Los autores desean agradecer a James Oesterling para citometría de flujo asistencia. Esta investigación fue apoyada por una subvención del Instituto Nacional del Cáncer (R01-CA178380, ZY) y una donación de US Nacional Institutos de formación postdoctoral (5T32CA009035-37, LWY).

El uso de animales en este protocolo se ha realizado de acuerdo con los procedimientos aprobados por el Comité del Centro de Cáncer de Cuidado de Animales y el empleo Fox Chase. 1. preparación de instrumentos, soluciones y Cubreobjetos <ol><li> Limpiar dos conjuntos de # 5 pinzas finas, un conjunto de # 7 pinzas curvas finas, una tijera quirúrgica grande, uno de tijera microdissecting, una espátula y una cuchara perforada. Coloque los instrumentos en una bolsa de esterilización autosellante y autoclave. Mantenga los instrumentos en la bolsa hasta que esté listo para su uso. </li><li> Preparar una solución de agarosa de baja temperatura de fusión 3%. <ol><li> Añadir 1,5 g de agarosa en 50 ml de tampón fosfato salino estéril de Dulbecco (DPBS) y luego coloque en el microondas y el calor hasta que aparezcan burbujas. Frasco Remolino y recalentar hasta que todo agarosa ha disuelto. Mezclar la solución con una barra de agitación antes del calentamiento si aparecen grumos. </li><li> Guarde la solución en un baño a 37 ° C hasta que se necesite. </li><li> Para storag largo plazoe, almacenar la solución de agarosa para uso futuro a temperatura ambiente o a 4 ° C durante meses. Vuelva a calentar en el microondas para licuar. Para el calentamiento repetido de la solución de agarosa, se pesa el matraz antes de calentar para mantener su peso inicial con agua caliente destilada estéril. </li></ol></li><li> Preparar las siguientes soluciones para la disociación de células basados ​​en papaína como se describió previamente 2: <ol><li> Una solución de papaína con papaína (100 U / ml), cisteína (0,2 mg / ml) y DNasa (250 U / ml) en fenol rojo-estéril que contiene DPBS. </li><li> Una solución de ovomucoide con albúmina de suero bovino (BSA; 8 mg / ml), inhibidor de tripsina de soja (8 mg / ml)) y DNasa (250 U / ml) en fenol-estéril que contiene rojo DPBS. Filtro esterilizar ambas soluciones y ajustar el pH a devolver el color original del fenol que contienen rojo DPBS. </li></ol></li><li> Preparar medios de cultivo celular (NB / B27): Suplemento medios basales con piruvato de sodio 1 mM, 2 mM L-glutamina, 1% de penicilina-estreptomicina (P / S) y suplemento B27. Filter esterilizar y protegerlo de la luz. Equilibrar los medios de comunicación a 37 ° C y 5% de CO 2 antes de su uso. </li><li> Preparar tampón FACS: suero bovino fetal al 5% (FBS) en DPBS. Añadir 2,5 ml de FBS al 47,5 ml de DPBS. </li><li> Preparar poli-D-lisina (PDL) recubiertas con cubreobjetos. <ol><li> Nuevos cubreobjetos de vidrio autoclave. </li><li> Escudo los cubreobjetos con PDL (100 ug / ml en agua destilada estéril) y se incuba durante 2 horas a 37 ° C o temperatura ambiente durante la noche. </li><li> Lavado cubreobjetos con agua destilada estéril. Lavar una vez con medio NB / B27. Deje cubreobjetos se sequen por completo en el tejido campana de cultivo antes de su uso. </li></ol></li></ol> 2. disección y preparación de cortes de tejido <ol><li> En el día de la disección, limpie la zona de disección con etanol al 70% y la cubierta con una almohadilla absorbente. </li><li> Retire las herramientas de disección de la bolsa de esterilización. Añadir hielo frío, DPBS