The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.

Alle Mäuse wurden nach Schweizer Tierschutzgesetz und in Übereinstimmung mit den Vorschriften des kantonalen Veterinäramt, Zürich gepflegt und behandelt werden. 1. Herstellung von Dissection Material, Kultur, Medien, Mikrofluidiksysteme <ol><li> Autoklaven Mikrodissektion Pinzette und Schere (121 ° C, Sterilisationszeit: 20 min) und speichert sie in einem sterilen Behälter. </li><li> Sterilisieren Deckgläser (24 mm x 24 mm) durch Inkubation in 1 M HCl 24 h lang auf einem Rundschüttler bei 37 ° C. Dreimal mit 99% Ethanol waschen sie mit sterilem, destilliertem H 2 O und dreimal. Dry dann die Deckgläser auf 37 ° C oder unter sterilen Flow-Haube. Schließlich Autoklaven oder setzen Sie die Deckgläser mit UV-Licht (30 min), um die Sterilisation zu vervollständigen. Deckgläser kann dann in Ethanol 70% gelagert werden. </li><li> Entfernen Sie vorsichtig die AXIS Axon Isolation Geräte aus der Verpackung mit Hilfe einer sterilen Pinzette und legen Sie sie in eine sterile Petrischale. </li&gt; <li> Mit einem sterilen Biopsie Stempel (ø: 1 mm) schaffen ein Loch pro Probe kultiviert werden (Abbildung 1) in Übereinstimmung mit den Kulturkammern. HINWEIS: stanzen nicht zu nahe an den Mikrorillen, da sie möglicherweise durch den Druck aufgebracht beschädigt werden. </li><li> Sterilisieren Sie die AXIS Axon Isolation Devices durch immerging sie in Ethanol 70%. Dry dann AXIS Axon Isolation Geräte und Deckgläser vollständig unter einer sterilen Flow-Haube. Warten Sie mindestens 3 Stunden, bevor Sie fortfahren. HINWEIS: unvollständige Trocknung wird in fehlerhaften Montage der mikrofluidischen Bauteilen führen. </li><li> Legen Sie jedes Deckglas in eine 35 mm Petrischale oder in einen Brunnen in einem 6-wells Platte. </li><li> Setzen Sie den AXIS Axon Isolation Gerät auf dem Deckglas und drücken Sie vorsichtig, aber fest mit einer Zange mit gebogenen Enden, um die volle Haftung zwischen der Trennvorrichtung und dem Deckglas zu ermöglichen. </li><li> In jeder Kulturkammer, einer Pipette 150 ul Poly-D-Lysin (0,1 mg / ml in sterilem destilliertem H 2O). Legen Sie die mikrofluidischen Vorrichtungen unter Vakuum für 5 Minuten, um die gesamte Luft aus den Kulturkammern zu entfernen. </li><li> Wenn Luft kann noch innerhalb der Kammern, re-Pipette die Poly-D-Lysin-Lösung in die Kammern zu sehen. </li><li> Die Vorrichtungen mit Poly-D-Lysin O / N bei 37 ° C. </li><li> Waschkammern dreimal mit sterilem destilliertem H 2 O. </li><li> Füllen Kammern mit 150 ul Laminin-Arbeitslösung (Sigma-Aldrich, 5 ug / ml in PBS oder Serum-freiem Medium), und Inkubation O / N bei 37 ° C. </li><li> Vorbereitung 50 ml Medium für Trigeminalganglien Kulturen 37, wie folgt zusammen: 48 ml Neurobasalmedium, 1 ml B-27, 100 U / ml Penicillin / Streptomycin, 2 mM L-Glutamin, 5 ng / ml Nervenwachstumsfaktor (NGF) , 12.25 Cytosinarabinosid. </li><li> Bereiten Sie 50 ml Medium für Zahnkeim Kulturen 37, wie folgt zusammen: 40 ml DMEM-F12, 10 ml fötalem Rinderserum (FBS, Endkonzentration: 20%), 100 U / ml Penicillin / Streptomycin, 2 mM L-Glutamin, 150 &amp; mgr; g / ml Ascorbinsäure. </li></ol> 2. Mouse Embryo Erzeugung und Dissection <ol><li> Bestimmen Sie embryonalen Alter nach Vaginalpfropf (Vaginalpfropf: embryonalen Tag der Entwicklung 0,5, E0.5) und bestätigen Sie es via morphologischen Kriterien. Für dieses Protokoll verwenden wir in der Regel E14.5-E17.5 Mäuseembryonen. </li><li> Reinigen Sie das Sezierung Bereich und die Stereoskop mit Ethanol 70%. </li><li> Opfer der schwangeren Mutter über Genickbruch. Blockieren Sie den Hals der Maus mit dem ersten und zweiten Finger auf ein Gitter, und ziehen Sie mit der Entscheidung der Schwanz. </li><li> Präparieren Sie die Haut um die Unterbauch, und öffnen Sie den Unterleib mit einer Schere. Suchen Sie die Gebärmutter: während dieser späten Stadien der Schwangerschaft, der Uterus reichlich die Bauchhöhle zu füllen. </li><li> Sezieren die Gebärmutter und in eine Röhre mit PBS