Este estudio fue financiado en parte por la Fundación Ipsen. IMV es un profesor Sociedad Americana del Cáncer de Biología Molecular, y el titular de la Cátedra Irwin y Joan Jacobs en Ciencias de la vida ejemplar. Este trabajo fue apoyado en parte por subvenciones del NIH, Fundación Leducq, Fundación Meriaux, Ellison Medical Foundation, Ipsen / Biomeasure, Sanofi Aventis, Prostate Cancer Foundation, y HN y Frances C. Berger Fundación. PAS es financiado por McKnight Fondo de Dotación para la Neurociencia y el Instituto Nacional sobre Abuso de Drogas (R01 DA019022). Los autores desean agradecer a Mark Schmitt. Arte gráfico fue diseñado por Jamie T. Simon. Patrocinio fue proporcionado amablemente por Invitrogen.

1. Organotípicos Culturas del cerebelo: <ol><li> Para generar éxito organotípicos culturas cerebelosa utilizar ratones entre P0-P3 o P8-P12. Para nuestro experimento utilizaron ratones en el rango de P8-P12 ya que esta etapa de desarrollo era más apropiado para nuestro estudio. </li><li> Prepare la disección y el medio de cultivo: <ol><li> La disección del cerebro y la preparación de corte se llevan a cabo en la baja artificial de sodio líquido cefalorraquídeo (ACSF), que contiene cloruro de calcio 1 mM, 10 mM D-glucosa, 4 mm de cloruro de potasio, cloruro de magnesio 5 mM, bicarbonato de sodio de 26 mm, 246 mm y una solución de sacarosa rojo fenol (1:1000) , pH 7,3. Para esterilizar la solución, se filtra a través de un filtro de 0,22 micras y se almacenan a 4 ° C. </li><li> Rodajas de cerebelo se cultivan en el 75% mínimo indispensable medio de Eagle (MEM), el 25% inactivado por calor suero de caballo, HEPES 25 mM, 1 mM glutamina, glucosa 5 mg / ml, penicilina (100 U / ml) y estreptomicina (100 U / mL). Filtrar el medio a través de un filtro de 0,22 micras y se almacenan a 4 ° C durante un máximo de dos semanas. </li></ol></li><li> Diseccionar el cerebro en el helado a medio ACSF previamente burbujeado con carbogen (95% de oxígeno y 5% de dióxido de carbono) Tenga en cuenta que la saturación con Carbogen va a cambiar el pH de la solución por lo que pasará de rojo a naranja. La disección se debe realizar en el menor tiempo posible, teniendo cuidado de no dañar la estructura del cerebro. </li><li> Rebanada del cerebro en las secciones sagitales de 400 m con un vibratome. <ol><li> Haga un corte sagital con una hoja de afeitar en un hemisferio, tratando de cortar lo menos posible del cerebelo, lo que proporcionará una superficie plana para cortar. La corteza se usa sólo para el apoyo para hacer el corte en lonchas más fácil, pero como el tiempo es un tema importante que es más conveniente para cortar la mitad rostral de la corteza con una cuchilla de afeitar. </li><li> El uso de cianoacrilato (Superglue) para fijar la posición del cerebro en la placa de metal vibratome. Es importante para enfriar la placa de hielo antes de añadir la cola y que se extendió también con la punta del tubo para formar una capa adhesiva delgada. Si hay un exceso de pegamento, que se desprenden de la placa cuando se entra en contacto con la ACSF y la muestra se perderá. </li><li> Una vez que el cerebro está conectado a la placa vibratome, añadir una cantidad suficiente para cubrir los medios de comunicación ACSF el cerebro y la puso debajo de la placa de hielo para mantenerlos fríos. </li><li> Cortar rodajas de 400 micras y la transferencia con una pipeta de plástico Pasteur (corte la punta para ampliarla y evitar daños a los cortes) a una placa de 6 celdas, y la cultura que contiene ACSF en el hielo. </li><li> Separar el cerebelo desde el resto del cerebro con la ayuda de dos jeringas de insulina (jeringas de pequeño diámetro de calibre 28 agujas) bajo el microscopio de disección. Algunos cortes se han unido a un pegamento de los bordes: este es el momento para tratar de eliminar, ya que puede afectar la viabilidad de la división. Es importante hacerlo con cuidado sin dañar la estructura del cerebelo. </li></ol></li><li> Transferencia de las rodajas de cerebelo a los 6 y placas que contienen de 30 mm se inserta la placa de cultivo con 0,4 micras poros. Añadir 1 ml de medio de cultivo por pozo, que se pre-condicionada por la incubación de al menos 2 horas a 37 ° C y 5% de CO 2 (en el cultivo celular incubadora). Colocar los insertos en la parte superior de los medios de asegurarse de que no hay burbujas de aire atrapadas debajo y luego colocar las rodajas de cerebelo (1-3 por inserto) en la parte superior de los insertos de asegurarse de que estén completamente extendidos y que el líquido no es cubierta ellos. Aspirar el exceso de los medios de comunicación si es necesario. Se incuba a 37 ° C y 5% de CO 2 cambia el medio de cultivo cada 2 o 3 días. Lo mejor es reemplazar sólo una parte del medio (por ejemplo, el volumen medio) a la vez, ya medio utilizado contiene derivados slice-factores tróficos que son importantes para la supervivencia celular. </li></ol> 2. Fas tratamiento y tinción de las rebanadas del cerebelo: <ol><li> Para el experimento de exposición a la apoptosis, que la cultura primero las rodajas durante 5 días cambiando los medios de comunicación en el día in vitro 3 (DIV3). En DIV5 culturas son tratados con 0,5 mg / mL de la Jo2 Fas anticuerpo agonista añadiendo directamente a los medios de comunicación a fin de tener un fondo limpio de la muerte celular ya que los cambios de los medios de comunicación pueden inducir a cierta mortalidad en los cultivos. La mitad de los pozos no se tratan como controles. </li><li> Después de 24 horas retire el material por debajo de los insertos por succión. Para solucionar los cortes, agregar 1 ml de helado un 4% por debajo de PFA y 1 ml por encima de la inserción. Incubar durante 10 minutos a temperatura ambiente. </li><li> Después de 10 minutos, retire la PFA y lavar brevemente con PBS frío. </li><li> A continuación, retire la PBS y añadir 1 ml y por debajo de 1 ml por encima de la inserción de helado de metanol al 20% en PBS e incubar durante 5 minutos a temperatura ambiente. </li><li> Lavar a continuación con PBS y añadir 1 ml por debajo de 1 ml y por encima de la inserción de un 0,5% de Triton X-100 en PBS durante la permeabilización. Se incuba durante un mínimo de 12 horas a 4 ° C. </li><li> Después de permeabilización, bloque con un 20% BSA en PBS durante un mínimo de4 horas a temperatura ambiente o la noche a 4 ° C. En esta etapa, las rebanadas se pueden mantener en solución de bloqueo durante al menos 3 días a 4 ° C. </li><li> Con el fin de manchar las rodajas de corte con cuidado la pieza de la membrana insertar adjunto a la parte del cerebelo y la transferencia a 24 y placas que contienen PBS. </li><li> La inmunotinción se hace de forma secuencial, primero con el reactivo TUNEL durante una hora y luego con el anticuerpo Calbidin noche a la mañana. <ol><li> Aspirar el