Estamos muy agradecidos a Lihong Yin para la asistencia técnica. NVD es un destinatario de un Premio al Investigador Joven NARSAD y también es apoyado por subvenciones del NIH (5 K99 MH095825-02). La investigación en el laboratorio Fishell es apoyado por el Instituto Nacional de Salud, Instituto Nacional de Salud Mental (5 R01 MH095147-02, 5 R01 MH071679-09), Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares (5 R01 NS081297-02, 1 P01 NS074972 -01A1) y la Fundación Simons.

Todos los animales fueron tratados de acuerdo con las normas y directrices del Comité de Cuidado y Uso de Animales institucional de la Escuela de Medicina de NYU. Las cepas de ratón Ratones hembra Swiss Webster proporcionadas por TACONIC se utilizaron para estos experimentos. Con el fin de centrarse específicamente en las interneuronas de la capa superficial, se utilizaron embriones E15.5. Nota: El plásmido usado en este trabajo (Dlx5 / plásmido 6.eGFP 3 g / l) se generó usando técnicas de clonación estándar. El ADNc de EGFP se clonó en un / 6-Pmin-poliA plásmido Dlx5. Este plásmido está disponible bajo petición (demarn02@nyumc.org). 1 Preparación de microinyección Pipetas <ol><li> Ajuste el extractor de calor # 2-860. </li><li> Coloque y fije el capilar de vidrio cerca del filamento. Compruebe que el diámetro exterior (OD) de la pipeta es más o menos 30 micras. </li><li> Pulse el botón &quot;tirar&quot;. </li><li> Retire con cuidado el capilar. La parte inferior es la aguja. Deseche la parte superior. </li></ol> 2. Procedimiento Anestesia <ol><li> Preparar una cámara que contiene isoflurano. Nota: El isoflurano es el anestésico preferido debido a su rápida acción y una baja incidencia de efectos secundarios. El pentobarbital es otra opción; Sin embargo, la tolerancia de este fármaco es variable y puede conducir a una tasa de mortalidad más alta que isoflurano. </li><li> Ajuste el flujo de isoflurano al 4-5% en 0,5-1 L / min de O 2. </li><li> Asegúrese de que la válvula de la cámara está abierta, mientras que la válvula para el adaptador de la cabeza está cerrado. </li><li> Coloca el ratón embarazada en la cámara. </li><li> Deje que el ratón para ir bajo el efecto de la anestesia. Esto tomará alrededor de 2-3 minutos. Compruebe la velocidad de la respiración. Si el ratón comienza a hiperventilar, disminuir la dosis de isoflurano. </li><li> Una vez anestesiado el ratón, con prontitud y eliminar de la cámara. Colóquelo del lado ventral hacia arriba sobre el cojín eléctrico y insert la cabeza en la máscara que continuará para suministrar isoflurano durante toda la cirugía. Asegúrese de cambiar el flujo de isoflurano desde la cámara a la tubería de conexión de la pieza de adaptador de la cabeza. Ponga una gota de lubricante ocular en cada ojo y cerrar los ojos. </li><li> Ajuste la almohadilla térmica a 36 ° C grados para minimizar la hipotermia. </li><li> Compruebe la ausencia de reflejos del pie y de la cola pellizcando las patas traseras y la cola. </li></ol> 3. Cirugía <ol><li> Limpiar la piel que cubre el abdomen con etanol al 70%. </li><li> Use unas pinzas para tirar de la piel hacia arriba. Después de cada cirugía, lavar todos los instrumentos con agua y detergente, y se esterilizó a 72 ° CO / N. </li><li> Hacer una pequeña incisión vertical (alrededor de 2 cm) a lo largo de la línea media de partida a medio camino entre el tercer y cuarto pares de glándulas mamarias. Con unas tijeras quirúrgicas finas. Tenga cuidado de no dañar la pared abdominal. </li><li> Separar suavemente la piel del peritoneo. </li><li> Visualice las arteriasy las venas en la pared abdominal. Hacer otro 2 cm incisión vertical a través del peritoneo (evitando los vasos) para exponer la cavidad abdominal. </li><li> Abrazadera de la piel y la pared abdominal en cada lado de sujeción evitando las arterias. Nota: Si se perfora accidentalmente las arterias, sacrificar al animal de inmediato mediante la realización de dislocación cervical o sobredosis de isoflurano. </li><li> Pre