Agradecemos a la Dra. Shirin Bonni para proporcionar la construcción pSil-GFP, el Dr. Alper Uzun para la ilustración de la Figura 1, y la instalación de Bioimagen Leduc para la microscopía confocal. EMM es apoyado por el premio a la Trayectoria para la ciencia médica con cargo al Fondo Burroughs Wellcome, un premio NARSAD de Jóvenes Investigadores, y el NIH CNRR COBRE RR018728 P20-01. SBL es apoyado por el NIH CNRR COBRE RR018728 P20-01, y ha recibido el apoyo de PHS NRSA 5T32MH019118-20.

1. Preparación de Soluciones de Cultura y Medios de Comunicación (no en el vídeo) <ol><li> Preparar 1 litro de solución salina equilibrada completa de Hank (HBSS) que contiene HBSS 1x, 2.5 mM HEPES (pH 7,4), 30 mM de D-glucosa, 1 mM CaCl 2, 1 mM de MgSO 4 y 4 mM NaHCO 3. Agregue el agua doblemente destilada (DDC 2 O). Filtrar con un filtro de esterilización de 0,2 micras y se almacena a 4 º C. </li><li> Para preparar el medio de cultivo utilizando rebanada 35 ml de Medio Basal de Eagle medios, 12,9 ml de HBSS completos, 20 mM de D-glucosa (1,35 ml de solución 1 M), 1 mM de Glutamax (0,25 ml de una solución 200 mM), 0,5 ml de penicilina -estreptomicina 100x de valores. Filtrar esterilizar con un filtro de 0,2-m, a continuación, añadir inactivado por calor suero de caballo a una concentración final de 5%. </li><li> Preparar una solución laminina de trabajo, haciendo un 1 mg / ml de solución madre laminina con agua desionizada estéril destilada. Preparar 100 ml de alícuotas de 0,5 ml tubos Eppendorf y se congela a -80 ° C. </li><li> Preparar poli-L-lisina de trabajo paralución mediante la adición de 5 ml de H 2 O estéril a 5 mg de poli-L-lisina para hacer un 1 mg / ml de solución de stock. Preparar alícuotas de 1 ml y congelar a -20 ° C. </li><li> Preparar una solución de revestimiento mediante la dilución de 1 ml de poli-L-lisina y 100 l de laminina a un volumen final de 12 ml con agua estéril. Haga esta solución fresca cada vez. </li></ol> 2. Preparación Inserta organotípicos Slice (no en video) <ol><li> Prepare dos seis placas con inserción de la cultura y uno por el uso de fórceps estériles. Añadir 2 ml de agua estéril O ddH 2 por debajo de las inserciones de la cultura. </li><li> Añadir 1 ml de la solución de revestimiento en la parte superior de la membrana teniendo cuidado de no perforar la membrana. Incubar toda la noche en un incubador humidificado a 37 º C y 5% de CO 2. </li><li> Extraiga el material de recubrimiento y lavar la membrana con estériles de H 2 0 tres veces. Que insertos en seco antes de usarlo. Añadir 1,8 ml de medio de corte cultura y el lugar en el 37 ° C incubadora. Envuelva las placas utilizadas con Parafilmy se almacena a 4 º C durante un máximo de 4 semanas. </li></ol> 3. Preparación para la electroporación (no en el vídeo) <ol><li> Preparar las construcciones de RNAi para la electroporación. Doble hebra insertos horquilla de ARN fueron clonados en un vector pSilencer. Los plásmidos Contiene: 1) un promotor U6 que impulsa la doble cadena de generación de ARN, y 2) un casete de expresión GFP dirigido por el promotor CMV 8,9. Este plásmido ha sido descrito previamente por Konishi y colegas 9 y otros 7,8. Los plásmidos se purificaron utilizando un Qiagen maxi-prep kit, y se utiliza a una concentración de 1 mg / ml. </li><li> Con el fin de visualizar ADN, mientras que la inyección, preparar una solución al 0,5% rápido colorante verde y utilizarlo a las 1:20 con el ADN que se inyecta (normalmente 20 l de ADN con 1 l de verde rápido). Quede sobre ADN-rápido mezcla de colorantes verde puede ser almacenada a -20 ° C durante hasta una semana. </li><li> Área de disección limpia, dissectiherramientas ng y vasos vibrátomo con etanol al 70%. Enfriar HBSS completas de modo que sea frío hielo. Chill vibrátomo nave de embalaje de hielo a su alrededor en el interior del vibrátomo con un poco de agua para el enfriamiento rápido. Preparar un 3% bajo punto de fusión de agarosa utilizando HBSS completos. Microondas durante 1 minuto. Evitar el exceso de ebullición. Mantener en un baño de agua a 42 ° C hasta su uso. </li><li> Electroporación parámetros se fijan de la siguiente manera. Para un uso de embriones E15 35 V, 5 pulsos de longitud, 100 m, 900 m de intervalo entre los pulsos. Para los animales mayores, para asegurar la electroporación, utilizar un voltaje más alto hasta 50 V o aumentar el número de pulsos de hasta 8 pulsos. Para evitar dañar el tejido en animales jóvenes, utilizar impulsos ya sea menos o pulsos de hasta un 2 y un menor voltaje de hasta 25 V. Estos parámetros pueden variar y determinar