PBS y añadir 250 l de reactivo TUNEL en cada pocillo asegurándose de que cubre toda la superficie del corte. Se incuba a 37 ° C durante una hora en la oscuridad. </li><li> Después de una hora remover la mezcla de TUNEL y lavar con PBS tres veces durante 10 minutos. </li><li> Después del último lavado, eliminar el PBS y añadir 250 l de una dilución 1 / 1000 de la calbindina anticuerpos en PBS e incubar durante la noche a 4 ° C en la oscuridad. </li><li> Eliminar la solución de anticuerpo primario y lavar con PBS tres veces durante 10 minutos. </li><li> Añadir 250 ml del anticuerpo secundario diluido 1 / 500 en PBS y se incuba durante al menos tres horas a temperatura ambiente en la oscuridad. El kit TUNEL usamos está marcado con rojo TMR, por lo tanto, nuestro anticuerpo secundario para calbindina se conjuga con Alexa-488. </li><li> Lavar tres veces durante 10 minutos con PBS y montar los cortes sobre un portaobjetos de vidrio utilizando un medio de montaje que contiene DAPI. </li><li> Imagen de las rebanadas con un microscopio confocal utilizando los 488 nm y 561 nm de longitud de onda de excitación de calbindina y TUNEL, respectivamente. </li></ol></li></ol> 3. Los resultados representativos: El principal reto de este protocolo es capaz de generar saludables culturas rebanada del cerebelo, sobre todo porque nuestra lectura final será la muerte celular. Una sana cultura aparece como uno donde la capa de células del cuerpo es transparente y permite ver la estructura foliada del cerebelo bajo el microscopio (Figura 1). Después de unos días en la cultura de las rodajas de empezar a células que son delgadas y no neuronales se puede observar la migración fuera de los márgenes de la división. Las células redondas oscuras (la mayoría de los macrófagos probable) que cubren los cortes después de unos días en la cultura, con el tiempo se reducirán en número después de 10-14 días in vitro (Figura 2) 5. La prueba definitiva para la viabilidad de los cortes es para teñir de diferentes marcadores celulares, tales como calbindina de las células de Purkinje. La Figura 3 muestra una imagen representativa microscopio confocal de la capa de células de Purkinje con varios núcleos TUNEL positivos. Es importante tener en cuenta que incluso si algunas rebanadas recibió un estímulo apoptótico, fueron expuestos sólo durante 24 horas. Esto permitió tiempo suficiente para observar la fragmentación del ADN en las células que mueren con la tinción de TUNEL, pero una capa de células de Purkinje definido todavía estaba presente. <img src="/files/ftp_upload/2564/2564fig1.jpg" alt="Figura 1" /> Figura 1. Parte sana del cerebelo en DIV2. Esta imagen muestra la capa de células del cuerpo translúcido y la estructura foliada del cerebelo en una porción saludable. <img src="/files/ftp_upload/2564/2564fig2.jpg" alt="Figura 2" /> Figura 2. Parte sana del cerebelo en DIV 7. En esta imagen todavía se puede observar la estructura foliada del cerebelo y la aparición de cuerpos de células oscuras. <img src="/files/ftp_upload/2564/2564fig3.jpg" alt="Figura 3" /> Figura 3. Imagen representativa de una rebanada de Fas-cerebelosa tratado. Esta imagen confocal muestra las células de Purkinje se tiñeron con calbindina (verde) y las células apoptóticas positivas para la tinción de TUNEL (rojo). Las células de Purkinje no aparecen en una sola fila, pero se agrupan formando una estructura similar a folio.