estériles / 1% P / S a una pequeña placa de Petri y el lugar en el hielo. </li><li> Decapitate P4 Math1-GFP / Nestin-PPC crías de ratón con grandes tijeras quirúrgicas a continuación, mantenga la cabeza hacia abajo con pinzas curvas finas. Quite la piel y que despegar el cráneo con # 5 pinzas finas Después vacíe el cerebro del cráneo con una espátula y lo transfieren a la placa de Petri que contiene DPBS / 1% P / S. <ol><li> Usando unas pinzas finas # 5, separar cuidadosamente el cerebelo del resto del cerebro. Asegúrese de diseccionar una cría a la vez lo más rápidamente posible. </li><li> Reunir un mínimo de 8 cerebelo con el fin de obtener suficientes NEP para los análisis moleculares y funcionales. </li></ol></li><li> Llenar un molde de inclusión de 2 x 2 x 2 cm con una solución de agarosa de baja temperatura de fusión 3%. Asegúrese de que la temperatura de agarosa es no más de 37 ° C. </li><li> Retire el exceso de líquido del cerebelo frotando suavemente sobre un pañuelo de papel de laboratorio. Coloque cerebelo en el molde en una posición vertical. Colocar el molde en hielo para permitir que se solidifique rápidamente. Utilice ~ 4 cerebella por bloque. </li><li> Obtener lisecciones de tejido ving con un vibratome cuchilla vibratoria. Recorte bloque de agarosa que contiene el cerebelo-con una hoja de afeitar. Pegar el bloque de agarosa a la placa de vibratome con orientado en una posición vertical cerebelo. </li><li> Llenar la bandeja del vibratome con heladas DPBS estériles / 1% P / S y el lugar de hielo debajo. <ol><li> Cortar 600 micras secciones a través de toda la longitud del cerebelo. Recoge las secciones de la bandeja de hielo utilizando una cuchara perforada. Se colocan los cortes en una placa de Petri llena de DPBS / 1% P / S en el hielo. </li></ol></li></ol> 3. Microdissection y disociación celular <ol><li> Coloque la placa de Petri que contiene cortes de tejido bajo un microscopio de disección fluorescente. <ol><li> Separar cuidadosamente el EGL del resto de la sección del cerebelo utilizando unas pinzas finas mediante la disección entre la PPC + capa molecular y la GFP + EGL (consulte la línea de puntos en la Figura 1). Tenga cuidado de no incluir ninguna parte de la capa molecular, que se traducirá encontaminación de Nestin-expresión de glía de Bergmann. </li><li> Completar este paso lo más rápidamente posible y mantener el tejido en hielo para evitar la muerte celular. </li><li> Retire la agarosa que rodea el tejido antes de proceder a la siguiente etapa. </li></ol></li><li> Digerir el tejido microdissected EGL utilizando un protocolo basado en papaína 2 como se describió previamente para obtener una suspensión de células individuales. <ol><li> Vuelva a suspender las células en tampón FACS para la recolección de células CFP-positivos mediante FACS. </li></ol></li></ol> 4. Cell Sorting y Plating <ol><li> Ordenar células para PPC fluorescencia utilizando un flujo de alta velocidad de citómetro estéril que contiene un filtro de PPC apropiado y recoger las células en tampón de FACS en hielo. Obtener aproximadamente 100.000 NEPs de cada cerebelo P4. </li><li> Centrifugar las células a 300 xg durante 5 min. Utilice células inmediatamente para el análisis molecular o de cultivo para experimentación adicional. </li><li> Para células de cultivo, vuelva a suspender las células en pre-calentado NB / B27 media y placa en cubreobjetos recubiertos-PDL como anteriormente descritos 2. </li></ol>