auf Eis gefüllt. Wenn auf Eis kann das Gewebe für mehrere Stunden verlassen werden. Entsorgen Sie die Leiche der Mutter gemäß gültiger Richtlinien </li> &lt;li&gt; sezieren die Embryonen aus der Gebärmutter und sie von ihrer extra Gewebe. Legen Sie die Embryonen in PBS auf Eis. </li><li> Enthaupten die Embryonen mit einer Schere, und trennen Sie den Unterkiefer aus dem Rest des Kopfes mit Mikrodissektion Schere (2A). Genau Entfernen Sie die Unterkiefer, ohne Beschädigung der Trigeminalganglien; dieser letzteren in der Nähe des Unterkiefers lokalisiert ist ihre versehentliche Beschädigung möglich. Erhalten Sie den Unterkiefer und den Rest des Kopfes in kaltem PBS, auf Eis. </li><li> Um TG sezieren, nehmen Sie den Kopf und legen Sie sie auf eine Dissektion Glaspetrischale, die zuvor mit kaltem PBS gefüllt. Unter Verwendung der Zange, entfernen Sie die Haut und den Schädel. Entfernen Sie dann das Telencephalon und das Kleinhirn, indem Zange unterhalb des Telencephalon und Aufzug; das Telencephalon und das Kleinhirn zusammen drehen, so dass die Unterseite des Schädels freigelegt. </li><li> Lokalisierung der trigeminalen Ganglien (in 2B gezeigt). Verwenden Sie die Pinzetteum die TG von den Trigeminus trennen. Beseitigen Sie die Reste der Trigeminus-Projektionen mit Hilfe der Dissektion Nadeln wie Messer. Legen Sie die seziert TG in einer Petrischale mit kaltem PBS gefüllt und halten sie auf Eis. </li><li> Embryonale Zähne sezieren, legen Sie den Unterkiefer, die zuvor aus dem Schädel getrennt auf die Dissektion Glaspetrischale mit kaltem PBS gefüllt. Verwendung Dissektion Nadeln wie Messer, entfernen Sie die Zunge und die Haut rund um den Kiefer. Trennen Sie den linken und den rechten Hemi-Backen durch Schneiden entlang der Mittellinie des Kiefers. Die Zahnanlagen sind gut sichtbar, wie in 1C gezeigt. Isolieren Sie die Zahnkeime mit Dissektion Nadeln und entfernen Sie den Überschuss an nicht-Zahngewebe. Legen Sie die seziert Zahnkeime in einer Petrischale mit kaltem PBS gefüllt und halten sie auf Eis. </li></ol> 3. Mikrofluidik-Co-Kulturen <ol><li> Nach der Sektion, entfernen Laminin aus den mikrofluidischen Systemen. Füllen der Kammern mit 200 ul der respective Medien. </li><li> Mit einer Pinzette, übertragen vorsichtig seziert die TG und Zahnkeime in die Löcher erzeugt durch Stanzen (1D). Stellen Sie sicher, dass die Zahnkeime nicht schwimmen, und dass sie sinken, bis sie die Deckgläser. </li><li> Kultur die Proben in Brutschrank bei 37 ° C, 5% CO 2. </li><li> Ändern Sie das Kulturmedium alle 48 Stunden. Die Kammern leeren nicht vollständig, und nicht direkt Pipette nicht in die Kulturkammern. Vollständige Entleerung der Kammern würde zur Bildung von Luftblasen in den Kammern führen; direkte Pipettieren in die Kammer würde in axonalen Schäden führen. Um diese Probleme zu vermeiden, entfernen Sie das Medium zeigt die Pipette in Richtung der Außenseite der Vertiefungen; Ähnlich pipettieren frischem Medium auf der Seite der Vertiefungen angeordnet gegenüber den Kammern. </li><li> Während der Kulturzeit kann Co-Kulturen leicht durch Zeitraffer-Mikroskopie abgebildet werden. Co-Kulturen für 10 Tage lang aufrechterhalten werden. </li><li> Nach der Kultur period, waschen Sie die Kammern durch Pipettieren von 150 ul PBS in einem gut pro Kammer und ließ PBS durch die Kammern fließen dreimal. </li><li> Entfernen Sie die PBS und fixieren Sie die Proben durch Pipettieren von 150 ul Para 4% (in PBS) in einer gut per Kammer; Inkubation bei Raumtemperatur für 15 min. </li><li> Zweimaliges Waschen der Kammern mit PBS, wie in 3.6 beschrieben. </li><li> Fahren Sie mit der weiteren Analyse. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in physiology. 5 (August), (2014).</li><li>Connor, R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+maxillary+factor,+a+maxillary+process-derived+chemoattractant+for+developing+trigeminal+sensory+axons.">Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons.</a> Neuron. 24, 165-178 (1999).</li></ol>