calentar el PBS estéril a 37 ° C. Cubra las abrazaderas con PBS húmedos Kim toallitas. </li><li> La hidratación de la cavidad abdominal expuesta con PBS. Repita este paso varias veces mientras se realiza la cirugía. </li><li> Con unas pinzas redondas, tire suavemente de la cadena de embriones (E15.5) de distancia de la cavidad. Que el resto de la cadena en las toallitas húmedas. Iniciar mediante la exposición de una mitad de la primera útero. Una vez que los embriones en que se electroporación, traerlo de vuelta en el interior, a continuación, trabajar en la segunda mitad. </li></ol> 4. electroporación <ol><li> Siga usando pinzas redondas para manipular los embriones. </li><li> Visualize los ventrículos laterales. Los ventrículos laterales corren a lo largo de la línea media (Figura 1b). </li><li> Añadir Fast Green (Stock: 10% w / v) a la solución de ADN para una mezcla 01:20 de visualizar durante la inyección. Preparar la solución de ADN disolviendo el sedimento obtenido a partir de un protocolo de purificación maxi-prep estándar en agua destilada. </li><li> Utilice micropipetas pre desmenuzado con un émbolo para inyectar 1 ml de solución de ADN (Dlx5 / 6-eGFP, 3 g / l) con Fast Green. Cortar la punta de la micropipeta con un fórceps fino # 5 (Dumont) para salir de 6 mm de longitud desde el comienzo del hombro hasta el extremo de la punta de la aguja antes de cargar la pipeta. Sostenga la cabeza del embrión con pinzas redondas. Introduzca el cerebro con la pipeta en un ángulo de 45 ° con el objetivo para el ventrículo lateral situado cerca de la línea media. Si la aguja penetra a través del ventrículo, retraiga lentamente la pipeta y cuando utilice el émbolo para inyectar ADN. El ventrículo se volverá color verde cuando elsolución comienza a llenarlo. En este punto, dejar de moverse la pipeta y se inyecta 1 ml de ADN. Con cuidado, retire la pipeta. </li><li> Ajuste el electroporador CUY21 a cinco 45V pulsos de 50 ms espaciadas por 950 ms. </li><li> Utilice 5 mm electrodos de pádel para los embriones E15.5. Sumergir los electrodos en PBS para asegurar la transmisión de corriente eficiente. </li><li> Coloque las paletas de electrodos alrededor de la cabeza del embrión con la paleta positivo en el ventrículo que contiene la solución de ADN. Ligeramente inferior de la paleta positiva con respecto a la negativa para crear una corriente ventral a través de las eminencias ganglionares. Esta manipulación se minimizará la expresión ectópica en las células piramidales. </li><li> Después de la colocación de los electrodos, aplicar un pulso al presionar el pedal una vez. </li><li> Retire los electrodos y límpielos. Repita los pasos 4.6 a 4.9 con cada embrión. </li><li> Después de la electroporación todos los embriones, vierta 3 ml de PBS en la cavidad abdominal y devolverlos a la cavidad abdominal. </li><li>Primera sutura de la pared abdominal con una aguja curva y sutura de seda 5-0 y luego la piel. Aplicar lidocaína (4%) sobre la herida. Administrar 120 mg / kg de aspirina peritoneo intra de la analgesia. Nota: Todo el procedimiento se debe realizar en 20 min o menos. Es aconsejable que los principiantes electroporar sólo unos pocos embriones para mantener el tiempo de funcionamiento corto. Cirugías prolongadas disminuirán la tasa de supervivencia de los embriones. </li><li> Retire el animal de la tubería de la anestesia y permitir la recuperación sobre el cojín eléctrico en una toallita de papel. </li><li> Animales Regresar a la jaula, manténgalo sobre el cojín eléctrico a 37 ° C y monitorear el estado de salud. Los animales toleran generalmente bien de la cirugía y comienzan a moverse lentamente poco después de que se retiran de la tubería de la anestesia. Si se observa sangrado excesivo o letargo, sacrificar al animal inmediatamente realizando IACUC aprobó proceder a la eutanasia como la dislocación cervical o sobredosis de anestesia. </li><li> Jaula Volver a la vivarium. Las hembras dan a luz de forma natural. </li></ol>

