfarbstoff. Geeigneten Kontrollen muss durchgeführt werden, um festzustellen, ob Injektion von Kochsalzlösung normale Entwicklung stört Neuralrohr werden. <img alt="Abbildung 1" src="/files/ftp_upload/4242/4242fig1.jpg" /> Abbildung 1. Zeitverlauf unterschiedlichen Molekulargewichts Farbstoffe. (A) Experimentelle Diagramm. ErsteWird Fluoreszenzfarbstoff in die Ventrikel injiziert. X = Position der Nadel für die Injektion. Weiter dorsal Bilder werden im Laufe der Zeit erfasst. Schließlich zog der Abstand durch den Farbstoff vor dem Vorderhirn Scharnier-Punkt gemessen (dargestellt durch eine rote Linie) ist. (BC) verschmolzen Hellfeld und Fluoreszenz dorsalen Bildern bei 22 hpf (t = 0 min, B) und 24 hpf (t = 120 min, C). Weiße Linie zeigt Abstand von dem Farbstoff Front von Vorderhirn Ventrikels. (D) Hypothetische Probe Durchlässigkeit Daten. Blau = Wildtyp oder Kontrollen, rot = Probe mit verminderte Permeabilität im Vergleich zur Kontrolle und grün = Probe mit erhöhten Permeabilität im Vergleich zur Kontrolle. <img alt="Abbildung 2" src="/files/ftp_upload/4242/4242fig2.jpg" /> Abbildung 2. Messung der Durchlässigkeit für neuroepithelialen unterschiedlichem Molekulargewicht Farbstoffe. (AE) Dorsal fusionierten Hellfeld und Fluoreszenzbilder von 22 hpf Wildtyp Embryonen bei t = 0 min nach Injektion mit FITC-dExtran der folgenden Molekulargewichte: 10 kDa (A), 40 kDa (B), 70 kDa (C), 500 kDa (D) und 2000 kDa (E). (A&#39;-E &#39;) Gleiches Embryo wie in (AE) bei t = 120 min bei 24 hpf. Anterior nach links. F = Vorderhirn, M = Mittelhirn, H = Hinterhirn. Asterisk = Ohr.

<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>

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Wir zeigen die Fähigkeit, die Durchlässigkeit des lebendigen embryonalen Zebrafischgehirn quantifizieren, da für eine injiziert Farbstoff einem bestimmten Molekulargewicht bestimmt. Unsere Beobachtung, dass die embryonale Zebrafisch Neuroepithel differentiell durchlässig Farbstoffe mit unterschiedlichen Molekulargewichten ist nahe, dass der Farbstoff über die parazelluläre Permeabilität zu bewegen. Wir können jedoch nicht ausschließen, die Möglichkeit einer transzellulären Beitrag zur beobachteten Durchlässi...

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Dextran, Fluorescein, anionische, Lysin Fixierbarer </td><td> Invitrogen </td><td> D7136, D7137, D1822, D1820, D1845 </td></tr><tr><td> Tricaine Pulver </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Kapillarrohre </td><td> FHC Inc. </td><td> 30-30-1 </td></tr></tbody></table>

