This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

HINWEIS: Alle Tierverfahren werden nach den Richtlinien und wurden von der Institutional Animal Care und Use Committee (IACUC) an der Universität von Connecticut genehmigt worden. HINWEIS: Für diese Experimente konstitutive NG2cre 18 (JAX # 008533) und induzierbare NG2creER 16 (JAX # 008538) transgenen Mäusen gekreuzt, um Z / EG 19 (JAX # 003920) oder gtRosa26: YFP Reporter 20 (JAX # 006148) Leitungen, die jeweils verwendet wurden Bild zu NG2 Zellen und deren Nachkommen. Um das Bild zu reifen Oligodendrozyten wurden PLPDsRed transgenen Mäusen 21 verwendet. Für eine konsistente Überlebens, können Scheiben von Mäusen bis zu postnatalen Tag 10 sowohl aus dem Vorderhirn und Kleinhirn isoliert werden. 1. Scheibe Vorbereitung <ol><li> In einer Kultur, Haube, legen Gewebekulturplatten in sechs Well-Platten und 1 ml Scheibe Kulturmedium (Rezept in Reagenzien Abschnitt). Platzieren Sie die Platten in Zellkulturbrutschrank bei 37 ° C mit 5% CO 2 bei mindestens 2 h vor der Sektion. </li><li> Sterilisieren alle Werkzeuge und Gewebe Chopper mit 70% Ethanol. </li><li> Blasen Dissektion Puffer (Rezept Reagenzien Schnitt) mit 95% O 2, 5% CO 2 auf Eis für mindestens 15 Minuten vor dem Beginn der Präparation. </li><li> Betäuben Maus Welpen, indem sie auf Eis für 5-10 min nach anerkannten Tier Protokolle. Ermitteln Sie die geeignete Narkosetiefe durch die Bestätigung, dass die Mäuse nicht bis zum Schwanz und Zehen Kneifen reagieren. </li><li> Enthaupten Tiere mit einer scharfen Schere folgenden Tier Protokolle. Entfernen Sie schnell den Schädel über dem Vorderhirn und Kleinhirn, indem zuerst eine sagittale Schnitt, gefolgt von seitlichen Schnitt, mit sterilisierten Werkzeugen. Geschnitten Hirnnerven von der ventralen Oberfläche des Gehirns und des Kleinhirns durch vorsichtiges Aufrollen des Gewebes an der Seite. </li><li> Zeigen Gewebe in einem sterilen 35 mm Schale mit eiskaltem sauerstoffhaltigen Dissektion Puffer. </li><li> Mit einer Rasierklinge, trennen das Kleinhirn und Vorderhirn. Dann machen Sie eine Mitte-Sagittalschnitt through das Vorderhirn und Kleinhirn, die Trennung der Hemisphären. </li><li> Schneiden Sie 300 um dicke koronale und sagittale Abschnitte des Vorderhirns und des Kleinhirns mit einem manuellen Gewebe Chopper. HINWEIS: Ein Handbuch Gewebe Chopper ermöglicht die schnelle Verarbeitung von Präparation zu Kultur Inkubation ohne die Notwendigkeit für Agarose-Einbettung. Ein Vibratom kann auch eingesetzt werden, wie zuvor 9 beschrieben. </li><li> Übertragen getrennt Scheiben in frisch sprudelte kalte Dissektion Puffer auf Eis. </li><li> Verwenden Sie kleine Sezieren oder einem Gewicht von Spachteln, einzelne Teile zu trennen und sie auf sechs und Gerichte mit Kultureinsätze vorinkubiert. </li><li> Überschüssiges Dissektion Puffer von der Oberfläche des Kultureinsatz mit einer Einwegübertragungspipette oder eine P 200 Pipettenspitze. Zwei bis drei Vorderhirn oder drei Kleinhirnscheiben können auf einem Kultureinsatz platziert werden. HINWEIS: Für die konsistente Ergebnisse mit Vorderhirn Kulturen, ist es ideal, um Scheiben nehmen überspannt den vorderen Drittel des Corpus callosum(1A), um die beiden grauen und weißen