Agradecemos al Dr. Takahiko Matsuda para la prestación del pCAG-mCherry construir. YCM está respaldada por un premio de Desarrollo de la Carrera de la Fundación Schweppe y una beca de la Fundación Whitehall.

1. Suministro preparación (no en vídeo) <ol><li> Preparar una dilución nueva 1X de penicilina / estreptomicina (Pen / Strep) en 1X PBS. No más de 50 mL es necesario. Filtrar con un filtro de 0,22 micras jeringa. </li><li> Preparar las construcciones de inyección mediante la combinación de construcciones de plásmidos deseados. Ponemos todas las construcciones en el vector pCAG-Bandera con estequiometría apropiada para un volumen final de ~ 10 l. Además, pCAG-GFP 14 o pCAG mCherry-se debe agregar para la eficiencia de la electroporación de seguimiento y células electroporadas. Usamos pCAG-mCherry para este experimento. Por último, añadir 0,22 micras-filtró colorante Fast Green en la mezcla de ADN del plásmido a una concentración final de 0,05% para visualizar la eficiencia de la inyección en midbrains de embriones de pollo. </li></ol> 2. La preparación de huevo (en vídeo) <ol><li> Con el fin de adquirir la etapa de HH 11 embriones, los huevos deben ser incubados a 100 ° F (37,8 ° C) durante aproximadamente 41 horas después de la fertilización con 25-40% de humedad. Lugarlos huevos de su lado y marcar la parte superior de cada uno con un toque de lápiz. Coloque las bandejas en un pre-calentado, humidificado incubadora. Poco a poco el rock los huevos mediante el establecimiento de la función de giro de las plataformas de la incubadora en &quot;Automático&quot;. Revise el nivel de humedad y el agua cada 24 horas. </li><li> Justo antes de empezar el experimento, eliminar los huevos de la incubadora y dejar a temperatura ambiente. Esto ayudará a frenar el desarrollo y prevenir sobre el desarrollo. </li><li> Limpie la superficie de cada huevo con un 70% de etanol y limpie con un Kimwipe. </li><li> Tomar dos piezas de 4 cm de cinta adhesiva y pegarlos de lado a lado en el óvulo para formar un rectángulo grande sobre el tablero lápiz. Alise la cinta contra el huevo. Aquí es donde la ventana se hará. La cinta evitará pequeños trozos de cáscara de huevo se caiga de la ventana cuando se corta. </li><li> Use una jeringa de 10 ml y 18 ½ aguja de calibre para dibujar a 5 ml de albúmina del huevo. Después de perforar la cáscara, el ángulo de la aguja fuera de la yema de no perturbaro dañar el embrión mientras se quita la albúmina. Limpie la aguja con etanol al 70% entre los huevos, pero asegúrese de que la aguja esté seco antes de perforar el siguiente huevo. </li></ol> 3. Inyección de cristal con aguja de preparación (en vídeo) <ol><li> Agujas de inyección se realizan con los siguientes ajustes en un Flaming / Brown Micropipeta extractor (Modelo de Instrumento de Sutter P-97):. Heat 670, Pull 120, Vel 40, Tiempo 165 World Precision Instruments (API) de 4 pulgadas de 1,0 mm de espesor de pared de vidrio capilar (API TW100F-4) se utiliza para fabricar agujas. </li><li> Coloque una aguja de vidrio tirado con una pipeta P10 y el largo de punta capilar de carga punta de la pipeta (Eppendorf 5.242.956,003). Cargar l 5-10, en función de cuántos huevos son para ser inyectado. Completa la aguja de cristal lentamente, creando una columna continua de solución plásmido ausente de burbujas de aire. </li><li> Colocar la aguja cargada en el soporte de aguja unida a la micromanipulador (Narishige MM3) y la microinyector Picospritzer III. Ver la aguja en un dissectien microscopio y recortar la aguja en el punto donde el líquido verde se detiene. Ayuda a utilizar un fondo oscuro para visualizar mejor la aguja. Correcta de la aguja de corte viene con la práctica. </li></ol> 4. Mesencéfalo / inyección del tubo neural (en vídeo) <ol><li> Con unas tijeras para cortar la primavera una ventana 2-2.5 cm de diámetro en el huevo. Inicio de la ventana del agujero de la aguja anterior realizada en la elaboración de albúmina, y girar el huevo en la mano mientras corta. Asegúrese de limpiar las tijeras con un Kimwipe saturado en etanol al 70% entre los huevos. </li><li> Ver embriones de pollo bajo un microscopio de