Nos gustaría dar las gracias al laboratorio de A. Hadjantonakis (Sloan-Kettering Institute) y el laboratorio de L. Niswander (U of Colorado-Denver) para consejos útiles con las técnicas de disección y la cultura. Este trabajo ha sido financiado por el NRSA NS059562 a JDG y RO1NS05897 de MER en colaboración con L. y J. Niswander Nadeau (Instituto de Biología de Sistemas).

1. Preparación de medios de cultivo - todos los reactivos se enumeran en la Tabla 1 <ol><li> Descongelar el suero de ratas machos a 37 ° C. </li><li> El calor inactiva suero de rata durante 30 minutos a 55 ° C </li><li> Centrifugar el suero de rata a 10 km por 5 minutos a temperatura ambiente </li><li> Quitar 50:50 sobrenadante y se mezcla con rojo fenol w DMEM libre / de glucosa </li><li> Esterilizar los medios de comunicación utilizando un filtro de 0,45 um jeringa </li><li> Por lo menos 1 ml de los medios de comunicación / embriones debe estar preparado </li></ol> 2. Aislamiento del útero de la madre embarazada <ol><li> Utilizando los procedimientos aprobados por el Cuidado de Animales institucional y el empleo (IACUC), la presa embarazada por primera vez anestesiados y luego sacrificados por dislocación cervical </li><li> Esterilizar el abdomen con un 70% EtOH </li><li> Una pizca de un pequeño camino de la piel a lo largo de la línea media, justo encima de la línea de los pezones, con unas pinzas grandes y abrir el abdomen por debajo de sus pinzas con unas tijeras grandes. Tenga cuidado de no dañar ninguna estructura internas </li><li> Use las tijeras para cortar lateralmente desde la apertura inicial a cada lado del ratón para que el abdomen está abierto </li><li> Intestinos y el exceso de depósitos de grasa a menudo pueden oscurecer el útero y estos deben ser trasladados a un lado para que la parte superior del útero y los ovarios se puede observar en cada lado </li><li> Apriete la parte superior del útero por debajo de los ovarios y el uso de las tijeras para separar el útero de la grasa, el corte de un extremo del útero para el otro ovario </li><li> Levante el útero con sus pinzas y colocar en una placa de Petri con solución salina </li></ol> 3. La eliminación de los embriones en el útero <ol><li> Enjuague el útero con una solución salina para eliminar cualquier exceso de sangre y recortar cualquier exceso de grasa desde el exterior </li><li> Transferir a un plato limpio con la temperatura ambiente. Tyrodes solución salina para mejorar la visualización </li><li> El uso de un microscopio estereoscópico, separe cada embrión utilizando unas tijeras pequeñas o si bien es necesario (# 5) pinzas para pelar cuidadosamente el útero de distancia. </li> &lt;li&gt; Insertar pinzas finas en el espacio entre la pared del útero y la decidua y despegue de la pared uterina, teniendo cuidado de no perforar el embrión </li></ol> 4. La eliminación de decidua a partir de embriones <ol><li> Orientar la decidua forma que la parte más oscura (la placenta) se enfrenta lejos de ti </li><li> Hacer una incisión con pinzas a lo largo de la línea que separa la parte oscura de la decidua de la parte más ligera, con cuidado de no cortar demasiado profundamente </li><li> Completar la separación de la placenta de la parte superior del saco vitelino, teniendo cuidado de no romper el ectoplacental cono (EPC). En este punto usted debería ser capaz de visualizar la membrana Reicherts en el saco vitelino </li><li> Continuar para eliminar toda la decidua (más claro de la porción) teniendo cuidado de no perforar el saco vitelino </li><li> Pellizque suavemente y eliminar el Reicherts retirándola de la EPC. Si el EPC está dañado o separado del saco vitelino, los embriones no podrán a su vez, normalmente, de E8.5-E9.5 </li><li&gt; Con la Reicherts quitado el embrión debe ser fácilmente visible a través del saco vitelino y puede ser montada por somities contar. </li></ol> 5. Configuración del sistema de cultivo <ol><li> Corte el extremo de una pipeta de transferencia de plástico con unas tijeras para aumentar