CYIY is supported by FAPESP (2012/14421-5) and FMV is supported by a fellowship from FAPESP (2009/53695-0). We thank the DNA Technologies Core at University of California, Davis for preparation and sequencing of the RNA-Seq libraries used in this work, the Galaxy team for providing an excellent and free interface for high-throughput sequencing data analysis tools and Dr. Marianne Bronner for allowing us to film in her laboratory space.

1. electroporar la construcción de interés en los tubos neurales de HH12-13 pollo embriones in ovo Utilice un indicador fluorescente, preferiblemente en una transcripción bicistrónico o fusionado a la proteína de interés con un intercalante péptido 2a viral 11, para permitir la identificación rápida de los individuos con niveles satisfactorios de la transfección. NOTA: El tiempo de incubación típico para esta etapa es de alrededor de 48 horas a 37,8 ° C. <ol><li> Abrir una pequeña ventana en los huevos después de la eliminación de 3 ml de albúmina con una jeringa acoplada a un 18 G x 1 1/2 aguja. Para mejorar la visualización de embriones, inyectar una pequeña cantidad de tinta china diluida 1:50 en solución de Ringer estéril debajo del embrión 1-4. </li><li> Después de cubrir el embrión con solución de Ringer estéril, inyectar una solución mM Tris-HCl 10, pH 8.2 y 0.1% del tinte de Alimentos (F, D &amp; C) que contiene 2,5 g / l de ADN a través del extremo posterior del tubo neural hasta que llenahasta la región posterior del cerebro. Para electroporar, electrodos de platino de posición (0.5 mm de diámetro) separados por 4 mm 1 a lo largo de la región de flanco 5 y entregar pulsos de 50 ms y 20 V con un intervalo de 100 mseg entre ellos 4. </li><li> Después de la electroporación, se incuban los embriones manipulados durante 48 horas más hasta que llegan a la etapa HH23 y identificar a las personas que presentan alto nivel de transfección en la región del flanco bajo un microscopio estereoscópico fluorescente. NOTA: La duración de la incubación después de la electroporación depende del objetivo del experimento. Los plásmidos electroporadas pueden generar niveles de expresión significativos después de 2 horas 2. Así, si el interés reside en genes de aparición temprana el período de incubación después de la electroporación puede ser reducido. </li></ol> 2. Cosecha transfectadas con éxito embriones y diseccionar las mitades de la médula espinal a electroporación NOTA: Este procedimiento dura 2-3 horas por cada 10 embriones y los rendimientos de 1-2 RNA total g por embrión. La disección de los tubos neurales requiere movimientos finos y algunas sesiones de práctica puede ser necesario antes de aplicarlo a las muestras experimentales. <ol><li> Descontaminar las herramientas metálicas disección con RNasa solución de descontaminación o de hornear a 150 ° C durante 4 h 12 y preparar PBS tratada con DEPC. Utilice sólo recipientes de plástico nueva RNasa libre de certificado. NOTA: Después de la cosecha, es importante seguir la atención estándar para prevenir la contaminación de ARNasa durante la disección. </li><li> Coseche los embriones utilizando un par de tijeras de disección finas y una cuchara colección tamizada. Mantener los embriones en PBS frío tratada con DEPC en un plato de Petri de 100 mm. </li><li> Con un par de pinzas finas, cortar toda la región del flanco (entre el límite posterior de la yema del miembro anterior y el límite anterior de la yema de la extremidad posterior) y la transferencia de esta sección a un nuevo plato que contiene frío PBS tratada con DEPC. </li><li> Utilice dos agujas finas de vidrio para perforar en tres diferentespuntos a cada lado dorsolateral del embrión entre el tubo neural y el mesodermo paraxial. </li><li> Introducir las dos agujas de cristal en la perforación central y mover lentamente uno de ellos de distancia a lo largo del eje antero-posterior del tubo neural. Repita esto para las perforaciones que no fueron alcanzados por los primeros movimientos. Retire el mesodermo paraxial unifamiliar con un par de pinzas finas. </li><li> Gire el embrión de lado, insertar las puntas de las pinzas entre el tubo neural y la notocorda y deslice en direcciones opuestas a lo largo del eje antero-posterior para separar las dos estructuras. NOTA: Esto también se debe eliminar la mayor parte del mesodermo todavía unido al tubo neural. </li><li> Si es necesario, retire todos los trozos más grandes de tejido mesodérmico que permanecen unidas tirando con un par de pinzas, mientras sujeta ligeramente el tubo neural con otro par de pinzas. Cuando