an assay for permeability of the zebrafish embryonic neuroepithelium

Das Gehirn ventrikuläre System unter den Wirbeltieren konserviert und besteht aus einer Reihe von miteinander verbundenen Hohlräumen Hirnkammern genannt, die in den frühesten Stadien der Entwicklung des Gehirns bilden und während der gesamten Lebensdauer des Tieres aufrechterhalten zusammengesetzt. Das Gehirn ventrikuläre System ist in Wirbeltieren, und die Ventrikel zu entwickeln, nachdem Neuralrohr Formation, wenn das zentrale Lumen mit Zerebrospinalflüssigkeit (CSF) 1,2 ausfüllt. CSF ist eine proteinreiche Flüssigkeit, die wichtig für die normale Entwicklung des Gehirns und Funktion 3-6 ist. Im Zebrafisch, beginnt Hirnventrikel Inflation bei etwa 18 Stunden nach der Befruchtung (HPF), nachdem das Neuralrohr ist geschlossen. Mehrere Prozesse sind mit Hirnventrikel Bildung, einschließlich Bildung einer Neuroepithels, tight junction Formation, die Durchlässigkeit und CSF-Produktion reguliert verbunden. Wir haben gezeigt, dass die Na, K-ATPase für Hirnventrikel Inflation erforderlich ist, beeinflussen alle diese Verfahrenes 7,8, während Claudin 5a notwendig tight junction Bildung 9 ist. Darüber hinaus haben wir gezeigt, dass &quot;Entspannung&quot; der embryonalen Neuroepithels, über eine Hemmung der Myosin, ist mit Hirnventrikel Inflation verbunden. Um die Regulation der Permeabilität im Zebrafisch Hirnventrikel Inflation zu untersuchen, entwickelten wir eine ventrikuläre Farbstoffretention Assay. Diese Methode verwendet Hirnventrikel Injektion in einem lebenden Zebrafisch-Embryos, eine Technik zuvor in unserem Labor 10 entwickelt, um Fluoreszenz-Markierung der Liquor. Embryonen werden dann im Laufe der Zeit den fluoreszierenden Farbstoff bewegt sich durch die Hirnkammern und Neuroepithel abgebildet. Der Abstand der Farbstoff vorne bewegt sich weg von der basalen (nicht-luminalen) Seite des Neuroepithel über die Zeit quantifiziert und ist ein Maß für neuroepithelialen Permeabilität (Abbildung 1). Wir beobachten, dass Farbstoffe 70 kDa und kleiner wird durch die Neuroepithel bewegen und kann detected außerhalb des embryonalen Zebrafischgehirn bei 24 hpf (Abbildung 2). Dieser Farbstoff Retention Assay kann verwendet werden, um neuroepithelialen Permeabilität in einer Vielzahl von unterschiedlichen genetischen Hintergründen zu analysieren, zu verschiedenen Zeiten während der Entwicklung, und nach ökologischen Störungen. Es kann auch nützlich sein bei der Prüfung pathologischen Anhäufung von CSF. Insgesamt ermöglicht diese Technik die Ermittler, um die Rolle und Regulierung der Durchlässigkeit während der Entwicklung und Krankheit zu analysieren.

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

Wir beschreiben eine Live ganze Tier quantitative Messung für die Durchlässigkeit des embryonalen Zebrafischgehirn. Die Technik analysiert die Fähigkeit, Zerebrospinalflüssigkeit und Moleküle unterschiedlichen Molekulargewichts in den neuronalen Schlauchlumen behalten und quantifiziert ihre Bewegung aus der Ventrikel. Diese Methode ist nützlich zur Bestimmung von Unterschieden in epithelialen Permeabilität und Reifung in der Entwicklung und Krankheit.

Ein Assay zur Durchlässigkeit des Zebrafisch-Embryos Neuroepithel

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

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Neuroscience

9.4K Aufrufe.  Massachusetts Institute of Technology. Wir beschreiben eine Live ganze Tier quantitative Messung für die Durchlässigkeit des embryonalen Zebrafischgehirn. Die Technik analysiert die Fähigkeit, Zerebrospinalflüssigkeit und Moleküle unterschiedlichen Molekulargewichts in den neuronalen Schlauchlumen behalten und quantifiziert ihre Bewegung aus der Ventrikel. Diese Methode ist nützlich zur Bestimmung von Unterschieden in epithelialen Permeabilität und Reifung in der Entwicklung und Krankheit.

Serie: 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.

Gründung Embryonale Maus Neural Stem Cell Culture Mit dem Neurosphäre 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 ...

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Neurowissenschaften

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.

Die Neuroblast Assay: Ein Test für die Erzeugung und Anreicherung von neuronalen Vorläuferzellen aus neuralen Stammzellen differenzieren Progeny mittels Durchflusszytometrie

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.

Hochauflösende Live-Bildgebung von Zell-Verhalten in der Entwicklung von Neuroepithel

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.

Manuelle Drainage der Zebrafisch Embryonale Hirnkammern

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 Live-Imaging von Single Cell Divisionen in Maus Neuroepithel

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.

Patch-Clamp-Aufnahmen aus embryonalen Zebrafisch-Mauthner-Zellen

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.

Live-Imaging der Mitose in der Entwicklung von embryonalen Maus-Cortex

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

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforated-Patch-Clamp-Aufzeichnung der Maus Riechzellen in Intact Neuroepithel: Funktionelle Analyse von Neuronen, die eine bestimmte Geruchsrezeptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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.

Ein Test zur Messung der Wirkung von Ethanol auf die Fortbewegungsgeschwindigkeit der<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.

Elektrophysiologische Verfahren zur Aufzeichnung von intrazellulärer Spannung Antworten von<em&gt; Drosophila</em&gt; Fotorezeptoren und Inter auf Lichtreize<em&gt; In Vivo</em