el diámetro de la apertura para que un embrión puede ser una pipeta sin dañar el saco vitelino </li><li> Llenar botellas limpias de rodillos con 1 ml de los medios de comunicación por embrión. Dependiendo del tipo de las botellas de rodillo utilizado un máximo de 3-6 embriones se pueden cargar en cada botella. </li><li> La transferencia de los embriones con la pipeta de corte a la botella llena de medios de comunicación de rodillos, llevando el cargo de Tyrodes menos posible para no diluir los medios de comunicación </li><li> Coloque el tapón de goma (tapón) de la parte superior de la botella de vidrio de rodillos </li><li> Introduzca los frascos rotatorios en el tambor la cultura rusa para que un sello hermético se forma entre el tapón y el tambor </li><li> Una vez que todas las botellas se han unido, y cualquier espacio vacío en el tambor han sido sellados wtapones de goma ITH, activar el rodillo </li><li> Abrir el CO 2 y O 2 tanques y ajustarlos a aproximadamente 2 psi, por lo que la válvula de salida es la liberación de una burbuja por segundo. Por E8.5-E10 embriones, el 20% de O 2 y el 5% de CO 2 se debe utilizar. </li><li> Asegúrese de que la incubadora se encuentra a 37 ° C y la cubierta o cerca de la incubadora para que los embriones están protegidos de la luz. </li><li> Los embriones deben ser revisadas periódicamente para evaluar su desarrollo mediante la adición de somities, la progresión de la gira, y en la medida de cierre del tubo neural </li><li> Después de 2-3 horas en la cultura, los inhibidores farmacológicos u otros tratamientos se pueden añadir a los medios de comunicación </li><li> El diseño del estudio debe incluir controles, tales como vehículos o análogos inactivos de reactivo de estudio. </li></ol> 6. Evaluación del desarrollo de cultivo de embriones después de todo <ol><li> Desconectar el gas, dejar de rodillos, y sacar las botellas de la incubadora </li><li> Embriones traslado de regreso a Tyrodes o PBS en una placa de Petri para su evaluación bajo microscopio estereoscópico </li><li> El saco vitelino debe aparecer en forma de globo, un latido del corazón debe ser visible y la frecuencia cardiaca debe ser&gt; 120 latidos por minuto, y la circulación debe ser evidente </li><li> Fotografía embriones antes de la yema de la eliminación del saco, si es necesario </li><li> Retire del saco vitelino del embrión mediante la reducción de un agujero cerca de la EPC y voltear el saco vitelino y el amnios en la cabeza y la cola del embrión. </li><li> El cordón umbilical se puede cortar para separar el saco vitelino del embrión y el saco vitelino se puede utilizar para determinar el genotipo del embrión, si es necesario. </li><li> Examinar el embrión para el número de somitas y defectos del tubo neural, tales como abrir los pliegues del cráneo, cierre incompleto de la cara y / o un neuroporo caudal abierto. Los embriones se pueden clasifican de acuerdo a la clasificación de los cambios morfogenéticos Theiler múltiples ( <a href="http://genex.hgu.mrc.ac.uk/Databases/Anatomy/MAstaging.html">EMAP http://www.emouseatlas.org/ / home.html</a> ) Una vez más, un registro fotográfico deel desarrollo del embrión es útil. </li></ol> 7. Notas <ol><li> Una disección de alta calidad es esencial, ya que cualquier nick o desgarros en el saco vitelino evitará convertir con éxito e inhiben el desarrollo normal. El EPC también pueden ser dañados durante la remoción de la membrana de Reichert (RM), y si el CPE es aún parcialmente separado del saco vitelino, los embriones no se desarrollan normalmente. Sin embargo, la eliminación incompleta de la RM puede causar embriones se peguen y / o células RM puede crecer en la cultura y la yema de constreñir la expansión del saco, tanto de lo que impediría el desarrollo normal. </li><li> Pinzas limpias y afiladas son el secreto para conseguir una buena disección, porque fórceps sordo o doblados que el procedimiento sea mucho más difícil. Depósitos de proteínas en los instrumentos que van a afectar los