Wir möchten Dr. Jeffrey Rothstein für die Bereitstellung von BAC GLT1 eGFP Mäusen und GLT1 Antikörper danken; Tufts Center for Neuroscience Research (NIH P30 NS047243; PI, Rob Jackson) für die Bereitstellung von wertvollen Core Facilities; New Fakultät Rekrutierung Zuschuss (NIH P30 5P30NS069254-02 , PI, Phil Haydon) in Tufts Neuroscience Department.

Ein. Versammlung der Mikrofluidik Kultur Kammer (MCP) <ol><li> MCP (Abbildung 1) offenen Kammern für kompartimentierten Kulturen verschiedener Zelltypen 4 ausgebildet ist. Es hat typischerweise zwei Abteile, die durch die mittleren Kanäle (3 um im Durchmesser) verbunden sind. Versammlung des MCP mit Glasboden-Gerichte ist zur Vorbereitung Kulturen und anschließende Bildanalyse. </li><li> Erstens Mantel sterilen Glasbodengondel Gerichte mit Polyornithin (Sigma-Aldrich, 1 mg / ml) in Natriumtetraborat (Sigma-Aldrich, 10 mM pH 8,5) gelöst und über Nacht bei 37 ° C inkubiert </li><li> Am folgenden Tag wurden die beschichteten Glasboden Gerichte dreimal mit ddH 2 O gewaschen und getrocknet unter der sterilen Abzug. Die Glasbodengondel Schalen wurden dann weiter mit Laminin (Sigma-Aldrich, 1 mg / ml) gewaschen und unter UV-Licht 1 Stunde. Beschichtete Platten sind gebrauchsfertig oder können bei -20 ° C bis zur Verwendung gelagert werden. Diese Beschichtung eineswieder notwendig, für die Beschichtung Neuronen und Astrozyten in der Co-Kultur. </li><li> Die Kultur Plattform zusammenzubauen, wurden mikrofluidischen Kultur Plattformen auf der beschichteten Glasbodengondel Gerichte mit zentralen Verbindungskanäle an seiner Unterseite angeordnet, um eine Abdichtung zu bilden. Regelmäßige Neuron oder Astrozyten Nährböden wurden zugesetzt (von den Zell Plattierungsflächen) auf beiden Seiten (Fig. 1) in der zusammengebauten MCP und inkubiert für 2-4 Stunden bei 37 ° C, um sicherzustellen, dass keine Undichtigkeit zwischen MCP und dem Glas- Boden Gericht. Wir haben mit Medium vor dem Plattieren Zellen getestet, um sicherzustellen, dass kein Leck. Die Montage der MCP auf dem Glasboden-Gericht sollte vor Gebrauch frisch hergestellt werden. </li></ol> 2. Vorbereitung neuronaler Kultur und Induktion der neuronalen Axonen in Assembled MCP <ol><li> Kortikalen neuronalen Zellkulturen wurden frisch aus embryonalen 14-16 Tage alten Maus Gehirn vorbereitet. Das Neuron Kulturmedium Neurobasalmedium, 2% B27 neurobasa zusammenl Ergänzung, 2 mM Glutamin durch Zugabe von 1% der 100x GlutaMAX und 1% Penicillin-streptomysin. Nach Dissektion der Maus Gehirn, wurde die Meningen aus der Rinde unter dem Binokular entfernt. Das Gewebe wurde dann mit einer Rasierklinge zu kleinen Blöcken Gewebe zu machen, um den Oberflächenbereich zu erhöhen ausgesetzt Trypsin zerkleinert. Tissue Blöcke wurden trypsiniert (Sigma-Aldrich, 0,05% Trypsin) für 10 min in einem 37 ° C Wasserbad und anschließend dissoziiert leicht durch Verreiben mit einem feuerpolierte Pasteur Pipette. Dissoziierten Zellen wurden durch eine 70 um Sieb gefiltert, um klare neuronalen Zellsuspension zu sammeln. </li><li> Neuronen (2-3 x 10 6 / ml, 150 ul) wurden auf die Plattierungsflächen Zelle auf der rechten Seite (Figur 1) des zusammengebauten MCP ausplattiert so daß Neuronen in der Zelle Retentionsbereich (Abbildung 1) befestigen kann. Frisch hergestellten Neuronen wurden mit Neuron Plattierungsmedium, welches mit 5% fötalem Rinderserum (Hyclone) in die Regulierung wird ausplattiertr Neuron Kulturmedium. Da nur die Neuronen, die in der Zelle Retentionsbereich befestigen können Axone in die andere Seite des zusammengebauten MCP durch zentrale Kanäle wachsen, ist es wichtig zu Platte hoher Dichte von Neuronen in der Zelle zu gewährleisten, Plattierungsfläche genug Neuronen zur gebunden sind Zellrückhaltung Bereich. Ein repräsentatives Bild von hoher Dichte von neuronalen Axonen ist in 2A gezeigt. </li><li> Am folgenden Tag wurde das Neuron Plattierungsmedium durch die regelmäßige Neuron Kulturmedium