disección. Puede que sea necesario para formar los ojos. Se puede ayudar a visualizar el embrión mediante la inyección de 0,22 m con filtro de 20% de tinta china en PBS en el embrión de pollo. Sin embargo, hemos encontrado que este paso debe ser evitado si es posible, ya que tiende a disminuir la supervivencia. </li><li> Inyección funciona mejor si el embrión se coloca en paralelo a la trayectoria de la aguja, con la cabeza de la aguja.Poco a poco perforar el tubo neural en un ángulo de 45 grados a la unión del cerebro medio-hindbrain. Es muy importante para llenar sólo la vesícula neuronal que representa el cerebro medio con el ADN del plásmido y el colorante Fast Green, sin una difusión significativa en las regiones del cerebro anterior y posterior del cerebro. Comenzar a inyectar con el conjunto de la duración de la microinyector Picospritzer de menos de 25 ms, lo que puede ser necesario cambiar dependiendo de la aguja de corte. En general, el uso entre 10-50 ms. </li></ol> 5. La electroporación (en vídeo) <ol><li> Recuperar la aguja de inyección. Colocar 3 gotas de Pen 1X / Strep en solución de PBS en el huevo sobre el embrión. </li><li> Compruebe la configuración de la toxina botulínica electroporador EMC830: <table border="1"><tbody><tr><td> TENSIÓN: </td><td> 20 V </td></tr><tr><td> Duración de impulso: </td><td> 25 ms </td></tr><tr><td> Número de Legumbres: </td><td> 3 </td></tr><tr><td> INTERVALO: </td><td> 500 ms </td></tr><tr><td> POLARIDAD: </td><td&gt; UNIPOLAR </td></tr></tbody></table></li><li> Colocar los electrodos de platino 2 mm de largo en forma de L en el mismo nivel horizontal que el fondo del tubo neural, con el cerebro medio embrionario sentado en el centro entre dos electrodos 3 mm entre sí. La alineación de los electrodos con la parte inferior del tubo neural es crítico para la electroporación eficiente del mesencéfalo ventral. Para alcanzar el cerebro medio específico de la electroporación, que reemplazaron a los electrodos de BTX originales con dos hechos a la medida de platino en forma de L con electrodos cortos 2 puntas mm de largo. Estos electrodos de platino son de 0,5 mm de diámetro alambres de platino (Alfa Aesar). Estos electrodos de platino son lo suficiente para cubrir el cerebro medio de un embrión en su fase de pollo 11, que es de aproximadamente 0,9 mm de largo, sin focalización gran parte del cerebro anterior o de las regiones del cerebro posterior. Pulse el pedal BTX una vez. Las burbujas de aire se deben generar alrededor de los electrodos con cada uno de electroporación éxito. Retire con cuidado los electrodos del huevo y limpie la electrodas con un Kimwipe 70% de etanol-cubierto. Coloque otros 3 gotas de Pen 1X / solución estreptococo en el huevo, en el embrión. </li><li> Cubra la ventana con unos 5 cm x 5 cm de la pieza de cinta de embalaje transparente. Asegúrese de sellar el huevo bien y que el contenido de huevo no entren en contacto con la cinta. </li></ol> 6. Incubar y la Cosecha <ol><li> Coloque los huevos de nuevo en la incubadora. Asegúrese de que el giro de las plataformas de la incubadora está en OFF. Incubar los huevos hasta que aparezca el estadio 23 HH se logra. </li><li> Cosecha de embriones vivos y colocarlos en PBS-comunes llenas de placas de Petri para cada condición. Pantalla de embriones cosechados bajo un microscopio de disección fluorescente, manteniendo sólo los embriones con la expresión mCherry. </li><li> Transferencia de embriones en los distintos pozos de una placa de 24 pocillos. Llene cada pocillo con 1 ml de paraformaldehído recién hecha el 2% (PFA) y que permiten fijar 2-3 horas a 4 ° C. </li><li> Lavar embriones fijos tres veces durante 10 minutos cada uno en PBS. Embriones lugar secuencialmente in 15% y 30% de sacarosa hasta equilibró. Los embriones inicialmente flotan en sacarosa, pero se hunde al llegar al equilibrio. </li><li> Compruebe embriones en virtud de un microscopio de disección fluorescente, tomando notas en el patrón de expresión mCherry y nivel de cada embrión. Insertar cada embrión en octubre Es muy crítico a los embriones orientar adecuadamente para generar óptimas secciones transversales del cerebro