<ol>
	<li>Fishell, G., Rudy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+inhibition+within+the+telencephalon:+&amp;#34;where+the+wild+things+are&amp;#34;.">Mechanisms of inhibition within the telencephalon: &amp;#34;where the wild things are&amp;#34;.</a> Annual review of neuroscience. 34, 535-567 (2011).</li><li>Stenman, J., Toresson, H., Campbell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+distinct+progenitor+populations+in+the+lateral+ganglionic+eminence:+implications+for+striatal+and+olfactory+bulb+neurogenesis.">Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis.</a> J Neurosci. 23, 167-174 (2003).</li><li>Eisenstat, D. D., 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=DLX-1,+DLX-2,+and+DLX-5+expression+define+distinct+stages+of+basal+forebrain+differentiation.">DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.</a> J Comp Neurol. 414, 217-237 (1999).</li><li>Batista-Brito, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+integration+of+cortical+interneurons+into+a+functional+network.">The developmental integration of cortical interneurons into a functional network.</a> Curr Top Dev Biol. 87, 81-118 (2009).</li><li>Miyoshi, G., Butt, S. J., Takebayashi, H., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologically+distinct+temporal+cohorts+of+cortical+interneurons+arise+from+telencephalic+Olig2-expressing+precursors.">Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors.</a> J Neurosci. 27, 7786-7798 (2007).</li><li>Miyoshi, G., 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=Genetic+fate+mapping+reveals+that+the+caudal+ganglionic+eminence+produces+a+large+and+diverse+population+of+superficial+cortical+interneurons.">Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.</a> J Neurosci. 30, 1582-1594 (2010).</li><li>Bulfone, 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=The+mouse+Dlx-2+(Tes-1)+gene+is+expressed+in+spatially+restricted+domains+of+the+forebrain,+face+and+limbs+in+midgestation+mouse+embryos.">The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos.</a> Mechanisms of development. 40, 129-140 (1993).</li><li>Bulfone, 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=Spatially+restricted+expression+of+Dlx-1,+Dlx-2+(Tes-1),+Gbx-2,+and+Wnt-3+in+the+embryonic+day+12.5+mouse+forebrain+defines+potential+transverse+and+longitudinal+segmental+boundaries.">Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries.</a> J Neurosci. 13, 3155-3172 (1993).</li><li>Robinson, G. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+a+member+of+a+new+family+of+murine+Distal-less+homeobox+genes+in+the+developing+forebrain.">Spatially restricted expression of a member of a new family of murine Distal-less homeobox genes in the developing forebrain.</a> The New biologist. 3, 1183-1194 (1991).</li><li>Close, 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=Satb1+is+an+activity-modulated+transcription+factor+required+for+the+terminal+differentiation+and+connectivity+of+medial+ganglionic+eminence-derived+cortical+interneurons.">Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons.</a> J Neurosci. 32, 17690-17705 (2012).</li><li>De Marco Garcia, N. V., Karayannis, T., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+is+required+for+the+development+of+specific+cortical+interneuron+subtypes.">Neuronal activity is required for the development of specific cortical interneuron subtypes.</a> Nature. 472, 351-355 (2011).</li><li>Petros, T. J., Rebsam, A., Mason, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> Journal of visualized experiments : JoVE. (31), (2009).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> Journal of visualized experiments : JoVE. (6), e239 (2007).</li></ol>

Los autores no tienen nada que revelar.

Limitaciones de la Técnica Si bien esta técnica permite la célula de análisis autónoma de procesos celulares, no es adecuado para el análisis de la población. Los electroporaciones son muy escasos con menos de mil células por electroporación cerebro. Como consecuencia, la técnica no se puede utilizar para evaluar las consecuencias de comportamiento derivados de la manipulación genética de las interneuronas derivados de CGE. Mientras electroporaciones ...