ersetzt. Ändern Medium wurde durch vorsichtiges Absaugen des Mediums von der Zelle Plattierungsfläche der Kammer erreicht, aber nicht aus der Zelle Retentionsbereich des zusammengebauten MCP., Um Luftblasen in der Zelle Retentionsbereich und den zentralen Verbindungskanäle vermeiden Luftblasen stark hemmen das Axon Auswuchs und anschließenden Eintritt in die zentralen Kanäle in der MCP. Glial-cell derived neurotrophic factor (GDNF, 10-20 ng / ml) wurde auch auf der anderen (linken) Seite angefügtder Kammer am selben Tag um die Induktion von Axon Auswuchs 5 aus dem neuronalen Seite und quer durch zentralen Kanäle der versammelten MCP (2A) zu erleichtern. Angemessene Menge der spontanen Neuritenwachstum ohne GDNF wird ebenfalls aus dem neuronalen Seite und quer durch zentralen Kanäle der zusammengebauten MCP beobachtet. Axone, die in den zentralen Kanal geben sind oft 2-3 Tage nach dem Ausplattieren beobachtet. Sobald die Axone in den zentralen Kanal geben, sie in der Regel geben die andere Seite innerhalb eines Tages. Axone sind normalerweise für mindestens eine Woche nach der Eingabe des intakten anderen Seite. </li></ol> 3. Zugabe von Astrozyten zu MCP eine unterteilte Co-Kultur zu etablieren Primäre Astrozytenkulturen wurden aus dem P1-3 Maus pup Gehirn vorbereitet. Das Gehirn Dissoziation Verfahren ist ähnlich dem neuronalen Zellisolation oben beschriebenen Verfahrensweise. Astrozyten wurden zunächst in 10 cm Schüssel, wurden vorab überzogenMit Polyornithin (Sigma-Aldrich, 1 mg / ml) beschichtet. Die Astrozyten Kulturmedium (DMEM, 10% fetales Rinderserum, 1% Penicillin-streptomysin) wurde jeden Tag für die nächsten zwei Tage, um die Ablagerungen zu entfernen. Danach wurde das Medium alle drei Tage gewechselt. <ol><li> Astrozyten 90% konfluent geworden und bilden eine Monoschicht nach 7 Tagen. Konfluente Astrozyten wurden mit Trypsin behandelt und 150 ul des resuspendierten Astrozyten (1x10 6 / ml) wurden in die Zelle Plattierungsfläche des linken Seite des zusammengebauten MCP erneut ausplattiert wenn Axone etwa einzugeben sind oder einfach in der linken Seite des eingegebenen die versammelte MCP. Astrozyten wurden in der Regel beschichtet 4-5 Tage nachdem die Neuronen waren vergoldet. GDNF wurde zuerst vor der erneuten Beschichtung der Astrozyten in die linke Seite des MCP entfernt. Re-plattierten Astrozyten wurden in der Zelle Retentionsbereich angebracht, wie durch die Immunfärbung GFAP (Abbildung 1) gezeigt. Nur minimale Strömung des Mediums zwischen beiden Seiten der Kammern (bestimmt durch Fluoreszenz dja) wurde auf die MCP gefunden, wie zuvor 4,6 gezeigt. </li><li> Astrozyten 90% konfluent geworden und bilden eine Monoschicht nach 7 Tagen. Konfluente Astrozyten wurden mit Trypsin behandelt und 150 ul des resuspendierten Astrozyten (1 x 10 6 / ml) wurden in die Zelle Plattierungsfläche des linken Seite des zusammengebauten MCP erneut ausplattiert wenn Axone zu betreten oder nur in den linken eingegeben sind Seite des zusammengebauten MCP. Astrozyten wurden in der Regel beschichtet 4-5 Tage nachdem die Neuronen waren vergoldet. GDNF wurde zuerst vor der erneuten Beschichtung der Astrozyten in die linke Seite des MCP entfernt. Re-plattierten Astrozyten wurden in der Zelle Retentionsbereich angebracht, wie durch die Immunfärbung GFAP (Abbildung 1) gezeigt. Nur minimale Strömung des Mediums zwischen beiden Seiten der Kammern (bestimmt durch Fluoreszenzfarbstoffe) wurde auf die MCP gefunden, wie zuvor 4,6 gezeigt. </li><li> Nach der erneuten Beschichtung der Astrozyten, Axone, die die linke Seite des zusammengesetzten MCP eingegebenen direkt mit dem astr interagierenocytes entweder durch direkten Kontakt oder axonalen Freisetzung von löslichen Faktoren. Immunfärbung von astroglialen Plasmamembran Glutamattransporter GLT1 und neuronalen βIII-Tubulin wurde durchgeführt, um die Wechselwirkung zwischen den Axonen und die Astrozyten visualisieren, wie in 2D-F dargestellt. </li></ol>