medio para análisis posteriores (Figura 1). </li><li> Preparar las secciones 18 micras de espesor del cerebro medio usando un criostato Leica CM3050S. Analizar el desarrollo del cerebro medio y la diferenciación de las neuronas dopaminérgicas por inmunotinción con las correspondientes marcadores de células de tipo. </li></ol> 7. Los resultados representativos Una representación esquemática de electroporación y analizar mesencéfalo de embriones de pollo se muestra en la Figura 1. En los embriones de pollo inyectados con éxito y la electroporación, la expresión mCherry debe ser visible en la región del cerebro medio después de un mayor desarrollo ( <strong&gt; Figura 2A, B, C). Suponiendo que mCherry se correlaciona con la expresión de otros co-electroporated los genes, los embriones exhiben expresión mCherry adecuado puede ser fijado y embebido para preparar cryosections mesencéfalo para el estudio adicional (Figura 2D). En general, aproximadamente el 50% de los progenitores del mesencéfalo ventral neurales pueden ser eficientemente transfectadas con el protocolo anterior. La especificación del destino dopaminérgica en electroporated progenitores neurales pueden ser examinadas por la co-localización de mCherry, Bandera de etiquetado de genes, con las neuronas dopaminérgicas marcadores tales como tirosina hidroxilasa (TH) (Figura 3). El patrón de los dominios de progenitoras en cerebro medio ventral puede ser investigado por cryosections immnostaining cooperación con diferentes marcadores progenitoras de dominio. Expresión mCherry insuficiente sugiere que los genes se ectópicos no es probable expresan eficientemente. Los embriones bajo esta condición no debe ser utilizado para estudios posteriores. Del mismo modo, los embriones queno sobrevivieron hasta el final del procedimiento experimental no puede ser utilizado para la recogida de datos y análisis. <img alt="Figura 1" src="/files/ftp_upload/4017/4017fig1.jpg" /> Figura 1. Ilustración del cerebro medio de embriones de pollo en el ensayo de electroporación in ovo. HH etapa 11 embriones de pollo embrionarias son inyectados con pCAG-mCherry junto con genes de interés mezclados con verde rápido para visualizar el proceso de inyección (A). Después de lleno con construcciones de ADN, se midbrains electroporación con un electroporador onda cuadrada. Los embriones se incuban hasta que se desea etapa HH 23 se alcanza. Luego mCherry positivos embriones son cosechadas, fijo, y embebido (B). Cryosections Mesencéfalo se preparan con cortar planos indicados por la línea blanca en la Figura 1B, y se analizaron por inmunotinción con marcadores mesencéfalo tales como tirosina hidroxilasa (TH) (C). <a href="http://www.jove.com/files/ftp_upload/4017/4017fig1large.jpg" target="_blank">Haga clic aquí</a>para ampliar la figura. <img alt="Figura 2" src="/files/ftp_upload/4017/4017fig2.jpg" /> Figura 2. Preparación de cryosections de mesencéfalo de embriones de pollo. Después de 41 horas de incubación, HH etapa 23 embriones de pollo que expresan mCherry y los genes de interés se cosechan (A, B y C), se fijaron con PFA, tratados con sacarosa, y sumergida en octubre de cryosectioning. Los embriones de pollo son cuidadosamente orientados a garantizar la preparación de las secciones del cerebro medio. Una típica morfología del embrión y el plano de corte para la obtención de las secciones del cerebro medio se muestran (D). <a href="http://www.jove.com/files/ftp_upload/4017/4017fig2large.jpg" target="_blank">Haga clic aquí para ver más grande la figura</a> . <img alt="Figura 3" src="/files/ftp_upload/4017/4017fig3.jpg" /> Figura 3. Análisis de las secciones del cerebro medio electroporadas. Criosecciones a partir de embriones con altos niveles de expresión mCherry (A) se preparan y se tiñeron con anticuerposs que reconocen la Bandera de etiquetado gen de interés (B) y el mesencéfalo dopaminérgica neuronal marcador de TH (C). Midbrains embrionarias electroporadas con pCAG-mCherry y pCAG-Bandera vector vacío como control. <a href="http://www.jove.com/files/ftp_upload/4017/4017fig3large.jpg" target="_blank">Haga clic aquí para ver más grande la figura</a> .