tejidos. </li><li> Botellas limpias, estériles de vidrio también son esenciales para evitar que los embriones se peguen a la pared lateral y se dañe. Antes de comenzar el experimento, las botellas deben ser scrubbed con agua y jabón, enjuagarse en dH 2 O, y el microondas con un poco de agua en cada botella hasta que el agua hierva. Las botellas se enjuagan con EtOH al 70% y dejar secar al aire. </li><li> El trabajo lo más rápido posible, porque cuanto antes el embrión puede ser transferido a los medios de cultivo y se calienta de nuevo a 37 ° C la mejor supervivencia. </li><li> Completa disección de toda la camada antes de transferir los embriones a las botellas de rodillos a fin de que cada embrión es a temperatura ambiente durante la misma cantidad de tiempo para evitar la creación de cualquier discrepancia en el crecimiento del embrión </li><li> Mantener un ambiente estéril para evitar la contaminación bacteriana de las culturas. Nuestros experimentos de cultivo se han realizado sin antibiótico, pero muchos laboratorios añadir antibióticos a los medios de comunicación para prevenir el crecimiento bacteriano. El medio debe aparecer tan clara en el final del experimento como lo fue al comienzo del experimento. Si los medios de comunicación se ha convertido en turbia o si es un precipitado visible, es probable que una estafacontaminación que ha infectado su cultura. </li><li> El intercambio gaseoso ineficaz también puede retrasar el desarrollo de embriones, así que asegúrese de tener un regulador fiable que garantice la prestación continua. </li><li> Embriones se desarrollan aproximadamente el 50% más lento en la cultura ex vivo, por lo tanto es imprescindible contar con somitas para asegurarse de que han alcanzado la fase de desarrollo apropiado para la realización del experimento. </li></ol> 8. Los resultados representativos: La aparición de pre-embriones y la cultura post-rusa se ilustra en la Figura 1. En el momento de la disección, los embriones debe estar en una configuración sin mover (Fig. 1A, D), donde la cola se encuentra detrás de los pliegues de la cabeza. Después de 36 horas en la cultura, los embriones deben haber completado vuelta para que estén en la C-curvas, posición fetal, donde la cola se encuentra en frente de la cabeza (Fig. 1B, C, E, F). La manipulación farmacológica con RhoA inhibidor de la cinasa (Y-27632), un conocido inhibidor de la extensión convergente enneurulación (Ybot-González, 2007), resulta en un acortamiento de los embriones a lo largo de su eje rostral-caudal (Fig. 1E, F) e inhibe el cierre del cráneo veces neural. Nuestros datos muestran que las dosis crecientes de Y-27632 progresivamente afecta el cierre veces craneal (Figura 2A) y se acorta el eje del cuerpo (Fig. 2B), en consonancia con el papel de RhoA aguas abajo de señalización en la neurulación del cráneo y la extensión convergente. <img alt="Figura 1" src="/files/ftp_upload/3132/3132fig1.jpg" /> Figura 1. La aparición de embriones y la manipulación de las culturas con el inhibidor farmacológico Y-27632. (A, D) los embriones disecados en E8.5 antes de que el cultivo de embriones enteros (B, E) Los embriones en E10 con el saco vitelino sigue intacta, después de 36hrs de la cultura rusa. (C, F) El saco vitelino se ha eliminado para ilustrar el éxito de inflexión y el cierre del tubo neural. Los embriones que fueron tratados con el inhibidor de la Rho quinasa Y-27632 (E, F) no someterse a la extensión convergente adecuada e ilustrarun cuerpo reducido eje. <img alt="Figura 2" src="/files/ftp_upload/3132/3132fig2.jpg" /> Figura 2. Efecto del inhibidor de la cinasa RhoA en el cierre del tubo neural craneal y el alargamiento del eje. (A) El porcentaje de embriones que fueron capaces de cerrar con éxito sus pliegues craneal (NTC% = porcentaje de cierre del tubo neural) se compara con la dosis de Y-27632 añadido al medio de cultivo. (B) La distancia entre la vesícula ótica y extremidad anterior se redujo significativamente a dosis crecientes de Y-27632 (p &lt;0,05).