empíricamente dependiendo de la edad del animal. </li></ol> 4. Disección y electroporación (en vídeo) <ol><li> Después de la eutanasia de una mujer embarazada, la disección de embriones a cabo en el hielo completos HBSS fría. Keep cada embrión en sus sacos placentarios individuales. </li><li> Disección del embrión y se cortará la cabeza después de la primera vértebra. Tenga en hielo completos HBSS fría. </li><li> Para inyección, poner la cabeza sobre un trozo de Parafilm en la parte superior de una placa de Petri. Usando una jeringa Hamilton por encargo (ver Tabla I) se inyectan de 6 a 8 l de ADN: mezcla rápida tinte verde a través del tercer ventrículo con el fin de completar los dos ventrículos laterales en las vesículas corticales. Alternativamente, la inyección puede hacerse directamente en cada ventrículo lateral. </li><li> Para la electroporación ex vivo, el uso de la BTX-pinza electrodos de platino. Colocar el electrodo positivo hacia el lado de la corteza que desea electroporate superior es decir, de la cabeza de la corteza dorsal. </li><li> Después de la electroporación, se incuban las cabezas en hielo durante al menos 5 minutos antes de la disección. </li><li> Dissect cerebros en HBSSby hielo frío haciendo una pequeña incisión en el lado de la cabeza y el pelado de la piel de los lados de la cabeza. A continuación, conunas pinzas finas pelar suavemente lejos de la pia del cerebro. Retire el cerebro intacto del cráneo, teniendo cuidado de no dañar la corteza. </li></ol> 5. Inclusión y corte de las cortezas electroporados (en vídeo) <ol><li> Transferir 3% de agarosa de bajo punto de fusión en un molde grande se coloca en hielo. La parte inferior del molde comenzar a solidificarse más rápido que evite los cerebros se hunda hasta el fondo del molde. Suavemente, transferir el cerebro con unas pinzas finas, uno por uno después de retirar el exceso de tampón con un Kimwipe o papel de filtro. Utilice una punta de pipeta de 10 l en forma de remolino los cerebros en el interior del molde para asegurar la máxima operatividad entre la agarosa y el tejido cerebral. </li><li> Oriente el cerebro para asegurar que todos los cerebros están en la misma orientación y en el mismo nivel en la agarosa. Que la agarosa solidificar durante aproximadamente 5 minutos. Utilice adhesivo (cola loca) para fijar los bloques de agarosa para que los bulbos olfativos están de pie. Una vez que los bloques están unidos, de inmediato añadoce HBSS fría y recortar la agarosa para asegurarse de que cada rebanada, se obtienen para cada cerebro. </li><li> Para cortar los bloques, establece la velocidad de la vibrátomo a una velocidad baja (aproximadamente la mitad del máximo) y establecer la frecuencia de vibración de los álabes en la posición más alta. Generar 250-micras rodajas gruesas coronal. Recuperar las rebanadas con una espátula fina doblada y transferirlos a los pozos de tejido con un pincel fino o con fórceps. </li><li> En la campana de cultivo de tejidos, transferir las rebanadas en los insertos recubiertos. Añadir 500 l de medio de cultivo a cada rebanada de inserción para facilitar los traslados. Hasta 5 rebanadas puede ser colocado por inserción. Quite el exceso de la parte superior de las rebanadas y se incuba a 37 ° C en un incubador humidificado. </li></ol> 6. Cultura y Análisis de Sectores organotípicos (en vídeo) <ol><li> A fin de mantener las rebanadas sanos, los medios frescos deben añadirse al menos cada dos días debajo de la membrana mediante la sustitución de la mitad de los medios de comunicación cada vez. </li><li> Con el fin de analizar las rebanadas después de la deseado día en la cultura, arreglar las rebanadas en la membrana. Lavar con 1 x tampón fosfato salino (PBS) a 37 ° C tres veces durante 10 minutos cada vez. A continuación, fijar con paraformaldehído al 4% (PFA) durante la noche a 4 ° C o durante 1 hora a temperatura ambiente. </li><li> Las rebanadas se pueden analizar con diferentes marcadores celulares o teñidas con Hoechst solo para visualizar las células electroporated y no electroporado. Permeabilizar y bloquear rodajas durante 2 horas a temperatura ambiente con suero de cabra al 10%, 0,1% de Triton en 1x PBS con agitación suave. </li><li> Stain con Hoechst durante 1 hora a temperatura ambiente, se lava 3 veces con PBS 1x 10 min cada vez con agitación suave. </li><li> Para montar rodajas cortar la membrana con un escalpelo, y utilizar un fórceps finas para transferir la membrana que contiene las rebanadas para un vidrio esmerilado se desliza en una cámara de agua. Hasta 5 rebanadas