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.

NOTA: Todos los animales procedimientos están siguiendo las directrices y han sido aprobados por el Cuidado de Animales y el empleo Comisión Institucional (IACUC) de la Universidad de Connecticut. NOTA: Para estos experimentos constitutivos NG2cre 18 (JAX # 008533) e inducibles NG2creER 16 (JAX # 008538) ratones transgénicos cruzó a Z / EG 19 (JAX # 003920) o gtRosa26: reportero YFP 20 (JAX # 006148) líneas se utilizaron respectivamente a imagen células NG2 y su progenie. Para imagen...

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La mielinización en el sistema nervioso central es esencial para la comunicación eficiente neuronal y la supervivencia axonal 22. Células NG2 generan continuamente oligodendrocitos myelinating en la edad adulta, mientras que el mantenimiento de una población residente en la mayoría de las regiones del cerebro 16,23 - 25. Algunos de los mecanismos genéticos y moleculares que regulan la diferenciación de estas células han sido descritas, pero mucho queda por descubrir. Cultivos de cortes orga...

Cultivos de corte Organoytpic del sistema nervioso central han demostrado ser de gran utilidad para el estudio de las neuronas y la biología de las células gliales en un sistema semiintact 1-4. Estos cultivos son relativamente fáciles de adoptar y mantener muchos beneficios de los cultivos de células disociadas primarios tales como la manipulación del ambiente extracelular y fácil acceso para imágenes de células vivas repetida a largo plazo y los registros electrofisiológicos 5-9. Además, cultivos de cortes de tejido mantienen citoarquitectura 3-dimensional, la conectividad neuronal regional, y la mayoría de tipos de células principales están presentes en el sistema. Estas propiedades hacen que estas culturas un sistema único y conveniente para investigar el comportamiento de una sola célula y fisiología con las interacciones celulares y ambientales. NG2 células son una población de células gliales en el sistema nervioso central de los mamíferos que continúan proliferando y generar myelinating oligodendrocitos durante el desarrollo embrionario y postnatal 10. Se han estudiado extensivamente en cultivos celulares primarios disociados, y reciente desarrollo de líneas de ratones transgénicos con NG2 expresión específica de célula de proteínas fluorescentes ha facilitado suerte de cartografía vivo y registros electrofisiológicos en preparaciones rebanada agudas en. Incluso con estos estudios, se sabe poco sobre la dinámica temporal de la proliferación celular y la diferenciación de oligodendrocitos NG2. Aunque el cultivo de células disociada es ampliamente utilizado para la instalación relativa en las manipulaciones farmacológicas y genéticas, que no es adecuado para interrogar a diferencias funcionales de estas células en diferentes regiones del cerebro en particular cuando es deseable mantener el contexto de la microambiente celular. Cultivos de cortes proporcionan una alternativa simple que es susceptible de manipulaciones farmacológicas y se han utilizado para investigar la mielinización de oligodendrocitos 11,12, célulaular respuesta después de lisolecitina (LPC) o el anticuerpo induce desmielinización 13,14, y la inducción de remielinización mediante tratamiento farmacológico 15. Un método para investigar y realizar imágenes en vivo y tejido fijado (o post-fijación) análisis de la proliferación celular y la diferenciación de oligodendrocitos NG2 en cultivos de cortes organotípicos tomadas tanto desde el cerebro anterior y el cerebelo se describe. Este es un poderoso método que se puede utilizar para estudiar el destino celular de las células individuales NG2 después de la división 16 y para descubrir diferencias de región y dependientes de la edad en el factor de crecimiento inducido por la proliferación de células NG2 17. Esta técnica es relativamente simple ampliamente accesible a investigar más a fondo celular intrínseco y / o ambiental mecanismos que regulan la fisiología de estas células gliales y su respuesta a la actividad neuronal o daño de mielina.

<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

Células que expresan NG2 (polydendrocytes, células precursoras de oligodendrocitos) son la cuarta población de células gliales importante en el sistema nervioso central. Durante el desarrollo embrionario y postnatal proliferan activamente y generar oligodendrocitos myelinating. Estas células han sido comúnmente estudiado en cultivos primarios de neuronas disociadas, cocultivos, y en tejido fijado. Uso de líneas de ratones transgénicos recientemente disponibles cortan sistemas de cultivo pueden usarse para investigar la proliferación y diferenciación de las células del linaje de oligodendrocitos en ambas regiones de materia gris y blanca del cerebro anterior y el cerebelo. Cultivos de corte se preparan a partir de ratones postnatales tempranas y se mantienen en cultivo hasta por 1 mes. Estos cortes se pueden visualizar varias veces durante el período de cultivo para investigar el comportamiento celular y las interacciones. Este método permite la visualización de la división celular NG2 y los pasos que conducen a la diferenciación de oligodendrocitos al tiempo que permite un análisis detallado de la región-dependenent células NG2 y la heterogeneidad funcional de oligodendrocitos. Esta es una técnica poderosa que se puede utilizar para investigar las señales intrínsecos y extrínsecos que influyen en estas células a través del tiempo en un entorno celular que se asemeja mucho a la que se encuentra in vivo.

Se dan ejemplos de datos representativos a continuación que se han obtenido usando cultivos de corte tanto del cerebro anterior y el cerebelo de P8 NG2cre: ZEG, NG2creER: YFP y PLPDsRed líneas de ratones transgénicos. NG2 células se pueden obtener imágenes en día en prosencéfalo y cerebelo rebanadas (Figura 1B-D, video 1) y el fenotipo de estas células se puede determinar después de la fijación y la inmunotinción con anticuerpos CC1 y NG2 (Figura 1E). Además de imágenes en ...

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.

Culturas Organotípicos Slice para Estudiar Oligodendrocyte Dinámica y mielinización

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

El organotípicos rebanada hipocampo modelo de cultivo para el examen de la lesión neuronal

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

Investigación

Neurociencias

Organotypic Hippocampal Slice Cultures

Organotípicos culturas rebanada hipocampo

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

Organotípicos Culturas del cerebelo: Retos y detección de apoptosis

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

Grabación de células enteras de una rebanada preparación organotípicos del neocórtex

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

Electrodos múltiples grabaciones matriz de avalanchas neuronal en cultivos organotípicos

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

Derivación de las Culturas enriquecido oligodendrocitos y neuronas oligodendrocitos / myelinating Co-culturas de Post-natal tejidos murinos

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

Culturas organotípicos rebanada de mesencéfalo ventral embrionario: Un sistema para estudiar el desarrollo neuronal dopaminérgica 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

El análisis de la morfología de células dendríticas Purkinje en cultivos de corte organotípicos

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 ...

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

Culturas de rebanada Hippocampal Organotypic como un modelo para estudio neuroprotección e invasividad de las células tumorales

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