<ol>
	<li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+ependymal+cells+in+tissue+cultures.">Human fetal ependymal cells in tissue cultures.</a> Anat Rec. 99, 523-529 (1947).</li><li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+studies+of+human+fetal+brain+cells+in+tissue+cultures.">Review of studies of human fetal brain cells in tissue cultures.</a> Etudes Neonatales. 1, 1-13 (1952).</li><li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+protocol+for+organotypic+hippocampal+slice+cultures.">Staining protocol for organotypic hippocampal slice cultures.</a> Nat Protoc. 1, 2452-2456 (2006).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Beat, H., G&#228;hwiler, S. M. T., McKinney, R. A., Debanne, D., Robertson, R. T., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic Slice Cultures of Neural Tissue in Culturing Nerve Cells. , 461-498 (1998).</li></ol>

Todos los animales de trabajo llevado a cabo se realizó de acuerdo con las políticas establecidas por el IACUC. El Instituto Salk es un centro acreditado AAALAC.

Este método describe una de las muchas aplicaciones posibles de cultivos organotípicos neuronal y que nos ha permitido estudiar el efecto de los estímulos de apoptosis de tipo salvaje y mutante rodajas de cerebelo. La parte más crítica de este experimento es ser capaz de generar consistentemente saludable culturas trozo del cerebelo. Los factores clave son la disección y el método de los cortes y la capacidad para llevar a cabo el protocolo en el menor tiempo posible, pero al mismo tiempo teniendo cuidado de no dañar los trozos. Otro factor crucial es la preparación y la composición de los medios de cultivo, estos cultivos son muy sensibles, así que tuvimos que probar varias recetas y las marcas de los medios de comunicación. Incluso los diferentes lotes de suero o cualquier otro componente de los medios de comunicación podría afectar la viabilidad de los sectores. Tenemos los mejores resultados con el suero de caballo de Lot # 480116 Invitrogen. Una vez que los cortes del cerebelo se generan se puede estudiar el desarrollo del cerebelo, la electrofisiología, la supervivencia de las células solas o en respuesta a estímulos apoptóticos, excitotoxicidad, o la falta de oxígeno. Los estudios comparativos con el tipo salvaje y mutante rebanadas del cerebelo han sido muy profundas en muchos aspectos de la función del cerebelo y el desarrollo.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11090</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/ Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Superglue</td>
<td>&nbsp;</td>
<td>Krazy Glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well / 24 well cell culture plates</td>
<td>&nbsp;</td>
<td>Corning Incorporated</td>
<td>3506/ 3527</td>
<td>&nbsp;</td>
<tr>
<td>30mm culture plate inserts (0.4-&mu;m pore)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM03050</td>
<td>&nbsp;</td>
<tr>
<td>Purified NA/LE Hamster anti-mouse CD95 (Jo2)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>554254</td>
<td>&nbsp;</td>
<tr>
<td>In situ cell death detection kit, TMR red</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>12 156 792 910</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-Calbindin D-28k antibody</td>
<td>&nbsp;</td>
<td>Swant</td>
<td>CB-38a</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 488-goat anti-rabbit IgG secondary antibody</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular probes)</td>
<td>A-11008</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield Mounting Medium with DAPI</td>
<td>&nbsp;</td>
<td>Vector labs</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Zeiss Stemi SV 11</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica TCS SP2 AOBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

organotypic cerebellar cultures: apoptotic challenges and detection

Cultivos organotípicos de tejido neuronal se introdujeron por primera vez por Hogue en 1947 de 1,2 y han constituido un gran avance en el campo de la neurociencia. Desde entonces, la técnica se desarrolló aún más y actualmente hay muchas formas diferentes de preparar cultivos organotípicos. El método presentado aquí es una adaptación de la descrita por Stoppini et al. Para la preparación de los cortes y de Gogolla et al. Por el procedimiento de tinción 3,4. Una característica única de esta técnica es que permite estudiar las diferentes partes del cerebro como el hipocampo y cerebelo en su estructura original, proporcionando una gran ventaja sobre los cultivos disociados en el que se interrumpen todas las de la organización celular y las redes neuronales. En el caso de que el cerebelo es aún más favorable, ya que permite el estudio de las células de Purkinje, muy difícil de obtener como cultivo primario disociados. Este método puede ser usado para estudiar algunas características del desarrollo del cerebelo in vitro, así como para los experimentos electrofisiológicos y farmacológicos, tanto de tipo salvaje y ratones mutantes. El método descrito aquí fue diseñado para estudiar el efecto de los estímulos apoptóticos como ligando de Fas en el cerebelo en desarrollo, mediante la tinción de TUNEL para medir la muerte celular por apoptosis. Si la tinción de TUNEL se combina con marcadores tipo específico de célula, como calbindina de las células de Purkinje, es posible evaluar la muerte celular de una manera la población de células específicas. La tinción calbindina también sirve el propósito de evaluar la calidad de las culturas del cerebelo.

Watch this Scientific Journal Video about Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection at JoVE.com

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Este método describe la generación de organotípicos culturas del cerebelo y el efecto de ciertos estímulos apoptóticos sobre la viabilidad de los diferentes tipos de células del cerebelo.

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. ...

organotypic-cerebellar-cultures-apoptotic-challenges-and-detection

Nanyang Technological University

Research

JoVE Journal

Neuroscience

18.2K vistas.  The Salk Institute for Biological Studies. Este método describe la generación de organotípicos culturas del cerebelo y el efecto de ciertos estímulos apoptóticos sobre la viabilidad de los diferentes tipos de células del cerebelo.

Video: Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

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 ...

Investigación

Neurociencias

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 ...

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 ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

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 ...

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 ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

El agotamiento selectivo de la microglía de cerebeloso gránulos Cultivos Celulares El uso de L-leucina metil éster

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

La investigación de la regeneración funcional en organotípicos médula espinal Co-culturas cultivadas en Arrays Multi-electrodos

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

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. ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex útero Electroporación y culturas Organotypic rebanada de cerebros embrionarios de ratón para vivir-la proyección de imagen de interneuronas GABAérgicas de migrando

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...