Substanz Regionen in der gleichen Schicht zu untersuchen. Jedes Tier erhält man in der Regel 6 Scheiben Vorderhirn und Kleinhirn 6 Scheiben schneiden. </li><li> Legen Sie die sechs Well-Platten im Brutschrank und mit 1 ml Medium Scheibe 1 Tag nach Schnittpräparat und dann jeden zweiten Tag, bis Scheibe Fixierung ersetzen Sie die Scheibe Medium. </li><li> Je nach Experiment, verwenden Scheiben nach 5-7 Tagen in Kultur. Verwenden Sie nur Scheiben, die transparent nach den ersten paar Tagen geworden, und entsorgen Sie die, die unebenen opaken Bereichen haben. HINWEIS: Opaque Regionen innerhalb Scheiben sind mit dem Auge sichtbar und erscheinen als ein Klumpen von dunklen Zellen unter Phasenkontrast. Nachdem die Technik bewältigt wurde, auftreten tot undurchsichtigen Scheiben selten, in weniger als 1-5% der Scheiben. </li></ol> 2. Time-Lapse-Imaging von NG2 Zellteilung und Differenzierung der Oligodendrozyten HINWEIS: Um Zeitraffer Bildgebung von NG2 Zellproliferation und Differenzierung führen wir verwenden NG2cre: ZEG-Mäusen 16die EGFP exprimieren in NG2 Zellen und deren Nachkommen (1C-E). Reporterexpression in dieser Zeile ist ausreichend robust, um Live-Bilder der GFP +-Zellen in Scheiben unmittelbar nach Schneiden für einen Zeitraum von mindestens vier Wochen erhalten. Die deutlichsten Bilder werden nach der ersten Durchforstung Zeit der Scheibe, die sich in den ersten 3-5 Tagen in Kultur anfällt. <ol><li> Während des Experiments halten die Scheiben in einem Zellkulturbrutschrank bei 37 ° C, und entfernen Sie nur für kurze Zeit (weniger als 15 min pro Scheibe), halten den Deckel auf die sechs Well-Platte, während Bildgebung. </li><li> Mit einem inversen Fluoreszenzmikroskop mit einer Digitalkamera ausgerüstet ist, zu erfassen Bilder von dem ersten Zeitpunkt mit einem 10X-Objektiv. HINWEIS: Zum ersten Mal Punkte werden durch das Experiment variieren und können den Tag, wenn die Scheiben hergestellt wurden oder einen Zeitpunkt nach sein. </li><li> Machen Sie Bilder von mehreren Regionen zum Zeitpunkt einer in beiden grauen und weißen Substanz Bereiche der Scheibe. Hinweis the Region durch spezifische Sehenswürdigkeiten innerhalb der Scheibe und durch die Abbildung die Lage des Bildes auf einem Schema, das für nachfolgende Imaging-Sitzungen verwendet werden können, abgebildet. Nützlich Sehenswürdigkeiten können Kanten der Scheiben und / oder Orientierung von der weißen Substanz enthalten. Bild vier Minuten vor acht Stellen auf jeder Scheibe zu jedem Zeitpunkt. </li><li> Erfassung nachfolgender Bilder in einem Intervall von 4-6 Stunden, wenn die Prüfung NG2 Zellteilung und Differenzierung Oligodendrozyten, idealerweise über 5-7 Tage. </li><li> Nehmen Sie ein endgültiges Bild von allen Regionen und fixieren die Scheiben sofort. 