<ol>
	<li>Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+signaling+in+the+development+and+plasticity+of+neural+circuits.">Neuron-astrocyte signaling in the development and plasticity of neural circuits.</a> Neuro-Signals. 16, 278-288 (2008).</li><li>Paixao, S., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+communication+and+synaptic+plasticity.">Neuron-astrocyte communication and synaptic plasticity.</a> Current opinion in neurobiology. 20, 466-473 (2010).</li><li>Fields, R. D., Stevens-Graham, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+neuron-glia+communication.">New insights into neuron-glia communication.</a> Science. 298, 556-562 (2002).</li><li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature. 1, 2128-2136 (2006).</li><li>Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuromuscular+synapse+development+by+glial+cell+line-derived+neurotrophic+factor+and+neurturin.">Regulation of neuromuscular synapse development by glial cell line-derived neurotrophic factor and neurturin.</a> The Journal of biological chemistry. 277, 10614-10625 (2002).</li><li>Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+regulation+of+astroglial+excitatory+neurotransmitter+transporter+GLT1.">Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1.</a> Neuron. 61, 880-894 (2009).</li><li>Regan, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+promoter+activity+reveal+a+differential+expression+and+physiology+of+glutamate+transporters+by+glia+in+the+developing+and+mature+CNS.">Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS.</a> The Journal of neuroscience. 27, 6607-6619 (2007).</li><li>Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+glutamate+transporter+subtype+expression+in+astrocytes.">Neuronal regulation of glutamate transporter subtype expression in astrocytes.</a> The Journal of neuroscience. 17, 932-940 (1997).</li><li>Schlag, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+glial+Na+-dependent+glutamate+transporters+by+cyclic+AMP+analogs+and+neurons.">Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.</a> Molecular pharmacology. 53, 355-369 (1998).</li><li>Ponomarev, E. D., Novikova, M., Maresz, K., Shriver, L. P., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+culture+system+that+supports+adult+microglial+cell+proliferation+and+maintenance+in+the+resting+state.">Development of a culture system that supports adult microglial cell proliferation and maintenance in the resting state.</a> Journal of immunological. 300, 32-46 (2005).</li><li>Espinosa-Jeffrey, A., Wakeman, D. R., Kim, S. U., Snyder, E. Y., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+system+for+rodent+and+human+oligodendrocyte+specification,+lineage+progression,+and+maturation.">Culture system for rodent and human oligodendrocyte specification, lineage progression, and maturation.</a> Current protocols in stem cell biology. Chapter 2, (2009).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li><li>Debanne, D., Rama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+shape+axonal+signaling.">Astrocytes shape axonal signaling.</a> Science signaling. 4, pe11 (2011).</li></ol>

Keine Interessenskonflikte erklärt.