<ol>
	<li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Wechsler-Reya, R. J., Scott, M. P. <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+neuronal+precursor+proliferation+in+the+cerebellum+by+Sonic+Hedgehog.">Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog.</a> Neuron. 22, 103-114 (1999).</li><li>Dahmane, N., Ruizi Altaba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+the+growth+and+patterning+of+the+cerebellum.">Sonic hedgehog regulates the growth and patterning of the cerebellum.</a> Development. 126, 3089-3100 (1999).</li><li>Riobo, N. A., Manning, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+signal+transduction+employed+by+vertebrate+Hedgehogs.">Pathways of signal transduction employed by vertebrate Hedgehogs.</a> Biochem J. 403, 369-379 (2007).</li><li>Dahmane, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sonic+Hedgehog-Gli+pathway+regulates+dorsal+brain+growth+and+tumorigenesis.">The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis.</a> Development. 128, 5201-5212 (2001).</li><li>Marino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma:+developmental+mechanisms+out+of+control.">Medulloblastoma: developmental mechanisms out of control.</a> Trends Mol Med. 11, 17-22 (2005).</li><li>Yang, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Goodrich, L. V., Milenkovic, L., Higgins, K. M., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neural+cell+fates+and+medulloblastoma+in+mouse+patched+mutants.">Altered neural cell fates and medulloblastoma in mouse patched mutants.</a> Science. 277, 1109-1113 (1997).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+of+Nestin-expressing+progenitors+in+the+cerebellum+exhibits+increased+tumorigenicity.">A population of Nestin-expressing progenitors in the cerebellum exhibits increased tumorigenicity.</a> Nat Neurosci. 16, 1737-1744 (2013).</li><li>Ben-Arie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390, 169-172 (1997).</li><li>Sutter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+stem+cells+act+as+medulloblastoma-initiating+cells+in+a+mouse+model+and+a+neural+stem+cell+signature+characterizes+a+subset+of+human+medulloblastomas.">Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas.</a> Oncogene. 29, 1845-1856 (2010).</li><li>Sotelo, C., Alvarado-Mallart, R. M., Frain, M., Vernet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+plasticity+of+adult+Bergmann+fibers+is+associated+with+radial+migration+of+grafted+Purkinje+cells.">Molecular plasticity of adult Bergmann fibers is associated with radial migration of grafted Purkinje cells.</a> J Neurosci. 14, 124-133 (1994).</li><li>Lumpkin, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1-driven+GFP+expression+in+the+developing+nervous+system+of+transgenic+mice.">Math1-driven GFP expression in the developing nervous system of transgenic mice.</a> Gene Expr Patterns. 3, 389-395 (2003).</li><li>Encinas, J. M., Vaahtokari, A., Enikolopov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoxetine+targets+early+progenitor+cells+in+the+adult+brain.">Fluoxetine targets early progenitor cells in the adult brain.</a> Proc Natl Acad Sci U S A. 103, 8233-8238 (2006).</li><li>Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+astroglial+morphology+and+proliferation+in+vitro.">Neuronal regulation of astroglial morphology and proliferation in vitro.</a> J Cell Biol. 100, 384-396 (1985).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> J Vis Exp. (23), (2009).</li><li>Emmert-Buck, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274, 998-1001 (1996).</li><li>Espina, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nat Protoc. 1, 586-603 (2006).</li></ol>