1 ml der Fixierlösung (4% PFA mit 0,1 M L-Lysin 0,01 M Natriummetaperiodat), um den Boden des Kultureinsatz, dann fügen Sie eine weitere 1 ml oben auf den Scheiben. Achten Sie darauf, die direkt auf dem Fixiermittel Scheibe spritzen, wie es aus der Membran zu lösen. Befestigen Sie das Gewebe für 30 Minuten und fahren Sie mit der Immunhistochemie mit Entwicklungsstadium spezifische Marker, wie in Kapitel 4 beschrieben. </li><li> Wasch Scheiben mit 0,2 M Natriumphosphatpuffer 3 x 10 m. Wenn nötig, speichern Scheiben bei 4 ° C in 0,2 M Natriumphosphat-Puffer für 1-3 Tage vor Immunfärbung durchzuführen, sondern im Idealfall innerhalb von 24 Stunden. Fahren Sie mit der Immunhistochemie, wie unten in Abschnitt 4 beschrieben, den Anteil der Zellen, die in Oligodendrozyten (2D-F) differenziert sind, zu bestimmen. </li></ol> 3. Fate Mapping von NG2 Zelle Nachkommen Mit induzierbare Reporter transgenen Mäusen in Schnittkulturen HINWEIS: Um das Schicksal von NG2 Zellen aus einem bestimmten Zeitpunkt in der Kultur zu verfolgen, induzierbare NG2creER: kann YFP transgenen Mäusen verwendet werden. <ol><li> Cre die Rekombination zu induzieren und Reporter-Expression durch Zugabe von 100 nM 4OHT in Ethanol zu dem Kulturmedium gelöst. HINWEIS: Reporter-positiven Zellen sollte innerhalb von 1-2 Tagen bei einem Induktionseffizienz von ~ 20-25% YFP + NG2 + / NG2 +-Zellen und zu diesem Zeitpunkt erscheinen alle Zellen an der NG2-Zell-Stadium (Abbildung 2a-c) </li><li> Fix und waschen Sie die Scheibeen zu verschiedenen Zeitpunkten nach unterschiedlichen Manipulationen (in den Abschnitten 2.5 und 2.6 beschrieben). </li></ol> 4. Scheibe Immunhistochemie <ol><li> Schneiden Sie die Membranen mit den Scheiben aus den Einsätzen mit einem Skalpell angebracht. </li><li> Übertragen Scheiben auf einzelne Vertiefungen einer 24-Well-Platte, die Phosphat-gepufferte Kochsalzlösung (PBS) mit einem Satz von Pinzetten, kümmert sich nicht um die Zusammensetzung der Scheibe stören bei der Abholung der Membran. </li><li> Transfer der Scheiben zu einer Platte mit 24 Vertiefungen mit Blockierungslösung, die 5% normales Ziegenserum (NGS) und 0,1% Triton-X100 in PBS für 1 Stunde. </li><li> Die Scheiben in primären Antikörper inkubieren O / N in 5% NGS in PBS bei 4 ° C ist. Zur Identifizierung von Zellen in der Zellstadium NG2, verwenden Sie Antikörper gegen NG2 und PDGFRα. Zur Identifizierung von differenzierten Oligodendrozyten, verwenden Sie ein Antikörpers an die adenomatöse Polyposis coli-Antigen (APC; Klon CC1). </li><li> Am zweiten Tag waschen Scheiben in PBS 3 x15 min, dann im Sekundär Antibo inkubierenstirbt in 5% NGS in PBS für 1 h bei RT. Scheiben waschen in PBS 3 x15 min und montieren auf Folien. Halterung mit Scheiben und Membran mit Blick auf den Objektträger, so dass die Membran nicht zwischen dem Ziel und dem Gewebe sein. </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine:+a+neuron-glial+transmitter+promoting+myelination+in+the+CNS+in+response+to+action+potentials.">Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials.</a> Neuron. 36 (5), 855-868 (2002).</li><li>Wake, H., Lee, P. R., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+local+protein+synthesis+and+initial+events+in+myelination+by+action+potentials.">Control of local protein synthesis and initial events in myelination by action potentials.</a> Science. 333 (6049), 1647-1651 (2011).</li><li>Rosenberg, S. S., Kelland, E. E., Tokar, E., De la Torre, A. R., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geometric+and+spatial+constraints+of+the+microenvironment+induce+oligodendrocyte+differentiation.">The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (38), 14662-14667 (2008).</li></ol>