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Los autores no tienen intereses en conflicto potencial a revelar.

La electroporación in ovo en el cerebro medio de los embriones de pollo ofrece una alternativa de bajo costo y rápida a la generación de animales transgénicos o knockout para realizar el estudio in vivo de las funciones de los genes en el cerebro medio dopaminérgico el desarrollo neuronal. Usando corto 2 mm de electrodos de platino largos en forma de L, junto con la inyección de ADN embrionario mesencéfalo-específico es la clave para lograr una expresión eficaz del gen de interés en ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Huevos de gallina fertilizados </td><td> Charles River Laboratories </td><td></td><td></td></tr><tr><td> Huevo de incubadora </td><td> GQF Manufactura </td><td> Deportista 1502 </td><td></td></tr><tr><td> BTX Electroporador </td><td> BTX Harvard Apparatus </td><td> ECM830 </td><td></td></tr><tr><td> Electrodos </td><td> BTX Harvard Apparatus </td><td> 45-0162 en forma de L genetrodes </td><td> Para el uso con ECM830 electroporador </td></tr><tr><td> De alambre de platino iridio </td><td> Alfa Aesar </td><td> # 10056 </td><td> 0,5 mm de diámetro Para hacer los electrodos 2 mm de largo </td></tr><tr><td> Vidrio tubo capilar </td><td> Mundial de instrumentos de precisión </td><td> TW100F-4 </td><td></td></tr><tr><td> Microloader punta de la pipeta </td><td> Eppendorf </td><td> 5242 956.003 </td><td></td></tr><tr><td> Tinta china </td><td> Staples </td><td></td><td> Filtrar 20% antes de su uso </td></tr><tr><td> Microinyector </td><td> Parker Automation, Parker Hannifin Cooperación </td><td> Picospritzer III </td><td></td></tr><tr><td> Microscopio de disección fluorescente </td><td> Leica </td><td> MZ16F </td><td></td></tr><tr><td> Micropipeta extractor </td><td> Sutter Instrument </td><td> D-97 </td><td></td></tr></tbody></table>