Los autores no tienen nada que revelar.

El método de microdisección se describe aquí es el primer procedimiento capaz de aislar una población pura de células vivas para su uso en análisis moleculares y funcionales. Como se ha demostrado por Li et al. 9, NEP obtenidos por este método pueden ser cultivadas y se incorporan en diversos experimentos y debido a su alta pureza, también se pueden utilizar para el análisis de microarrays. Es muy importante tomar el tiempo para alcanzar la competencia...

El cerebelo se compone de múltiples capas de células, cada uno con distintos tipos de células. Durante el desarrollo, el EGL contiene la proliferación de precursores neuronales de gránulos (PNB), mientras que la capa molecular y la capa de Purkinje contienen neuronas Bergmann neuroglia y de Purkinje, respectivamente. En lo profundo de cerebelo se encuentra la sustancia blanca, que contiene células madre neurales (NSC) y oligodendrocitos 1. Sonic hedgehog (Shh) juega un papel crítico en la regulación de desarrollo del cerebelo y, en particular, promueve la proliferación de PNB través de la unión a su receptor Patched (Ptc), un regulador negativo de la señalización de Shh 2-4. La activación aberrante de la señalización de Shh genera meduloblastoma (MB), el tumor cerebral maligno más común en los niños 5,6. MB mutantes modelos de ratones transgénicos de PTC son herramientas poderosas para el estudio de la MB y se desarrollan los tumores se asemejan MB humana 7,8. El uso de estosratones, se descubrió que PNB son la célula de origen de hedgehog de ​​tipo MB 7. Además, en adición a PNB, recientemente hemos identificado una población única de células progenitoras neuronales dentro de la EGL del cerebelo en desarrollo que también puede dar lugar a MB. Estas células expresan altos niveles de la proteína de filamentos intermedios VI tipo, Nestin, y se denominan NEP 9. NEP se encuentran dentro de la parte profunda de la EGL y existen transitoriamente sólo durante el desarrollo del cerebelo neonatal. Están comprometidos con el linaje de células granulares como PNB, pero son distintas, ya que son de reposo y no expresan Math1, un marcador bien establecido para la PNB convencionales 10. Además, NEP dan lugar a MB más eficientemente que PNB después de la activación de la vía de señalización Shh 9, lo que les una novela origen para MB tumorigénesis hace. Se aislaron previamente NEPs del cerebelo EGL en el día postnatal 4 (P4) mediante la técnica de microdisección9 describe aquí. Microdisección mediante visualización microscópica directa del tejido permite la disección específica de la cerebelosa EGL. Esto es necesario ya Nestin también se expresa por NSC en la sustancia blanca del cerebelo y por glía de Bergmann en la capa molecular 1,7,11,12 y era fundamental que estas células no se incluirán, como lo harían confundir el análisis. Las células aisladas de tejido microdissected se pueden utilizar inmediatamente para el análisis molecular o pueden ser cultivadas para otras aplicaciones. Para ayudar en microdissecting específicamente el EGL y para purificar aún más los NEP, los ratones Math1-GFP (proteína verde fluorescente) se cruzaron con ratones Nestin-PPC (proteína fluorescente cian). El factor de transcripción Math1 se expresa específicamente por PNB y la expresión de GFP es claramente visible en el EGL 9,13. Ratones Nestin-PPC expresan una forma nuclear de la PPC y se pueden visualizar fácilmente en la capa molecular 9,14. En conjunto, las buenas prácticas agrarias y la expresión PPC crean límites para microdisección del EGL (ver Figura 1). NEP CFP-positivas se aislaron por FACS, después de la disociación enzimática de las EGLS disecados. Actualmente, el método más bien utilizada para aislar las células de la EGL es centrifugación en gradiente de Percoll de todo el tejido cerebelar 15,16. Este método, sin embargo, no es capaz de excluir completamente las poblaciones de células nestina + de la capa molecular y la materia blanca y por lo tanto no se puede utilizar para el estudio de NEP. Microdisección de captura por láser, que utiliza la transferencia de energía de láser para eliminar las células de interés, es otro método usado para aislar las células específicas dentro de un tejido heterogéneo 17,18 pero las células capturadas sólo se pueden utilizar para la recuperación de ADN, ARN y proteínas y no son capaces de cultivarse. Este protocolo proporciona una forma de aislar específicamente una población pura vida de los PIJ de la EGL.Esta técnica también se puede aplicar a diferentes tipos de tejidos que tienen ya sea la arquitectura anatómica reconocible o células marcadas fluorescentemente / regiones. Por lo tanto, la principal ventaja de la incorporación de esta nueva técnica de microdisección en los protocolos de aislamiento de células es que las células pueden ser aisladas de regiones específicas de tejido para eliminar la contaminación de los tejidos vecinos y las células se pueden recoger inmediatamente para su análisis o se cultivaron durante otras aplicaciones.