The authors declare that they have no competing financial interests

Myelinisierung im zentralen Nervensystem ist wesentlich für eine effiziente Kommunikation und neuronalen Axonen Überleben 22. NG2-Zellen zu generieren kontinuierlich myelinisierenden Oligodendrozyten in das Erwachsenenalter, während eine Wohnbevölkerung in den meisten Hirnregionen 16,23 - 25. Einige genetische und molekulare Mechanismen die Differenzierung dieser Zellen beschrieben worden, aber es bleibt noch viel zu entdecken. Organotypischen Kulturen sind ein geeignetes Mittel, um diese Mecha...

Organoytpic Schnittkulturen des zentralen Nervensystems haben sich als sehr nützlich für das Studium Neuron und Glia Zellbiologie in semiintact System 1 - 4. Diese Kulturen sind relativ einfach zu übernehmen und behalten viele Vorteile der primären dissoziierten Zellkulturen, wie Manipulationen der die extrazelluläre Umgebung und einfachen Zugang für wiederholte langfristige Live Cell Imaging und elektrophysiologischen Ableitungen 5-9. Außerdem Schnittkulturen pflegen 3-dimensionalen Gewebe Zellarchitektur, regionalen neuronalen Konnektivität, und die meisten großen Zelltypen in dem System vorhanden sind. Diese Eigenschaften machen diese Kulturen ein einzigartiges und bequemes System zur Einzelzellverhalten und Physiologie mit zellulären und Umweltwechselwirkungen zu untersuchen. NG2 Zellen eine Population von Gliazellen im Zentralnervensystem von Säugetieren, die zur Proliferation und erzeugen weiterhin myelinating Oligodendrozyten während der embryonalen und postnatalen Entwicklung 10. Sie sind ausführlich in dissoziierten primären Zellkulturen untersucht, und die jüngsten Entwicklung transgener Mauslinien mit NG2 zellspezifische Expression von fluoreszierenden Proteinen in vivo Schicksal Mapping und elektrophysiologische Ableitungen im spitzen Schnittpräparate erleichtert. Auch bei diesen Studien ist wenig über die zeitliche Dynamik der NG2 Zellproliferation und Differenzierung Oligodendrozyten bekannt. Obwohl dissoziierten Zellkultur in großem Umfang zur relativen Anlage in pharmakologischen und genetischen Manipulationen verwendet werden, ist es nicht geeignet für die Abfragefunktionsunterschiede dieser Zellen in verschiedenen Hirnregionen insbesondere wenn es wünschenswert ist, den Rahmen der zellulären Mikroumgebung aufrechtzuerhalten. Schnittkulturen bieten eine einfache Alternative, die zugänglich für pharmakologische Manipulationen ist und verwendet worden, um Oligodendrozyten Myelinisierung 11,12 untersuchen, Zelldere Reaktion nach Lysolecithin (LPC) oder Antikörper-induzierte Demyelinisierung 13,14, und die Induktion von Remyelinisierung über pharmakologische Behandlung 15. Ein Verfahren zur Untersuchung und Analyse von NG2 führen Zellproliferation und Differenzierung in Oligodendrozyten organotypischen Schnittkulturen sowohl aus dem Vorderhirn und Kleinhirn getroffen wird beschrieben Live-Bildgebung und fixiertem Gewebe (oder Nachfixierung). Dies ist eine leistungsfähige Methode, die verwendet werden können, um die Zelle Schicksal der einzelnen Zellen nach NG2 Abteilung 16 zu studieren und regionale und altersabhängige Unterschiede in der Wachstumsfaktor induzierte Proliferation NG2 17 entdecken. Diese relativ einfache Technik ist allgemein zugänglich, weiter zu untersuchen Zelle intrinsischen und / oder Umweltregulierungsmechanismen die Physiologie dieser Gliazellen und ihre Reaktion auf die neuronale Aktivität oder Myelinschäden.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Slice Culture Medium</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>11090</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Invitrogen</td>
			<td>14175</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H-4034</td>
			<td>25mM</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>1mg/L</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>0.4mM</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Hyclone</td>
			<td>SH30074.03</td>
			<td>25%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>124mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>3.004mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>1.25mM</td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>4.004mM</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>26mM</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>2.0mM</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>0.075mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O2 5%CO2 before use</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents and Supplies</td>
		</tr>
		<tr>
			<td>4-hydroxy tamoxifen (4OHT)</td>
			<td>Sigma-Aldrich</td>
			<td>H7904</td>
			<td>100nM</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>EM Sciences</td>
			<td>19200</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L-6027</td>
			<td>0.1M</td>
		</tr>
		<tr>
			<td>Sodium Metaperiodate</td>
			<td>Sigma-Aldrich</td>
			<td>S-1878</td>
			<td>0.01M</td>
		</tr>
		<tr>
			<td>Rabbit anti NG2 antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>AB5320</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Mouse anti CC1 antibody</td>
			<td>Calbiochem (Millipore)</td>
			<td>OP80</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Mouse anti Ki67 antibody</td>
			<td>Vision Biosystems</td>
			<td>NCL-Ki67-MM1</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Mouse anti NeuN antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>MAB377</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Species specific secondary antibodies</td>
			<td>Jackson Immunoresearch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td></td>
			<td></td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture membrane inserts 0.45&#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle top filters</td>
			<td>Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 5% CO2 gas mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile six well plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting (hippocampal) or weighing spatulas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manual or automatic tissue chopper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm and 60mm sterile Petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Phase Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microcope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures to study oligodendrocyte dynamics and myelination