pueden ser colocados por cada portaobjetos de vidrio. Retire el exceso de agua, y añadir una gota de solución Flourmount a cada sector del cerebro. Con suavidad, coloque un cubreobjetos encima de las rodajas de cerebro y eliminar uny las burbujas de aire. Analizar las rebanadas con el microscopio confocal. </li></ol> 7. Inclusión en parafina de las rebanadas organotípicos alternativa (no en el vídeo) <ol><li> Rebanadas organotípicos también puede ser embebido en parafina para un análisis más preciso morfológica, a este fin, la membrana que contiene las rebanadas organotípicos puede ser fijado en 4% PFA como se describió anteriormente. </li><li> Las rodajas fijas están incrustados en% de agarosa 1 (precalentado a 37 ° C), y se solidifica en hielo durante 30 minutos. Los bloques de agarosa puede ser post-fija en el 4% PFA a 4 ° C durante 30 min. </li><li> El bloque de agarosa que contiene la rebanada organotípico luego se incluyeron en parafina y se procesaron para inmunofluorescencia como se ha descrito previamente 10. </li></ol> 8. Los resultados representativos Una representación esquemática de la electroporación de la corteza murino y la cultura de rebanadas organotípicos se muestra en la Figura 1. Este método es una estrategia útil para rapid evaluación de la función de genes implicados en el desarrollo neuronal 11. Dependiendo de la cantidad de ADN y electroporado la etapa embrionaria en la electroporación, la eficacia de transfección puede variar. Rebanadas comenzará expresando GFP por lo menos 8 horas después de la electroporación y las células se someterá a la secuencia normal de eventos neurogénicos (proliferación, migración y diferenciación neuronal temprana) en la cultura. La figura 2 muestra un corte de cerebro electroporado que está expresando un control pSilencer-GFP vectorial y se puede observar progenitores neuronales, las neuronas que migran y las neuronas diferenciadas en la división. Rebanadas organotípicos mantendrá su morfología, siempre que se mantengan en un buen medios de interfaz de aire en las membranas y se puede utilizar hasta por lo menos 5 días en cultivo. <img alt="Figura 1" src="/files/ftp_upload/3621/3621fig1.jpg" /> Figura 1. Ilustración de la electroporación ex vivo y cortar en rodajas organotípicoensayo de cultivo. E14.5 embriones disecados, y de forma individual inyectados con ADN mezclado con colorante verde rápido para visualizar el sitio de la inyección. ADN se puede inyectar en ambos los ventrículos laterales como se muestra en la ilustración o en el tercer ventrículo con el fin de llenar los ventrículos laterales. Después de la inyección, los cerebros se electroporación con un electroporador onda cuadrada, colocando el electrodo positivo en el lado deseado del cerebro. Los cerebros están incorporados en el 3% de agarosa de bajo punto de fusión y se seccionaron utilizando un vibrátomo. 250 rodajas de cerebro micras se colocan en 0,4 micras inserta y se cultivaron hasta una semana. GFP se puede observar después de las 8 horas después de la transfección. <img alt="Figura 2" src="/files/ftp_upload/3621/3621fig2.jpg" /> Figura 2. El análisis de las secciones de cerebro electroporación. Rodajas de cerebro electroporados se tiñeron de Hoescht. La corteza dorsal muestra electroporadas progenitores neuronales en la zona ventricular (VZ). Las neuronas de la cortiCal placa (CP) están delimitados por la zona marginal (MZ). Las flechas blancas muestran las neuronas que migran. En este caso, los cerebros fueron inyectados en E15.5 y electroporación con un vector de control pSilencer GFP. Las secciones representan explantes corticales 4 días después de la electroporación. Barra de escala de 100 micras. Solución de problemas: <ol><li> Eficacia de transfección bajo: ajustar la concentración de ADN utilizado para al menos 1 g / l. Siempre usar el ADN muy limpia a partir de una preparación máxima, si es necesario, utilice una endo-libre de Quiagen kit para purificar el ADN. </li><li> Las células transfectadas en un área del cerebro diferente a la deseada: Asegurarse de que los electrodos están colocados correctamente con la posición del electrodo positivo hacia el lado del cerebro que se electroporación. </li><li> Rebanadas organotípicos pierde la morfología: los medios de comunicación cambian todos los días y garantizar los cortes no están flotando en los medios de comunicación </li><li> Rebanadas salir de la agarosa, ya que se están cortando en la vibratome: Asegúrese de que una buena interfaz se hace al incrustar el cerebro en el bajo punto de fusión de agarosa. </li></ol>