<table border="0" cellpadding="0" cellspacing="0" width="572">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">16520-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DPBS</td>
			<td style="width:71px;">Gibco</td>
			<td align="right" style="width:119px;">14190144</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of distinct cell populations from the developing cerebellum by microdissection

Microdissection es una nueva técnica que puede aislar regiones específicas de un tejido y eliminar la contaminación de fuentes celulares en las zonas adyacentes. Este método fue utilizado por primera vez en el estudio de la expresión de nestina progenitoras (NEP), una población recientemente identificado de células en la capa germinal externa cerebelosa (EGL). Uso de microdisección en combinación con la clasificación de células fluorescentes activadas (FACS), una población pura de NEP se recogió por separado a partir de precursores neuronales de gránulos convencionales en la EGL y de otras células que expresan nestina contaminantes en el cerebelo. Sin microdisección, análisis funcionales de los PIJ no habría sido posible con los métodos actualmente disponibles, como la centrifugación en gradiente de Percoll y captura microdissection láser. Esta técnica también se puede aplicar para su uso con diversos tejidos que contienen cualquiera de las regiones reconocibles o células marcadas con fluorescencia. Lo más importante, una gran ventaja de este microdissectiotécnica n es que las células aisladas viven y pueden ser cultivadas para la experimentación, que en la actualidad no es posible con otros métodos descritos.

Como se muestra en la Figura 1A, rebanadas del cerebelo se prepararon a partir P4 Math1-GFP / ratones Nestin-PPC, en el que el cerebelo EGL estaba dominado por PNB que expresan GFP y la capa molecular fue enriquecida por las células gliales CFP-positivas. Para aislar los PIJ que se encuentran en la parte profunda de la EGL, rebanadas se microdissected entre el EGL y la capa molecular (por la línea discontinua; Figura 1A). EGLS diseccionado (Figura 1B)</strong...

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Progenitores nestina que expresan son una población recientemente identificado de progenitores neuronales en el cerebelo en desarrollo. Utilizando la técnica de microdisección se presenta aquí en combinación con la clasificación de células activadas por fluorescencia-, esta población de células puede purificarse sin contaminación de otras regiones del cerebelo y puede ser cultivado para estudios posteriores.

El aislamiento de las poblaciones celulares distinto del cerebelo en desarrollo por Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

isolation-distinct-cell-populations-from-developing-cerebellum

Research

JoVE Journal

Neuroscience

9.5K vistas.  Temple University Health System.  Progenitores nestina que expresan son una población recientemente identificado de progenitores neuronales en el cerebelo en desarrollo. Utilizando la técnica de microdisección se presenta aquí en combinación con la clasificación de células activadas por fluorescencia-, esta población de células puede purificarse sin contaminación de otras regiones del cerebelo y puede ser cultivado para estudios posteriores. 

Video: Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Caracterización electrofisiológica de las buenas prácticas agrarias que expresan las poblaciones de células en la retina intacta

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Investigación

Neurociencias

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Captura de microdisección láser poblaciones enriquecidas de neuronas o neuronas individuales para análisis de expresión génica después de una lesión cerebral traumática

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Producción De ARN Para El Análisis Transcriptómico De Los Cuerpos Celulares De La Neurona Motora De La Médula Espinal De Ratón Por Microdisección De Captura Láser

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Microdisección de captura por láser - Una demostración del aislamiento individual de dopamina neuronas y el Área ventral tegmental completo

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

Análisis fisiológico en profundidad de poblaciones de células definidas en el infarto agudo del tejido Rebanadas del ratón órgano vomeronasal

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

La proyección de imagen seguida de la célula de seguimiento para la biología de la célula de Monitor y la progresión de linaje de poblaciones neuronales múltiples en vivo

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

Optimización del Microdissection de la captura de láser para el aislamiento de los ganglios entéricos de tejido humano fresco congelado

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Un protocolo de acero inoxidable para el aislamiento de ARN de alta calidad del Laser capturar las células Microdissected de Purkinje en el cerebelo humano Post-Mortem

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultivo primario de neuronas aisladas del cerebelo del ratón embrionario

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...