The authors declare that they have no competing financial interests.

Vorherige in vitro-Studien der Zahn Innervation wurden auf herkömmliche Kokulturen Trigeminalganglien und Zahngewebe oder Zellen 26,28,29 basiert. Diese Studien wurden durchgeführt, um in erster Linie untersuchen die attraktive Wirkung dieser Zellen oder Gewebe auf sensorische Axone 38. Zwar bringen bedeutende Fortschritte auf dem Gebiet wurden mehrere technische Fragen aufgeworfen. Zahnkeime beginnen, nach ein paar Tagen der Kultur 37 degenerieren. Basierend auf diesen Beobac...

Innervation spielt eine Schlüsselrolle in der Entwicklung, Homöostase und Regeneration von Organen und Geweben, 1,2. 5 - Weiterhin ist Innervation bei der Regulation der Stammzellproliferation, Mobilisierung und Differenzierung 3 beteiligt. In der Tat, die jüngsten Studien in Geweben des orofazialen Komplexes realisiert haben gezeigt, dass parasympathischen Nerven sind für epithelialen Vorläuferzellen Funktion notwendig bei der Entwicklung und Regeneration der Speicheldrüsen 6,7. Ebenso hat sich gezeigt, dass Innervation für die Entwicklung und Wartung von Gaumen 8 notwendig - 11. Somit ist es wichtig, die noch vernachlässigt Rollen Innervation bei der Entwicklung anderer wichtiger orofacial Organen und Geweben wie Zähne analysieren. Trotz der Innervation reichen zweiten Zähne und im Gegensatz zu allen anderen Organen und Geweben des Körpers, developing Zähne beginnen, in den frühesten Stadien der postnatalen innerviert werden. Zähne entwickeln als Folge der sequentiellen und gegenseitige Wechselwirkungen zwischen oralen Ektoderm und Schädel Neuralleiste stammenden Mesenchym. Diese Wechselwirkungen führen zu Epithelzellen abgeleitete Adamantoblasten und Mesenchym abgeleitete Odontoblasten, die für die Bildung von Zahnschmelz und Dentin, die jeweils 12 verantwortlich sind. Sensorischen Nerven vom Trigeminalganglien und sympathischen Nerven aus der oberen Halsganglien innervieren die Erwachsenen die Zähne 13-15. Während der Embryogenese, Nervenfasern aus dem Trigeminalganglien Projekt gegenüber den Entwicklungszahnkeime ausgeht und progressiv umgeben sie aber nicht in die Papille Mesenchym 13 eindringen. Nervenfasern in das Zahnmark Mesenchym in fortgeschrittenen Entwicklungsstadien, die mit odontoblast Differenzierung und Dentin Matrixablagerung 16 zu korrelieren. Pulpa Innervation ist komplETED bald nach dem Zahndurchbruch in der Mundhöhle 13. Frühere Studien haben gezeigt, dass verschiedene Semaphorine und Neurotrophine sind an der Regulation der Innervation bei Odontogenese 16 beteiligt - 19. Frühere Studien haben klar gezeigt, dass Innervation ist eine Voraussetzung für die Zahnbildung bei Fischen 20. Neuere Studien haben gezeigt, daß die Homöostase der Zahn Mesenchym-Stammzellen in Maus-Schneidezähne wird durch sensorische Nerven über Sekretion von Sonic Hedgehog (Shh) 21 reguliert wird. Dennoch ist die Rolle der Innervation in Zahn Initiierung, Entwicklung und Regeneration immer noch sehr umstritten in Säugetieren 22-24. Eine Vielzahl von in-vivo-Studien haben wichtige Informationen über die Muster der Innervation der Zahngewebe während der Entwicklung und Reparaturprozesse