<ol>
	<li>Cockroft, D. L., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+culture+of+post+implantation+embryos.">Dissection and culture of post implantation embryos.</a> Mammalian Postimplantation Embryos A Practical Approach. , (1990).</li><li>Gray, J. D., Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+folate+supplementation+from+Crooked+tail+and+other+NTD-prone+mutant+mice.">Mechanistic insights into folate supplementation from Crooked tail and other NTD-prone mutant mice.</a> Birth Defects Res. A. Clin. Mol. Teratol. 85, 314-321 (2009).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1992).</li><li>Ybot-Gonzalez, P., Savery, D., Gerrelli, D., Signore, M., Mitchell, C. E., Faux, C. H., Greene, N. D., Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension,+planar-cell-polarity+signalling+and+initiation+of+mouse+neural+tube+closure.">Convergent extension, planar-cell-polarity signalling and initiation of mouse neural tube closure.</a> Development. 134, 789-799 (2007).</li><li>Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-environment+interactions,+folate+metabolism+and+the+embryonic+nervous+system.">Gene-environment interactions, folate metabolism and the embryonic nervous system.</a> Wiley Interdiscip Rev Syst Biol Med. 2, 471-480 (2010).</li></ol>

No hay conflictos de interés declarado.

La capacidad de separar los factores maternos, intrauterina de los que son intrínsecos al embrión durante su desarrollo es una importante herramienta para el estudio de todas las etapas de la embriogénesis. Aquí hemos analizado los efectos de un inhibidor de molécula pequeña de quinasa RhoA en el útero craneal ex neurulación, eliminando así la variable del metabolismo de la madre de la droga. Esta manipulación farmacológica tiene un profundo efecto en la neurulación del cráneo y la extensión convergente. La sensibilidad a este compuesto se puede comparar entre los diferentes mutantes de ratón genético. El método presentado aquí también se puede aplicar a los estudios de otras vías moleculares en el desarrollo, permitiendo la manipulación directa de la función celular en embriones usando una variedad de reactivos.

<table border="1"><tbody><tr><th> Nombre </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Estereomicroscopio de disección </td><td> Leica </td><td> M165 </td><td></td></tr><tr><td> Incubadora de precisión </td><td> BTC Ingeniería (Reino Unido) </td><td> BTC 001 </td><td></td></tr><tr><td> Botella girando Unidad de Cultura </td><td> BTC </td><td> BTC 003 </td><td></td></tr><tr><td> Botellas de vidrio para la Rotación de la Unidad </td><td> BTC </td><td> BTC 005 </td><td></td></tr><tr><td> Tapón de caucho de silicona </td><td> BTC </td><td> BTC 007 </td><td></td></tr><tr><td> Silicone Rubber Cork </td><td> BTC </td><td> BTC 008 </td><td></td></tr><tr><td> Gas pelele de entrada </td><td> BTC </td><td> BTC 012 </td><td></td></tr><tr><td> Gas pelele trampa de entrada </td><td> BTC </td><td> BTC 013 </td><td></td></tr><tr><td> Gas pelele de salida </td><td> BTC </td><td> BTC 014 </td><td></td></tr><tr><td> Cilindro de gas </td><td> TechAir </td><td></td><td> 20% de O 2, 5% de CO 2 </td></tr><tr><td> Regulador de gas </td><td> TechAir </td><td></td><td></td></tr><tr><td> Suero de rata macho </td><td> Harlan Labs. </td><td> 4520 </td><td></td></tr><tr><td> DMEM w / o rojo fenol </td><td> Invitrogen </td><td> 31053 </td><td></td></tr><tr><td> 10 ml jeringa </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> Filtro de jeringa </td><td> Nalgene </td><td> 190-2545 </td><td></td></tr><tr><td> Grandes tijeras de disección (Blunt punta) </td><td> VWR </td><td> 82027-594 </td><td></td></tr><tr><td> Extra Fino Tijeras Bonn </td><td> Bellas Herramientas Científicas (FST) </td><td> 14084-08 </td><td></td></tr><tr><td&gt; Dumot # 5 fórceps </td><td> FST </td><td> 11252-50 </td><td></td></tr><tr><td> Tyrode Saline </td><td></td><td></td><td> Como se describe en Cockroft 1990 </td></tr><tr><td> 10 cm desechables Petri </td><td> BD </td><td> 351029 </td><td></td></tr></tbody></table>