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No tenemos nada que revelar.

Estos métodos implican la electroporación ex vivo de los plásmidos que codifican ARN bicatenario horquillas 8 y la cultura de rebanadas organotípicos 4 proporcionan varias ventajas distintas. En primer lugar, estos métodos permiten una evaluación rápida de RNAi derivados de fenotipos. La inclusión de una expresión que codifica GFP cassette en el vector pSilencer mismo que contiene el promotor U6 que impulsa la doble cadena de ARN horquilla permite una rápida identificación y car...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Hamilton jeringa </td><td> HAMILTON </td><td> 80008 </td><td> 31 de calibre 0,5 pulgadas de largo, PT-4 (nivel de biselado punto), el volumen de 10ul </td></tr><tr><td> Tweezertrodes Platinum </td><td> BTX </td><td> 45-0489 </td><td> Tamaño de 5mm </td></tr><tr><td> ECM830 electroporador </td><td> BTX </td><td> 45-0002 </td><td></td></tr><tr><td> BTX pedal </td><td> BTX </td><td> 45-0208 </td><td> Para el uso con ECM830 electroporador </td></tr><tr><td> Vibrante microtomo la hoja </td><td> LEICA </td><td> VT1000 S </td><td></td></tr><tr><td> 6 - plato bien para su uso con insertos </td><td> FALCON </td><td> 353502 </td><td> Contiene muescas para encajar insertos </td></tr><tr><td> Insertos de cultivo de tejidos </td><td> FALCON </td><td> 353090 </td><td> 0,4 micrómetros </td&gt; </tr><tr><td> Fast Green </td><td> SIGMA </td><td> F7252 </td><td></td></tr><tr><td> Agarosa de bajo de fusión </td><td> FISHER </td><td> BP165-25 </td><td> ADN de grado </td></tr><tr><td> La laminina </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Poli-L-lisina </td><td> Sigma-Aldrich </td><td> P5899 </td><td></td></tr><tr><td> Medio Basal de Eagle </td><td> Sigma Aldrich </td><td> B-1522 </td><td></td></tr><tr><td> 10x HBSS sin Ca y Mg </td><td> GIBCO </td><td> 14180-046 </td><td></td></tr><tr><td> HEPES libre de ácido </td><td> Sigma-Aldrich </td><td> H4034 </td><td></td></tr></tbody></table>

methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex

La corteza cerebral dirige las funciones cognitivas superiores. Esta estructura seis capas se genera en el interior de un primer, fuera-última forma, en el que las neuronas primeros nacidos permanecen más cerca al ventrículo, mientras que las neuronas últimos nacidos migran más allá de las neuronas primeros nacidos hacia la superficie del cerebro 1. Además de la migración neuronal 2, un proceso clave para la función cortical normal es la regulación de la morfogénesis neuronal 3. Si bien la morfogénesis neuronal puede ser estudiado in vitro en cultivos primarios, hay mucho que aprender de cómo estos procesos están regulados en los entornos de tejidos. Se describen las técnicas para analizar la migración neuronal y / o la morfogénesis en rodajas organotípicos de la corteza cerebral 4,6. Un vector pSilencer modificado se usa que contiene tanto un promotor U6 que impulsa el ARN de doble hebra de horquilla y un casete de expresión independiente que codifica la proteína GFP impulsado bya promotor CMV 7-9. Nuestro enfoque permite la evaluación rápida de los defectos en el crecimiento de las neuritas en caída específica de genes candidatos y ha sido utilizado con éxito en una pantalla para que los reguladores de crecimiento de las neuritas 8. Debido a que sólo un subconjunto de células se expresan las construcciones RNAi, las rebanadas organotípicos permitir un análisis del mosaico de los fenotipos posibles. Además, debido a este análisis se realiza en una aproximación cercana del entorno in vivo, que proporciona un bajo coste y rápida alternativa para la generación de animales transgénicos o knockout para los genes de función desconocida cortical. Finalmente, en comparación con la tecnología de electroporación in vivo, el éxito de los ex experimentos in vivo electroporación no depende de desarrollo proficiente habilidad cirugía y se puede realizar con un tiempo de formación más cortos y habilidad.

Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Para llevar a cabo una evaluación rápida de la función de los genes en el desarrollo de la corteza cerebral, se describen procedimientos que implican la<em&gt; Ex vivo</emElectroporación&gt; de plásmidos co-expresión de inhibitoria ARN (RNAi), y las buenas prácticas agrarias en la corteza de embriones de ratón. Este protocolo se presta al estudio de diversos aspectos de neurodesarrollo, tales como la neurogénesis, la migración neuronal y la morfogénesis neuronal incluida la dendrita y axón.

Métodos de estudio de la morfogénesis neuronal:<em&gt; Ex vivo</em&gt; RNAi electroporación en la corteza cerebral embrionario murino

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

11.7K vistas.  Brown University. Para llevar a cabo una evaluación rápida de la función de los genes en el desarrollo de la corteza cerebral, se describen procedimientos que implican la Ex vivo de plásmidos co-expresión de inhibitoria ARN (RNAi), y las buenas prácticas agrarias en la corteza de embriones de ratón. Este protocolo se presta al estudio de diversos aspectos de neurodesarrollo, tales como la neurogénesis, la migración neuronal y la morfogénesis neurona...

Video: Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

La transfección de células de ratón del ganglio de la retina por In vivo La electroporación

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Investigación

Neurociencias

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En electroporación in vivo de la retina del ratón en desarrollo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando<em&gt; En el útero</em&gt; La electroporación

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Electroencefalografía simultánea, en tiempo real Medición de la concentración de lactato y Manipulación optogenético de la actividad neuronal en la corteza cerebral de roedores

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dos registros electrofisiológicos de sinápticamente respuestas evocadas neuronal y astroglial en agudas rodajas de hipocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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 ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

La electroporación en el útero métodos para estudiar la excitabilidad de las subpoblaciones neuronales y Conectividad Una sola célula

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Inducción de la recombinación del Cre-lox en la corteza Cerebral de ratón a través de electroporación en el útero

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

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 ...