haya terminado, transferir suavemente el tubo neural a otro plato que contiene fría tratada con DEPC PBS utilizando unaRNasa libre Pasteur de vidrio pipeta acoplado a una bomba de pipeta manual y mantener en hielo hasta que se diseccionan todos los tubos. </li><li> Divida a los tubos neurales en dos mitades. Para ello, inserte la punta afilada de un par de pinzas de cierre en el lumen y abra la chapa de techo moviendo las pinzas a través de ella. Después de esto, separe las mitades por el corte de la placa de suelo con las puntas de pinzas. </li><li> Ordenar electroporación de mitades no electroporación bajo un microscopio de disección fluorescente y la transferencia a un tubo de 1,5 ml lleno con PBS tratada con DEPC enfriado con hielo. Si es necesario, chasquear los microtubos suavemente para liberar cualquier tejido unido a las paredes de plástico y para promover la sedimentación de los tubos. </li><li> Centrifugado a 100 xg durante 3 segundos y retirar muy lentamente tanto PBS como sea posible con una micropipeta de 1 ml. Haga esto mientras mira a una fuente de luz que brilla a través del microtubo para monitorear si cualquier material embrionario se aspira a la punta de la pipeta; si esto sucede, se descargan lentamente y repita la centrifugación. </li><li> Añadir 70 l de solución de lisis para la extracción de ARN por medio de tubo neural y la película del tubo para mezclar el contenido. Incubar a temperatura ambiente durante 5 min y agitar vigorosamente. Repetir la incubación de 5 min y agitación en vórtex dos veces más. Almacenar a -80 ° C. </li><li> Recoge al menos ocho mitades del tubo neural por muestra para asegurar un rendimiento adecuado para el siguiente paso. Reúnase muestras más pequeñas si se requieren varias sesiones de electroporación / disección amasar número suficiente de tubos neurales. Almacene las muestras suficientes para duplicados o triplicados para cada condición. </li></ol> 3. Purificar ARN y Preparar Bibliotecas para Illumina secuenciación <ol><li> Retire las muestras de -80 ° C almacenamiento y colocar en hielo durante 15-30 min. Reanudar la descongelación a temperatura ambiente hasta que se complete. </li><li> Vortex bien y dejar a temperatura ambiente durante 5 min. Vortex de nuevo y se centrifuga a 15.000 xg durante 1 min para sedimentar el material insoluble. </li><li> Transferir el sobrenadante a un tubo nuevoy proceder a la extracción de RNA de acuerdo con las instrucciones de aislamiento de ARN total del manual del kit. Eluir en el volumen mínimo recomendado para aumentar la concentración. NOTA: después de tratamiento DNasa limpieza se recomienda con el fin de evitar la contaminación potencial de secuencias de ADN genómico en los conjuntos de datos de RNA-Seq. </li><li> Flick el tubo para mezclar el contenido y eliminar 5 l para el análisis de control de calidad. Conservar la muestra restante a -80 ° C hasta que la preparación de la biblioteca; estas muestras deben descongelarse sólo una vez para evitar la degradación del ARN. </li><li> Ejecutar ARN purificado en un instrumento Bioanalyzer utilizando el kit RNA 6000 Nano; RIN valor debe ser al menos por encima de 7 para continuar. </li><li> Generar bibliotecas utilizando un kit de preparación de la biblioteca de ARN-Seq compatible Illumina y multiplexar hasta 4 bibliotecas en un carril de un instrumento HiSeq configurar para generar 100 pb solo lee. Si es posible, incluir todas las muestras en el mismo plazo para reducir al mínimo los posibles artefactos técnicos. NOTA: Esto debería producir hasta 50 millones de rEADS por muestra, lo cual es más que suficiente para obtener la cobertura para la mayoría de los genes 13. </li></ol> 4. Comparación de conjuntos de datos de RNA-Seq en el servidor de la galaxia público 10. NOTA: el espacio de almacenamiento del servidor está limitada y experimentos que comparan muchas muestras puede requerir la eliminación de los archivos intermedios generados en los pasos de filtrado. <ol><li> Crear una cuenta de usuario en el servidor público de la galaxia ( <a href="https://usegalaxy.org/">https://usegalaxy.org</a> ) y FTP subir los conjuntos de datos en formato de Illumina FASTQ. </li><li> Preparar los archivos cargados con la herramienta FastqGroomer 14 bajo la NGS: menú de control de calidad y la manipulación; si las puntuaciones de calidad ya están codificados en Phred + 33 (Sanger), omita este paso, simplemente cambiando el tipo de archivo a fastqsanger bajo los atributos de cada conjunto de datos. </li><li> Analizar la calidad general de cada conjunto de datos