Die MCP basierte Neuron und Astrozyten Co-Kultur-System ermöglicht Dissektion detaillierte Neuron Astroglia Signalwege, indem nur die Axone leiten die zentralen Kanäle und Interaktion mit den Astrozyten. Diese Co-Kultur-System bequem eingerichtet werden mit herkömmlichen Neuron und Astrozyten Kultur Verfahren. Beschrieben wir auch eine praktische Anwendung dieser Co-Kultur-System durch den Einsatz eines eGFP basierte Reporter für den Nachweis Axon-abhängige GLT1 Promotoraktivierung in Astrozyten. <p class="jove...

<table border="1"><tbody><tr><td> Fötales Rinderserum </td><td> Hyclone </td><td> SH30070.03 </td><td> für die Beschichtung Neuron für Neuron cutlure Medium </td></tr><tr><td> Fötales Rinderserum </td><td> Sigma-Aldrich </td><td> F4135 </td><td> für Astrozyten Kulturmedium </td></tr><tr><td> Glialem Nerven Faktor </td><td> R &amp; D-Systeme </td><td> 212-GD </td><td> Anwenden 10-20 ng / ml bis Neuron Seite der Kammer </td></tr><tr><td> Dulbecco modifiziertes Eagle-Medium hoher Glukose </td><td> Sigma-Aldrich </td><td> 11995 </td><td></td></tr><tr><td> 70 mm Zellsieb </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Sterile Glasboden Gericht </td><td> MatTek Konzern </td><td></td><td></td></tr><tr><td> Mikrofluidische Kultur Plattformen </td><td> Xona Microfluidics LLC </td><td> SND150 </td><td></td></tr><tr><td> 6 Vertiefungen der Kulturplatte </td&gt; <td> CELLSTAR </td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> Neuron Kulturmedium <ul><li> Neurobasalmedium </li><li> 2% B27 Neurobasal Ergänzung </li><li> 2 mM Glutamat durch Zugabe von 1% 100x GlutaMAX </li><li> 1% Penicillin-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Neuron Nährboden für Galvanisierzelle <ul><li> Neurobasalmedium </li><li> 2% B27 Neurobasal Ergänzung </li><li> 2 mM Glutamat durch Zugabe von 1% 100x GlutaMAX </li><li> 1% Penicillin-streptomysin </li><li> 5% Fötales Rinderserum SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> Astrocyte Kulturmedium <ul><li> Dulbecco modifiziertem eagle mittel hoch Glukose </li><li> 10% Fötales Rinderserum F4135 </li><li> 1% Penicillin-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Tabelle 1. Materialien in der mikrofluidischen Kultur plattformbasierte neuronalen Axonen und Glia Co-Kultursystem benutzt. </td></tr></tbody></table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

