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 oligodendrocitos maduros, se utilizaron ratones transgénicos PLPDsRed 21. Para la supervivencia consistente, rebanadas pueden ser aislados de los ratones hasta el día postnatal 10 tanto desde el cerebro anterior y el cerebelo. 1. Preparación rebanada <ol><li> En una campana de cultivo, colocar insertos de cultivo de tejidos en placas de seis pocillos y se añade 1 ml de medio de cultivo de cortes (receta en la sección reactivos). Poner las placas en la incubadora de cultivo celular a 37 ° C con 5% de CO 2 al menos 2 horas antes de la disección. </li><li> Esterilizar todas las herramientas y cortador de tejidos con etanol al 70%. </li><li> Tampón de disección de la burbuja (receta en la sección reactivos) con 95% de O 2, 5% de CO 2 en hielo durante al menos 15 min antes del inicio de la disección. </li><li> Anestesiar crías de ratón, colocándolos en hielo durante 5 a 10 min de acuerdo con los protocolos de los animales aprobados. Determinar la profundidad adecuada de la anestesia mediante la confirmación de que los ratones no responden a la cola y de pellizco del dedo del pie. </li><li> Decapita animales utilizando tijeras afiladas siguientes protocolos de animales. Quite rápidamente el cráneo sobre el cerebro anterior y el cerebelo, haciendo primero un corte sagital seguida de incisión lateral, utilizando herramientas esterilizadas. Cortar los nervios craneales desde la superficie ventral del cerebro y el cerebelo enrollando cuidadosamente el tejido a un lado. </li><li> Coloque el tejido en una placa de 35 mm que contiene tampón de disección estéril oxigenada enfriado con hielo. </li><li> Utilizando una hoja de afeitar, cortar el cerebelo y el cerebro anterior. A continuación, hacer un corte sagital medial throuf el cerebro anterior y el cerebelo, la separación de los hemisferios. </li><li> Cortar 300 micras de espesor secciones coronal y sagital del cerebro anterior y el cerebelo con un cortador de tejidos manual. NOTA: Un cortador de tejidos manual permite un rápido procesamiento de la disección de la incubación de cultivo sin la necesidad de incrustación de agarosa. Un vibratome también se puede utilizar como se describió anteriormente 9. </li><li> Transferir las rebanadas separadas en tampón de disección en frío recién burbujeó en hielo. </li><li> Utilice pequeña disección o con un peso espátulas para separar secciones individuales y transferirlos a preincubada seis pocillos con insertos de cultivo. </li><li> Eliminar cualquier exceso de tampón de la disección de la superficie de la pieza de inserción de cultivo utilizando una pipeta de transferencia desechable o una punta de pipeta P 200. De dos a tres prosencéfalo o tres rebanadas cerebelosas se puede colocar en un inserto de cultivo. NOTA: Para obtener resultados consistentes con las culturas del cerebro anterior, que es ideal para las rebanadas que abarca el tercio anterior del cuerpo calloso(Figura 1 A) con el fin de estudiar ambas regiones de materia gris y blanca en el mismo segmento. Cada animal por lo general rinde 6 rebanadas del cerebro anterior y 6 rebanadas cerebelo. </li><li> Coloque las placas de seis pocillos en la incubadora y reemplazar el medio rebanada con 1 ml de medio rebanada 1 día después de la preparación rebanada y luego cada dos días hasta que la fijación de la rebanada. </li><li> Dependiendo del experimento, utilizar rebanadas después de 5-7 días en cultivo. Utilice únicamente las rebanadas que se convierten en transparentes después de los primeros días y descartar aquellas que tienen regiones opacas irregulares. NOTA: las regiones opacas dentro de rebanadas son visibles a simple vista y aparecen como un grupo de células oscuros bajo contraste de fase. Después de la técnica se ha dominado, rebanadas opacos muertas ocurren rara vez, en menos de 5.1% de las rebanadas. </li></ol> 2. Time-lapse de imágenes de la división de células NG2 y Oligodendrocyte Diferenciación NOTA: Para realizar la proyección de imagen de lapso de tiempo de la proliferación celular y la diferenciación NG2 utilizamos NG2cre: ratones ZEG 16que expresan EGFP en células NG2 y su progenie (Figura 1C-E). Expresión reportero en esta línea es suficientemente robusto para obtener imágenes en directo de las buenas prácticas agrarias + células en rodajas inmediatamente después de la sección por un período de tiempo de al menos cuatro semanas. Las imágenes más claras se obtienen después del período de adelgazamiento inicial de la rebanada, que se produce durante los primeros 3-5 días en cultivo. <ol><li> Durante todo el experimento, mantener las rodajas en un incubador de cultivo celular a 37 ° C y retirar sólo durante breves períodos de tiempo (menos de 15 min por rebanada), manteniendo la tapa en la placa de seis pocillos mientras que las imágenes. </li><li> El uso de un microscopio de fluorescencia invertido equipado con una cámara digital, la captura de imágenes en el primer punto de tiempo utilizando un objetivo de 10X. NOTA: los puntos de primera vez pueden variar por el experimento y puede ser el día en que se prepararon los cortes o cualquier punto de tiempo después. </li><li> Capturar