utilizando la herramienta de FastQC bajo la NGS: QC y la manipulación menu. NOTA: Especial atención se debe dar a los gráficos resultantes para cada secuencia de bases de la calidad y por base el contenido de la secuencia, ya que éstos proporcionarán estimaciones de los parámetros que se utilizan para recortar los extremos de las lecturas. </li><li> Recortar lee descartar 3 &#39;termina con datos de baja calidad utilizando la herramienta FASTQ Calidad Trimmer 14 bajo la NGS: menú de control de calidad y la manipulación. Ajuste la herramienta para procesar sólo 3 &#39;termina para el nivel de calidad por debajo de 20. </li><li> Retire secuencias adaptadoras de lee con la función Clip ( <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ) bajo la NGS: menú de control de calidad y la manipulación. Ajuste la herramienta para descartar lee con menos de 45 pb a la izquierda, la entrada de la secuencia del adaptador utilizado y elija el resultado de incluir ambas secuencias recortadas y no recortadas. Además, optar por no descartar las secuencias con bases desconocidos desde la mayoría de las bases de baja calidad ya fueron retirados en el último paso. </lyo&gt; <li> Con la herramienta Recortar secuencias ( <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ) bajo la NGS: control de calidad y el menú de la manipulación, corte las primeras bases de las lecturas para eliminar 5 &#39;sesgo de secuencia detectada por FastQC (típicamente 10). NOTA: En este punto, es una buena idea ejecutar FastQC de nuevo y comparar con el análisis previo para determinar si la calidad de conjunto de datos se ha mejorado. </li><li> Descargar y descomprimir la última asamblea del genoma de pollo para la UCSC genoma navegador de Illumina iGenomes ( <a href="http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn">http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn</a> ) y cargar el archivo en formato fasta todo el genoma y el archivo de formato gtf genes anotación a la historia de la galaxia con la función de carga de archivos en el menú Obtener datos. </li><li> Mapa lee para cada muestra que el genoma de pollo usando el alineador TopHat2 15 bajolos NGS: menú Análisis de ARN. Elija utilizar un genoma de Historia (seleccione el archivo fasta cargado en el paso 4.7). </li><li> Construir un transcriptoma predicho para cada muestra usando Gemelos 16 bajo los NGS: menú de análisis de ARN. Elija utilizar la anotación de referencia como guía (seleccione el archivo gtf cargado en el paso 4.7) y encender las opciones Realizar sesgo de corrección (de la Historia, archivo fasta desde el paso 4.7) y uso correcto de lectura múltiple. </li><li> Combinar transcriptomas previstos para todas las muestras utilizando Cuffmerge 16 bajo la NGS: menú Análisis de ARN. Elija utilizar la anotación de referencia (archivo gtf desde el paso 4.7) y datos de la secuencia (de Historia, archivo fasta desde el paso 4.7). </li><li> Comparar archivos BAM generados por TopHat2 para cada muestra usando Cuffdiff 16 bajo la NGS: menú Análisis de ARN. En las transcripciones de campo seleccione el metranscriptoma rged generada por Cuffmerge y activar las opciones Realizar sesgo de corrección (de la Historia, archivo fasta desde el paso 4.7) y uso correcto de lectura múltiple. </li><li> Ordenar expresión diferencial probar tablas numérico ascendente en la columna 13 (valor q) utilizando la herramienta Ordenar debajo del filtro y el menú Ordenar. NOTA: Los genes / transcripciones muestran significativa expresión diferencial entre las condiciones experimentales se presentarán los valores q más bajos. </li><li> Para comprobar los resultados visualmente con gráficos de cobertura, primero convertir archivos a formato BAM bedGraph utilizando la herramienta Crear un BedGraph de cobertura del genoma 17 bajo el menú BEDTools. Desactivar la presentación de informes de las regiones sin cobertura, optar por tratar las entradas empalmados como intervalos distintos y la escala de la cobertura por un factor que normaliza profundidad secuenciación de todos los conjuntos de datos que se comparan. </li><li> Convertir el archivo bedGraph resultante a formato utilizando el pez gordoherramienta peluca / BedGraph-a-pez gordo ( <a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ) en el menú Formatos convertir. Sube los archivos bigwig como pistas personalizadas en la UCSC genoma navegador ( <a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ) para el montaje galGal4 y la búsqueda de un gen de interés para visualizar la cobertura en los conjuntos de datos. </li></ol>