Ordnungsgemäße Neuron zu Glia-Wechselwirkung ist entscheidend für physiologische Funktion des zentralen Nervensystems (ZNS). Diese bidirektionale Kommunikation wird raffiniert durch spezifische Signalwege zwischen Neuron und Glia 1,2 vermittelt. Identifizierung und Charakterisierung dieser Signalwege ist wesentlich für das Verständnis, wie Neuron zu Glia-Wechselwirkung Formen CNS Physiologie. Zuvor haben Neuron und Glia Mischkulturen weithin zum Testen und Charakterisieren Signalwege zwischen Neuronen und Glia ausgenutzt. Was wir von diesen Präparaten und anderen Werkzeugen in vivo gelernt hat jedoch vorgeschlagen, dass die gegenseitige Signalisierung zwischen Neuronen und Glia häufig in speziellen Kompartimenten innerhalb von Neuronen (dh Axon, Dendrit oder Soma) 3 aufgetreten ist. Daher ist es wichtig, eine neue Kultur System, Trennung der Fächer ermöglicht neuronalen Entwicklung und speziell untersucht die Wechselwirkung zwischen Glia und neuronal Axone / Dendriten. Darüber hinaus ist das herkömmliche System Mischkultur nicht fähig Differenzieren der löslichen Faktoren und direkte Membrankontakt Signalen zwischen Neuronen und Gliazellen. Außerdem fehlt die große Menge von Neuronen und Gliazellen im herkömmlichen Co-Kultursystem die Auflösung notwendig, die Wechselwirkung zwischen einem einzelnen Axon und einer Gliazelle beobachten. In dieser Studie beschreiben wir eine neue Axone und Glia Kokultur System mit der Verwendung eines mikrofluidischen Kulturplattform (MCP). Auf diese Co-Kultur-System sind Neuronen und Gliazellen in zwei getrennte Kammern, die durch mehrere zentrale Kanäle angeschlossen sind, kultiviert. In diesem mikrofluidischen Kultur-Plattform, kann nur neuronale Prozesse (insbesondere Axone) die Gliazellen Seite durch den zentralen Kanälen geben. In Kombination mit leistungsfähigen fluoreszierendes Protein Etikettierung, ermöglicht dieses System eine direkte Untersuchung von Signalwegen zwischen axonalen / dendritischen und Glia Interaktionen, eine solches Axon-vermittelten Transkriptionsregulation in Glia, Glia-vermittelte Rezeptor-in neuronalen Terminals und Glia-vermittelten Axon Wachstum. Die schmalen Durchmesser der Kammer ebenfalls signifikant verbietet die Strömung des Neurons angereicherten Mediums in die Kammer gliale, Erleichterung Sondieren des direkten Membran-Protein-Wechselwirkung zwischen Axone / Dendriten und glialen Oberflächen.

Zeitraffer-Aufnahmen Analyse der Axon-induzierte GLT1 Promotoraktivierung in Astrozyten Der Fächerwagen Neuron und Astrozyten Kokultur System erlaubt nur die neuronalen Prozesse, insbesondere die Axone, um selektiv mit Astrozyten interagieren. Nach der erfolgreichen Etablierung der Axon und Astrozyten (oder andere Gliazellen) Co-Kultur im zusammengebauten MCP, können verschiedene Arten von Axon-Glia Interaktionen wie studiert werden; Axon-induzierte Aktivierung von Astrozyten-Gen-Promotor-Akt...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

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

Diese Studie beschreibt die Verfahren für die Einrichtung einer neuartigen neuronalen Axon und (astro) Glia Co-Kultur-Plattform. Auf diese Co-Kultur-System wird Manipulation direkte Wechselwirkung zwischen einem einzelnen Axon (und einzigen Gliazelle) durchführbar ist, so dass mechanistischen Analyse des gegenseitigen Neuron zu glialen Signalisierung.

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 ...

imaging-analysis-neuron-to-glia-interaction-microfluidic-culture

Research

JoVE Journal

Neuroscience

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

14.9K Aufrufe.  Tufts University. Diese Studie beschreibt die Verfahren für die Einrichtung einer neuartigen neuronalen Axon und (astro) Glia Co-Kultur-Plattform. Auf diese Co-Kultur-System wird Manipulation direkte Wechselwirkung zwischen einem einzelnen Axon (und einzigen Gliazelle) durchführbar ist, so dass mechanistischen Analyse des gegenseitigen Neuron zu glialen Signalisierung.

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

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 ...

Forschung

Neurowissenschaften

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

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

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 ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Die indirekte Neuron-Astrozyten Cokultur Assay: Ein<em&gt; In Vitro</em&gt; Für die detaillierte Untersuchung von Neuron-Glia Interaktionen Set-up

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Isolation und Kultur der primär neuronen und Glia von Adult Ratte Harnblase

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

Gezielte neuronale Verletzung für die nicht-invasive Trennung von Gehirn-Schaltung

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissektion und Isolierung von murinen Glia aus mehreren Regionen des zentralen Nervensystems

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ein Zellkulturmodell zum Studium der Rolle von Neuron-Glia-Interaktionen in Ischämie

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging in Maus Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Visualisierung von Verschiebungen auf dem Neuron-Glia-Schaltkreis mit der Kalzium-Bildgebungstechnik

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Transplantation von humaninduzierten pluripotenten Stammzell-abgeleiteten Mikroglia im immunkompetenten Mäusegehirn über nicht-invasiven transnasalen Weg

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...