in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

Las neuronas dopaminérgicas en el movimiento del mesencéfalo ventral de control, el comportamiento emocional y los mecanismos de recompensa 1-3. La disfunción de las neuronas dopaminérgicas del mesencéfalo ventral, está implicado en la enfermedad de Parkinson, la esquizofrenia, la depresión y la demencia 1-5. De este modo, el estudio de la regulación de la diferenciación de las neuronas dopaminérgicas del mesencéfalo, no sólo podría proporcionar información importante sobre los mecanismos que regulan el desarrollo del cerebro medio y la especificación progenitoras neurales destino, sino también ayudar a desarrollar nuevas estrategias terapéuticas para el tratamiento de una variedad de trastornos neurológicos humanos. Las neuronas dopaminérgicas diferenciarse de progenitores neurales que recubren la zona ventricular del cerebro medio ventral embrionario. El desarrollo de progenitores neurales está controlado por programas de expresión génica 6,7. Aquí mostramos las técnicas que utilizan la electroporación para expresar los genes específicamente en el cerebro medio de Hamburger y Hamilton (HH) la 11 ª etapa (tsomitas irteen, 42 horas), los embriones de pollo 8,9. El desarrollo de embriones de pollo externa permite convenientes manipulaciones experimentales en determinadas etapas embrionarias, con los efectos determinados en los puntos de tiempo posteriores del desarrollo 10-13. Polluelo de tubos neurales embrionarias antes del estadio HH 13 (diecinueve somitas, 48 ​​horas) consistirá en multipotenciales progenitoras neuronales que son capaces de diferenciarse en distintos tipos de células del sistema nervioso. El vector pCAG, que contiene tanto un promotor de CMV y un polluelo β-actina potenciador, permite la expresión robusta de la bandera o otras construcciones etiquetados epítopo en tubos de embriones de pollo neural 14. En este informe, hacemos hincapié en las medidas especiales para lograr la expresión de genes regional restringido en mesencéfalo dopaminérgicas embrionarias progenitoras neuronales, incluyendo cómo inyectar ADN construye específicamente en la región del cerebro medio embrionario y cómo identificar electroporación con pequeños electrodos hechos a la medida. El análisis de ccerebro medio cateto en etapas posteriores ofrece un excelente sistema in vivo para el plásmido vector-mediado por el aumento de la función y la pérdida de la función de los estudios del desarrollo del cerebro medio. La modificación del sistema experimental puede extender el ensayo a otras partes del sistema nervioso para realizar análisis destino mapeo y para investigar la regulación de la expresión génica.

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Para evaluar la función y la regulación de los genes durante el desarrollo de las neuronas del cerebro medio dopaminérgico, se describe un método que consiste en<em&gt; In ovo</em&gt; Electroporación de construcciones de ADN plásmido en células progenitoras embrionarias de pollo ventral del cerebro medio de neuronas dopaminérgicas. Esta técnica puede utilizarse para lograr la expresión eficiente de genes de interés para estudiar diferentes aspectos del desarrollo y diferenciación de las neuronas del mesencéfalo dopaminérgica.

En electroporación in ovo en el mesencéfalo polluelo para estudiar la función génica en el desarrollo neuronal dopaminérgica

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

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In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

11.3K vistas.  Children's Hospital of Chicago Research Center. Para evaluar la función y la regulación de los genes durante el desarrollo de las neuronas del cerebro medio dopaminérgico, se describe un método que consiste en In ovo Electroporación de construcciones de ADN plásmido en células progenitoras embrionarias de pollo ventral del cerebro medio de neuronas dopaminérgicas. Esta técnica puede utilizarse para lograr la expresión eficiente de genes de interés para estudiar diferentes aspectos del desarrol...

Video: In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

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

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Aplicación de una conductancia del receptor NMDA en las neuronas dopaminérgicas del mesencéfalo de rata con la técnica de sujeción dinámica

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Del ratón en el útero de electroporación: Transefection controlada Gene espacio-temporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

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 ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

En Expression in ovo de microARN en ventral del polluelo Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dirigida dopaminérgica Neurona La diferenciación de las células madre pluripotentes humanas

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

En proyección de imagen del calcio Vivo de las células de pelo de la línea Lateral en el pez cebra larvas

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Estudiar la función cerebral en niños utilizando Magnetoencefalografía

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Análisis cinemático 3D para la evaluación funcional en el modelo de rata de lesión de aplastamiento de nervios ciáticos

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...