neural tube closure in mouse whole embryo culture

Modelos genéticos de ratón son una herramienta importante en el estudio de los mamíferos cierre del tubo neural (Gray &amp; Ross, 2009; Ross, 2010). Sin embargo, el estudio de los embriones de ratón en el útero está limitada por nuestra incapacidad de manipular farmacológicamente directamente a los embriones en el aislamiento de los efectos del metabolismo materno en el reactivo de interés. Si el uso de una pequeña molécula, la proteína recombinante, o siRNA, la entrega de estas sustancias a la madre, a través de la dieta o por inyección someterá estos compuestos inestables a una variedad de defensas corporales que podrían impedir que lleguen al embrión. Las investigaciones en cultivos de embriones enteros se pueden utilizar para separar la madre de la intrínseca efectos sobre el feto en desarrollo. A continuación, presentamos un método para el cultivo de embriones de ratón utilizando los medios de comunicación altamente enriquecido en un aparato giratorio incubadora que permite el cierre normal del tubo neural después de la disección (Crockett, 1990). Una vez en la cultura, los embriones puedenser manipulado utilizando técnicas in vitro convencional que de otro modo no sería posible si los embriones estaban todavía en el útero. Los hermanos de embriones se pueden recoger en diversos puntos de tiempo para estudiar diferentes aspectos de la neurulación, que se producen a partir de E7-7.5 (formación de placa de los nervios, justo antes del inicio de la neurulación) a E9.5-10 (en la conclusión del neuroporo craneal y caudal veces cierre, Kaufman, 1992). En este protocolo, demostramos nuestro método para la disección de embriones en los puntos de tiempo que son óptimas para el estudio de la neurulación craneal. Los embriones serán disecados en E8.5 (aprox. 10-12 somities), después del inicio del cierre del tubo neural, pero antes de cumplir los embriones y el cierre del cráneo veces neural, y se mantienen en cultivo hasta E10 (26-28 somities), cuando craneal neurulación debe ser completa.

Watch this Scientific Journal Video about Neural Tube Closure in Mouse Whole Embryo Culture at JoVE.com

Neural Tube Closure in Mouse Whole Embryo Culture

Un método que permite la manipulación directa farmacológico de embriones de ratón durante la neurulación que no pasa por el metabolismo materno se describe. La técnica se puede adaptar a estudiar diferentes aspectos de la neurulación variando el punto en el tiempo y el agente farmacológico.

Cierre del tubo neural en el cultivo de embriones de ratón entero

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

neural-tube-closure-in-mouse-whole-embryo-culture

Research

JoVE Journal

Neuroscience

21.4K vistas.  Weill Cornell Medical College. Un método que permite la manipulación directa farmacológico de embriones de ratón durante la neurulación que no pasa por el metabolismo materno se describe. La técnica se puede adaptar a estudiar diferentes aspectos de la neurulación variando el punto en el tiempo y el agente farmacológico.

Video: Neural Tube Closure in Mouse Whole Embryo Culture

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

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

La combinación del comportamiento de Endocrinología Experimental y Economía: Decisión de la testosterona y sociales que

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

El establecimiento de embriones de ratón con células madre neurales Cultura mediante el ensayo de neuroesfera

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

La localización de la proteína en 3D Cultura de células madre neurales: una metodología de visualización híbrida

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

La detección de la hipoxia Microrregional en corteza cerebral de ratón por la imagen de dos fotones de fluorescencia endógena NADH

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Ratón reproducible aplastamiento del nervio ciático y posterior evaluación de la regeneración mediante el análisis de músculo entero Monte

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Aislamiento y cultivo de células de la cresta neural de embriones murinos del Tubo Neural

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embriones de pollo de la Médula Espinal Slice Cultura Protocolo

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Todo el montaje de imágenes de embriones de ratón Proyecciones sensorial Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Aislamiento y cultivo de células madre adultas neural de la Zona subcallosal Ratón

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...