von verschiedenen Tiermodellen 13,25,26 vorgesehen. Jedoch sind die meisten von diesen auf angemesseneSchmerzen nicht optimal sind, um die molekularen Grundlagen der Wechselwirkungen zwischen Nervenfasern und Ziel Organe und Gewebe zu markieren. Co-Kulturen stellen eine wertvolle Methode zu untersuchen und zu manipulieren, die Wechselwirkungen zwischen den Nervenfasern und die Zähne in eine kontrollierte und isolierte Umgebung 26-29. Zugleich ist Kokultivierung unterliegen verschiedenen technischen Anpassungen. Zum Beispiel, Nerven und spezifische Zahngewebe (zB Pulpa, Zahnsäckchen, Zahn Epithel) erfordern oft unterschiedliche Kulturmedien, um Gewebeüberlebenszeit für längere Zeit garantieren 30 - 32. In den letzten Jahrzehnten haben sich herkömmliche Co-Kulturen mit dem gleichen Kulturmedium für sehr kurze Zeiträume (zB zwei Tage) durchgeführt wurde, um die anziehende oder abstoßende Wirkung der Entwicklungs Mund- und Zahngewebe an sensorischen Nervenfasern 27 zu untersuchen - 29.Jedoch Verlängerung der Kultivierungsperiode erforderlich, um die Auswirkungen der Innervation auf Zahn Morphogenese und Zelldifferenzierung zu untersuchen und die Dynamik von Nervenfasern innerhalb Verzweigungszielorgane zu untersuchen. Deshalb würde nicht zusammenhängende Co-Kulturen besser geeignet, um Studien über neuronale-Zahngewebe Interaktionen durchzuführen. Mikrofluidik-Systeme ermöglichen Co-Kulturen von Neuronen und verschiedene Zelltypen in ihren geeigneten Kulturmedien. In diesen Vorrichtungen werden Zahngeweben und Neuronen in verschiedenen Kammern getrennt, während gleichzeitig das Wachstum der Axone der neuronalen Zellkörper durch Mikrokanäle in Richtung der Kammer ihre Zielgewebe 33 enthält. Mikrofluidik-Vorrichtungen wurden bereits verwendet, um die Interaktionen zwischen Neuronen und Mikroglia 34,35 sowie von Zelle zu Zelle-Wechselwirkungen bei Krebs und Neovaskularisation 35 zu studieren. Darüber hinaus sind diese Systeme sind verwendet worden, um die Wechselwirkungen zwischen dors studierenal Wurzelganglien und Osteoblasten 36. Wir haben kürzlich gezeigt, dass trigeminalen Ganglien (TG) und der Zähne in der Lage sind, für längere Zeiträume zu überleben, wenn co-kultiviert in Mikrofluidik-Vorrichtungen 37. Darüber hinaus haben wir gezeigt, dass die Zähne von verschiedenen Entwicklungsstadien zu erhalten in diesen in vitro Bedingungen die gleichen abstoßend oder anziehend Auswirkungen auf Trigeminus innerviert, dass sie in vivo 37 zeigen. Dieses Protokoll enthält Informationen über eine einfache, leistungsfähige und flexible Möglichkeit, Co-Kultur-Ganglien / Nerven und Zielgewebe und die Rollen von spezifischen Molekülen auf solche Wechselwirkungen in einer kontrollierten und isolierten Umgebung zu studieren.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AXIS Axon Isolation Devices</td>
			<td>Millipore</td>
			<td>AX15010-TC</td>
			<td>Microchannels of different lenght are available</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse beta-NGF</td>
			<td>R&#38;D Systems</td>
			<td>1156-NG-100</td>
			<td>Human and Rat beta-NGF (R&#38;D Systems) are equivalent</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

