Name: organotypic slice cultures to study oligodendrocyte dynamics and myelination
Uploaded: 2014-08-25T15:00:00
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

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 ...

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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 ...

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining 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

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

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

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

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

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

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

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

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

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

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. ...