NG2-exprimierenden Zellen (polydendrocytes, Oligodendrozyten-Vorläuferzellen) sind die vierte große glialen Zellpopulation im zentralen Nervensystem. Während der embryonalen und postnatalen Entwicklung sie aktiv vermehren und erzeugen myelinisierenden Oligodendrozyten. Diese Zellen haben häufig in der Grund dissoziiert Kulturen, Neuron Kokulturen untersucht worden, und in fixiertem Gewebe. Mit neu verfügbaren transgenen Mauslinien schneiden Kultursysteme können verwendet werden, um die Proliferation und Differenzierung von Oligodendrozyten-Linie Zellen in beiden grauen und weißen Substanz Regionen des Vorderhirns und des Kleinhirns zu untersuchen. Schnittkulturen sind aus der frühen postnatalen Mäusen hergestellt und in Kultur für bis zu 1 Monat gehalten. Diese Scheiben kann mehrfach über die Kulturzeit abgebildet werden, um das Zellverhalten und Wechselwirkungen zu untersuchen. Diese Methode ermöglicht die Visualisierung von NG2 Zellteilung und die Schritte, die zu Oligodendrozyten-Differenzierung und ermöglichen detaillierte Analyse der Region-abhängigent NG2 Zelle und Oligodendrozyten funktionelle Heterogenität. Dies ist eine leistungsfähige Technik, die verwendet werden können, um die innere und äußere Signale in einer zellulären Umgebung, die sehr ähnlich, die in vivo gefunden Beeinflussung dieser Zellen mit der Zeit zu untersuchen.

Beispiele für repräsentative Daten sind unten angegeben, die mit Schnittkulturen sowohl aus dem Vorderhirn und Kleinhirn der P8 NG2cre erhalten worden sind: ZEG, NG2creER: YFP und PLPDsRed transgenen Mauslinien. NG2 Zellen über Tage in Vorderhirn und Kleinhirn Scheiben (1B-D, Video 1) und der Phänotyp dieser Zellen abgebildet werden kann, nach der Fixierung bestimmt und Immunfärbung mit NG2 und CC1 Antikörper (1E). Neben der Echtzeit-Bildgebung kann die Zellproliferation auch mit ...

Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotypischen Kulturen zu Oligodendrozyten Dynamik und Myelinisierung Studieren

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

organotypic-slice-cultures-to-study-oligodendrocyte-dynamics

Research

JoVE Journal

Neuroscience

18.2K Aufrufe.  University of Connecticut. A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Serie: Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Die Organotypische hippokampalen Slice-Kultur-Modell für die Prüfung neuronaler Schädigung

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Forschung

Neurowissenschaften

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotypischen hippokampalen Slice-Kulturen

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypische Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

Whole Cell Aufnahme von einem organotypischen Vorbereitung der Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multi-Elektroden-Array Recordings neuronaler Lawinen in organotypischen Kulturen

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Ableitung von Enriched Oligodendrozyten Kulturen und Oligodendrozyten / Neuron myelinisierenden Co-Kulturen von Post-Natal Mausgeweben

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotypischen Kulturen von embryonalen ventralen Mittelhirn: Ein System zur Dopaminerge Neuronale Development Study In-vitro-</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Die Analyse von Purkinje Zelle Dendritische Morphologie in organotypischen Kulturen

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Die Analyse der neurovaskuläre Remodeling in Entorhino-Hippocampus Organotypischen Kulturen

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypischen Hippocampal Slice Kulturen als Modell zur Studie Neuroprotektion und Invasivität der Tumorzellen

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...