Innervation spielt eine Schlüsselrolle in der Entwicklung, Homöostase und Regeneration von Organen und Geweben. Allerdings sind die Mechanismen, die diese Phänomene sind nicht gut noch verstanden. Insbesondere wird die Rolle der Innervation in der Zahnentwicklung und Regeneration vernachlässigt. Mehrere In-vivo-Studien haben wichtige Informationen über die Muster der Innervation der Zahngewebe während der Entwicklung und Reparaturprozesse von verschiedenen Tiermodellen zur Verfügung gestellt. Allerdings sind die meisten dieser Ansätze nicht optimal, um die molekulare Grundlage der Wechselwirkungen zwischen den Nervenfasern und die Zielorgane und Gewebe zu markieren. Co-Kulturen stellen eine wertvolle Methode zu untersuchen und zu manipulieren, die Wechselwirkungen zwischen den Nervenfasern und die Zähne in einer kontrollierten und isolierten Umgebung. In den letzten Jahrzehnten haben sich herkömmliche Co-Kulturen unter Verwendung des gleichen Kulturmediums für sehr kurze Zeiträume durchgeführt wurden (beispielsweise zwei Tage)um die anziehende oder abstoßende Wirkung der Entwicklungs Mund- und Zahngewebe an sensorischen Nervenfasern zu untersuchen. Jedoch Verlängerung der Kultivierungsperiode erforderlich, um die Auswirkungen der Innervation auf Zahn Morphogenese und Zelldifferenzierung zu untersuchen. Mikrofluidik-Systeme ermöglichen Co-Kulturen von Neuronen und verschiedene Zelltypen in ihren geeigneten Kulturmedien. Wir haben kürzlich gezeigt, dass Trigeminalganglien (TG) und die Zähne sind in der Lage, für eine lange Zeit überleben, wenn co-kultiviert in Mikrofluidvorrichtungen, und dass sie zu erhalten in diesen Bedingungen die gleiche Innervationsmuster, dass sie in vivo zeigen. Auf dieser Basis beschreiben wir, wie zu isolieren und Co-Kultur entwickeln Trigeminalganglien und Zahnkeime in einem mikrofluidischen Co-Kultur system.This Protokoll beschreibt eine einfache und flexible Möglichkeit, Co-Kultur-Ganglien / Nerven und Zielgewebe und die Rollen zu studieren spezifischer Moleküle auf solchen Wechselwirkungen in einem controlled und isolierten Umgebung.

Diese Ergebnisse zeigen, dass isolierte Trigeminalganglien kann in einem Abteil der mikrofluidischen Vorrichtung zu wachsen, und zusätzlich, dass die Entwicklung von den isolierten Zahnanlagen ist für einen langen Zeitraum in der anderen Kammer der mikrofluidischen Vorrichtung aufrechterhalten. Verschiedenen Kulturmedien sind in den zwei Kammern verwendet, und die Mikrorillen zwischen den beiden Kammern zu ermöglichen Verlängerung der Axone vom Ganglion trigeminale gegenüber den Entwicklungszahnanlagen dar....

Watch this Scientific Journal Video about Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices at JoVE.com

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Die Analyse der Entwicklung von Zahnkeim Innervation Mit Microfluidic Co-Kultur-Geräte

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

analysis-developing-tooth-germ-innervation-using-microfluidic-co

Research

JoVE Journal

Neuroscience

8.2K Aufrufe.  University of Zurich. Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Serie: Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In-vivo-Elektroporation von Entwicklung Maus Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Forschung

Neurowissenschaften

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminäre Co-Kultur von primären Ratten-kortikalen Neuronen und Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Ein Functional Motor Unit in der Kulturschale: Co-Kultur von Spinal Cord Explantate und Muskelzellen

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualisierung und genetische Manipulation von Dendriten und Dornen in der Maus Hirnrinde und im Hippocampus mit<em&gt; In utero</em&gt; Die Elektroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Imaging Analyse von Neuron zu Glia Interaktionen in Microfluidic Kultur Platform (MCP)-basierte Neuronale Axon und Glia Co-Kultur-System

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

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 ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funktionellen und morphologischen Bewertung der Membran Innervation durch phrenicus Motoneuronen

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

Hochauflösende Volume Imaging von Neuronen durch den Einsatz von Fluoreszenz-Ausschluss Methode und mikrofluidischen Geräten gewidmet

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Datenerfassung und -analyse in Brainstem Evokierte Reaktion Audiometrie bei Mäusen

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Die Analyse der Entwicklung von Zahnkeim Innervation Mit Microfluidic Co-Kultur-Geräte

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