La mayor parte de las interneuronas GABAérgicas corticales se originan a partir de dos estructuras embrionarias transitorias nombradas las eminencias ganglionares medial y caudal (MGE y CGE, respectivamente) 4. Parvalbúmina y somatostatina expresar interneuronas se originan en el MGE mientras Calretinina (Cr), vasointestinal péptido (VIP) y Reelin (Re) que expresa interneuronas son originarios de la CGE. Estos subtipos de interneuronas pueden ser distinguidos por sus fechas de nacimiento. MGE subtipos derivados nacen entre el día embrionario 9.5 (E9.5) y E16.5 5,6. En contraste, las interneuronas CGE derivadas nacen de E12.5 E18.5 a través con su producción alcanzando un máximo de 6 E15.5. La selección genética de esta población nació tarde, sin embargo, sigue siendo difícil de alcanzar. Los distal-less genes murinos (Dlx) se expresan exclusivamente en el prosencéfalo ventral desarrollo 3. Interneuronas GABAérgicas y las neuronas de proyección estriatales pero las células piramidales corticales no expresan Dlx1, 2,5, y 6 genes en las etapas tempranas del desarrollo 3. De hecho, los genes Dlx se expresan en la zona MGE y CGE subventricular (SVZ) en todos los progenitores GABAérgicas. La expresión de estos genes se restringe para seleccionar subtipos en las etapas postnatales 7-9. Evidencia experimental anterior mostró que el elemento potenciador Dlx5 / 6 permite el señalamiento selectivo de linajes GABAérgicas en modelos de ratones transgénicos 2. Hemos probado el uso de uno de estos elementos potenciadores en el contexto de expresión episomal en el cerebro de ratón en desarrollo. Se subclonó el elemento potenciador Dlx5 / 6 junto con un promotor mínimo y la proteína fluorescente verde mejorada (EGFP) en un Bluescript (BS) plásmido columna vertebral (Figura 1). Hemos introducido el plásmido mediante electroporación en el útero en E15.5 para atacar selectivamente Cr-, VIP e Investigación subtipos 3,8,10. Nuestra técnica permite la electroporación escasa, lo que facilitala reconstrucción de las características morfológicas de las células chamusquina. Además, los niveles excepcionalmente altos de expresión génica en neuronas GABAérgicas corticales permite para los estudios funcionales. Hemos llevado a cabo la pérdida y ganancia de estudios de la función utilizando varios tipo silvestre y genes dominantes negativos 11.

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subtype-selective electroporation of cortical interneurons

El estudio del sistema nervioso (CNS) central de la maduración depende de la orientación genética de poblaciones neuronales. Sin embargo, la tarea de restringir la expresión de genes de interés para los subtipos neuronales específicas ha demostrado ser notablemente difícil debido a la escasez relativa de los elementos específicos del promotor. Interneuronas GABAérgicas constituyen una población neuronal con amplia diversidad genética y morfológica. De hecho, más de 11 subtipos diferentes de interneuronas GABAérgicas se han caracterizado en la corteza del ratón 1. Aquí presentamos un protocolo adaptado para la orientación selectiva de poblaciones GABAérgicas. Hemos logrado subtipo dirección selectiva de las interneuronas GABAérgicas utilizando el elemento potenciador de la transcripción homeobox factores Dlx5 y Dlx6, homólogos de la (DLL)-gen distal Drosophila menos 2,3, para conducir la expresión de genes específicos a través de la electroporación en el útero.

Se adaptó la técnica de electroporación en el útero para lograr el tipo de célula orientación específica de las neuronas maduras. Para conducir la expresión de EGFP en interneuronas derivados de CGE, se utilizó el elemento potenciador Dlx5 / 6 y restringido nuestro inyecciones a E15.5, la etapa en que la mayoría de las interneuronas derivados de CGE se generan. Hemos llevado a cabo el análisis en P8 y P15 11 (Figuras 1 y 2). Se confirmó el origen...

Watch this Scientific Journal Video about Subtype-selective Electroporation of Cortical Interneurons at JoVE.com

Subtype-selective Electroporation of Cortical Interneurons

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

-Subtipo selectivo electroporación de interneuronas corticales

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

subtype-selective-electroporation-of-cortical-interneurons

Research

JoVE Journal

Neuroscience

8.8K vistas.  New York University School of Medicine. This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Video: Subtype-selective Electroporation of Cortical Interneurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

En el útero de electroporación seguido por cultivos neuronales primarias para estudiar la función génica en el subconjunto de neuronas corticales

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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 ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Aislamiento y cultivo de astrocitos corticales de ratones

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

La electroporación neonatal zona subventricular

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Ex utero electroporación y explantes enteros hemisferio: un método simple Experimental de Estudios del Desarrollo Cortical Temprana

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Neonatal electroporación superficie pial

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Plenario de células patch-clamp grabaciones de Morphologically- e identificados-neuroquímicamente interneuronas del hipocampo

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Una guía para<em&gt; En vivo</em&gt; Single-unidad de grabación de Optogenetically Identificado cortical inhibitorio interneuronas

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Método electrofisiológico para registrar las respuestas de voltaje intracelular<em&gt; Drosophila</em&gt; Los fotorreceptores y interneuronas a la luz Estímulos<em&gt; En Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Visualización de módulos corticales en cortezas mamíferos aplanados

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...