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The authors have nothing to disclose.

Aquí proporcionamos directrices para el análisis de los efectos después de la electroporación de la gallina de la médula espinal. Aunque la electroporación de vectores de ADN se utiliza con más frecuencia para sobreexpresar un gen de interés, se puede también utilizar construcciones que codifican dominantes negativos, proteínas o precursores para quimeric siRNAs para generar las condiciones de la función de genes knock-down 19-21. De hecho, la comparación de los perfiles resultantes de transcripto...

Los estudios genéticos en los organismos vivos utilizan con frecuencia el embrión de pollo como modelo porque en la electroporación in ovo representa un modo rápido y barato para transfectar parcialmente estructuras embrionarias in vivo con construcciones de ADN 1-5. Vectores de expresión bicistrónicos que codifican una transcripción que contiene un indicador fluorescente junto con el gen de interés, tales como pCIG 6 o 7 PMES, permiten una rápida verificación de la calidad de la transfección en la región deseada bajo un estereomicroscopio embriones antes de procesar para el análisis de aguas abajo. Con los recientes avances en la secuenciación del ADN, ahora es posible obtener los perfiles de expresión del transcriptoma enteros digitales a partir de muestras de ARN usando ARN-Seq 8,9. Por lo tanto, en lugar de los métodos que requieren mucho tiempo que permiten el análisis de los pocos productos de genes en paralelo, tales como la hibridación in situ, inmunohistoquímica y qPCR, preparación de bibliotecas de ADNc parasecuenciación de alto rendimiento puede proporcionar información de todo el transcriptoma. La observación de los efectos experimentales sobre los niveles de expresión de cada gen puede a su vez proporciona una visión de gran alcance en las vías alteradas por la construcción utilizada. Esto es particularmente fácil si un conjunto genómico para el organismo utilizado está disponible, por lo tanto el embrión de pollo de nuevo es un modelo adecuado. Si el usuario tiene acceso a un centro de secuenciación del genoma fiable, la cuestión principal es la obtención de muestras de ARN de alta calidad. Este trabajo demuestra un método para la obtención de muestras de ARN a partir de tubos neuronales transfectadas adecuadas para la RNA-Seq, así como los pasos aguas abajo para análisis in silico de los conjuntos de datos resultantes. Después de la electroporación en HH12-13 seguido de 48 horas de incubación, tronco transfectadas mitades de la médula espinal fueron disecados en condiciones libre de ribonucleasa, inmediatamente zadas y más protegidos de la degradación por la ultra-bajo de congelación hasta la purificación de ARN y la biblioteca preparatde iones. El servidor público de la galaxia 10 proporciona acceso a recursos gratuitos para el análisis de datos de secuenciación de alto rendimiento. La cantidad de espacio que ofrece a los usuarios registrados es suficiente para pequeños experimentos, eliminando así la necesidad de una infraestructura computacional local. Análisis de datos RNA-Seq incluye filtrado, la alineación y la cuantificación / comparación de la expresión génica. Aunque existen pautas generales para el análisis de datos RNA-Seq, parámetros de filtrado dependerá en gran medida de la calidad de la secuencia y se deben determinar después de un análisis de control de calidad de los datos en bruto. Este protocolo describe los pasos para obtener y comparar los perfiles de ARN-Seq de pollo embrionario médulas espinales transfectadas in vivo. Como un resultado representativo, la comparación de conjuntos de datos obtenidos a partir de dos muestras de control independientes transfectadas con un vector vacío mostró que el método es reproducible y debería permitir la identificación de expres diferencialmentetranscripciones de sed en las muestras experimentales. Por lo tanto, la aplicación de este método tiene el potencial de aumentar enormemente el número de descubrimientos después de un solo experimento y por lo tanto proporcionar un conjunto grande de datos para la investigación posterior.

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			<td height="20" style="height:20px;">Truseq RNA Sample Preparation Kit v2</td>
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rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

Para validar el método descrito, dos muestras de control se generan a partir de embriones a electroporación con las PME vector vacío 7 y una muestra a partir de embriones transfectadas con el mismo vector que contiene el inserto construir SCRT2-ZNF, que codifica los dominios de dedos de zinc de 18 SCRT2 pollo. Las muestras que producen ARN total 11-22 mg cada se obtuvieron de las piscinas de 8 a 12 embriones. Las tres muestras se calificó como un valor óptimo de RIN 10 en un control de calidad...

Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq Análisis de Expresión diferencial de genes en electroporación de embriones de pollo de la médula espinal

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

rna-seq-analysis-differential-gene-expression-electroporated-chick

Research

JoVE Journal

Neuroscience

14.4K vistas.  Universidade de São Paulo. This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

Video: RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Investigación

Neurociencias

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Médula Espinal Electrofisiología II: Fabricación de electrodos de aspiración extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

La disección del ganglio y Cultura polluelo Statoacoustic y explantes médula espinal en geles de colágeno para ensayos neurite

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII-etiquetado de las neuronas DRG para el Estudio de ramificación axonal en una preparación Todo el montaje del cable del ratón embrionario espinal

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using 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 ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Inyección estereotáxica de un vector viral para la manipulación genética condicional en la médula espinal de ratón

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

La disección y de montaje lateral de embriones de pez cebra: Análisis para el Desarrollo de la médula espinal

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

La transección de la médula espinal en el pez cebra larval

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Una<em&gt; Ex Vivo</em&gt; Médula espinal inducida por láser Lesiones modelo para evaluar los mecanismos de degeneración axonal en tiempo real

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Expresión de transgenes de mediada por Virus adeno-asociado en neuronas genéticamente definidas de la médula espinal

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...