imágenes desde múltiples regiones en el punto de una vez en las dos áreas de materia gris y blanca de la división. Nota ªe región fotografiada por puntos de referencia específicos dentro de la rebanada y mediante la asignación de la ubicación de la imagen en un esquema que puede ser utilizado para las sesiones de formación de imágenes subsiguientes. Puntos de referencia útiles pueden incluir bordes de las rebanadas y / o la orientación de tractos de sustancia blanca. Image siete y cincuenta y seis ubicaciones en cada rebanada en cada punto de tiempo. </li><li> Capturar las imágenes siguientes en un intervalo de 4-6 horas si el examen de la división celular y la diferenciación de oligodendrocitos NG2, idealmente más de 5-7 días. </li><li> Capturar una imagen final de todas las regiones y fijar las rebanadas de inmediato. Añadir 1 ml de fijador (4% PFA con 0,1 M de L-lisina 0,01 M metaperyodato de sodio) a la parte inferior de la inserción del cultivo, a continuación, añadir otros 1 ml en la parte superior de las rebanadas. Tenga cuidado de no rociar el fijador directamente en el segmento, ya que puede desprenderse de la membrana. Fijar el tejido durante 30 minutos y proceder con inmunohistoquímica utilizando marcadores específicos de la etapa de desarrollo, como se describe en la sección 4. </li><li> Rodajas de lavado con tampón fosfato sódico 0,2 M 3 x 10 m. Si es necesario, rebanadas almacena a 4 º C en tampón de fosfato de sodio 0,2 M durante 1-3 días antes de la inmunotinción pero lo ideal es realizar la tinción dentro de las 24 horas. Proceda con la inmunohistoquímica, como se describe a continuación en la sección 4 para determinar la proporción de células que se han diferenciado en oligodendrocitos (Figura 2D-F). </li></ol> 3. Destino Cartografía de la progenie de células NG2 Utilizando ratones Inducible Periodista transgénicos en cultivos de corte NOTA: Para realizar el seguimiento del destino de las células NG2 desde un punto de tiempo particular en la cultura, NG2creER inducibles: se pueden usar ratones transgénicos YFP. <ol><li> Inducir Cre recombinación y expresión del indicador mediante la adición de 100 nM 4OHT disuelto en etanol al medio de cultivo. NOTA: células positivas Reporter deben aparecer dentro de 1-2 días con una eficiencia de inducción de ~ 20-25% YFP células NG2 + + / + NG2 y en este punto el tiempo todo será células en la etapa de células NG2 (Figura 2A-C) </li><li> Fijar y lavar la rebanadas en diferentes puntos temporales después de varias manipulaciones (descritos en las secciones 2.5 y 2.6). </li></ol> 4. rebanada inmunohistoquímica <ol><li> Cortar las membranas con las rodajas de adjuntos de los insertos utilizando un bisturí. </li><li> Transferir las rebanadas a pocillos individuales de una placa de 24 solución salina tamponada que contiene bien fosfato (PBS) usando un conjunto de pinzas, teniendo cuidado de no alterar la composición de la rebanada al momento de retirar la membrana. </li><li> Transferencia de las rebanadas a una placa de 24 pocillos con solución de bloqueo que contenía 5% de suero normal de cabra (NGS) y 0,1% de Triton-X100 en PBS durante 1 hr. </li><li> Incubar las rebanadas en anticuerpo primario O / N en un 5% en NGS en PBS a 4 ° C. Para identificar las células en la etapa de células NG2, utilizar anticuerpos contra NG2 y PDGFR. Para la identificación de los oligodendrocitos diferenciados, utilizar un anticuerpo para el antígeno de la poliposis coli adenomatosa (APC; CC1 clon). </li><li> En el segundo día de las rebanadas de lavado en PBS 3 x15 min, luego se incuban en Antibo secundariamuere en 5% de NGS en PBS durante 1 hora a TA. Lávese las rebanadas en PBS 3 x15 min y montar en portaobjetos. Montar con rodajas de arriba y de membrana que se enfrenta la diapositiva para que la membrana no será entre el objetivo y el tejido. </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

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 ...

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

Research

JoVE Journal

Neuroscience

18.2K vistas.  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.

Video: 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.

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

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.

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

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.

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

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.

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

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.

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

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).

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

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.

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

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.

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 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.

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.

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. ...