Los autores desean reconocer NanoVentures Australia para el apoyo financiero de viajes y el Prof. John Haycock por haber acogido parcialmente esta investigación en la Universidad de Sheffield y la Sra Jaimee Mayne por su ayuda durante el rodaje.

1. Preparación Au rupias Nota: Au NR se puede sintetizar por una serie de recetas 16, o comprados de vendedores comerciales. <ol><li> Medir la densidad óptica inicial (OD) de la solución de Au NR través de espectroscopía de UV-Vis, mediante el registro de los valores de absorción de 300 nm a 1000 nm con una resolución de 0,5-2 nm. Variar el volumen de la solución a ser utilizado con la cubeta disponible. </li><li> Evaluar la concentración molar inicial NP con una técnica 17 adecuado (por ejemplo, la espectroscopia UV-Vis, partícula única espectrometría de masas con plasma acoplado inductivamente, microscopía electrónica de transmisión) o utilizar los valores de concentración proporcionados por el vendedor. </li><li> Preparar una solución madre de 5 ml por dilución de la muestra inicial Au NR llegar a un OD = 1. Para el mejor repetibilidad, mantener constante la OD para todas las muestras ensayadas. Siga el protocolo del proveedor comercial para la composición del disolvente de dilución. Si no está seguro, Use agua destilada. </li><li> Centrifugar 1 ml de la solución de Au NR dos veces durante 15 minutos a 7800 xg para eliminar cualquier exceso de sustancia química de la solución. Ciclos de centrifugación pueden variar en fuerza y el tiempo (por ejemplo, 20 min a 5600 × g) 18. </li><li> Eliminar el sobrenadante y volver a suspender los NR en agua desionizada. Como partículas resuspendidas podrían formar agregados en solución, prepararlos diariamente para tener los mejores resultados. Alternativamente, almacenar en un refrigerador por no más de 1 semana. No lo congele. </li><li> Antes de usar en condiciones de cultivo celular, sonicar la solución Au NR durante 5 min y luego esterilizar con luz UV durante 30 min (intensidad de la radiación UV no menos de 400 mW ∙ m -2 a 254 nm). </li></ol> 2. NG108-15 célula neuronal Línea Cultura y Diferenciación <ol><li> Para el medio de cultivo celular, preparar 500 ml de medio estéril de Dulbecco modificado Eagle (DMEM) que contiene 10% (w / v) de suero de ternera fetal (FCS), 1% (w / v) L-glutamina, 1%(W / v) de penicilina / estreptomicina y 0,5% (w / v) de anfotericina B. Nota: Los suplementos pueden ser alicuotaron, almacenado a -20 ° C y se añaden a los medios de comunicación en el día requerido. Medio de cultivo celular se puede refrigerar en una condición estéril para un máximo de 1 mes. </li><li> Para el medio de diferenciación celular, preparar 50 ml de DMEM estéril que contenía 1% (w / v) L-glutamina, 1% (w / v) de penicilina / estreptomicina y 0,5% (w / v) de anfotericina B. </li><li> Crecer NG108-15 células neuronales en 10 ml de medio de cultivo de células en matraces T75 de poliestireno en una incubadora con atmósfera humidificada (5% de CO2 a 37 ° C). Normalmente, las semillas 1.5-2 × 10 5 células de cada matraz para estar listo en 3-4 días. Cambio de medio de cultivo de células cada dos días. Nota: Para evitar derivas genéticas o variación, no utilice células mayores de paso 21 para los experimentos. </li><li> Cuando el 70-80% de confluencia en la cultura, cambie el medio con una cálida medio de cultivo celular fresco. Mecánicamente separar las células suavemente knocrey el fondo del matraz confluente. No utilice la tripsina. </li><li> Centrifugar la suspensión celular durante 5 min a 600 xg y volver a suspender el sedimento celular en 2 ml de medio de diferenciación celular caliente. </li><li> Seed 2 × 10 4 células / cm 2 en una placa de cultivo de tejidos de poliestireno de 96 con 200 l de medio de diferenciación celular. Incubar el experimento durante 1 día a 5% de CO 2/37 ° C. </li><li> Añadir entre 3,2 × 10 9 -4,2 × 1 0 10 partículas / ml de solución de Au NR en el día 2 y se incuba durante 24 h adicionales. No agregue las partículas para los experimentos de control. Nota: A modo de control alternativo, Au NPs con una longitud de onda de absorción máxima bien diferenciado puede ser utilizado para propósitos de comparación 14. Para un comportamiento celular más consistente, mantener la densidad celular constante y de no modificar la superficie bien antes de la siembra de células. </li></ol> 3. neuritas Enhancement <ol><li> Parejael láser con una fibra óptica monomodo (apertura numérica = 0,13) y terminar con un conector de fibra (FC conectores son convenientes y comúnmente disponibles). Medir la potencia del láser de salida con un medidor de potencia estándar. Para obtener los resultados más eficaces, que coincida con la longitud de onda máxima del láser al pico de resonancia de plasmón de los NR. </li><li> En el día 3 después de la incubación NR, fijar el conector FC al pozo. Irradiar muestras y los controles a TA durante 1 min en onda continua, para diferentes potencias de láser. Deje que la cultura de proceder durante 3 días adicionales en el 5% de CO 2/37 ° C. Repetir la irradiación con láser durante un mínimo de 3 mediciones independientes. Nota: Los diferentes tiempos de irradiación y frecuencias de impulsos se pueden seleccionar, dependiendo de la aplicación. </li><li> Caracterizar el láser en términos de diámetro del haz y la irradiación láser (W ∙ cm -2). <ol><li> Para una fibra monomodo estándar, utilice NA = n ∙ pecado θ,donde n es el índice de refracción del medio en el uso y θ es el medio-ángulo del cono de luz que sale de la fibra (ver Figura 1A). De trigonometría, r = tan θ ∙ d, donde r es el radio del haz y d es la distancia entre la fibra y la muestra. El conector FC coincida con el diámetro del pozo, por lo tanto, la iluminación de la muestra a una distancia d = 2,70 ± 0,20 mm. Luz sale de la fibra en el agua (n = 1.33), lo que r = tan (sin-1 (NA / n)) ∙ d. </li><li> Usando la última ecuación, calcular el radio del haz láser, el área del haz correspondiente y la irradiancia media de láser (láser de potencia dividida por el área del haz) en el objetivo (véase el ejemplo gráfico de la Figura 1B). Estos valores representan la irradiancia media de la zona iluminada. </li><li> Evaluar los errores de las mediciones con la teoría general de la propagación de errores. Nota: La distancia bntre el conector FC y la muestra se pueden medir por las fotografías de la disposición experimental y post-procesamiento de la imagen utilizando el software apropiado (por ejemplo ImageJ). </li></ol></li><li> En el día 5, retire medio de diferenciación celular a partir de los experimentos y fijar las muestras con 3,7% (v / v), solución de formaldehído durante 10 min, a continuación, permeabilizar las células con 0,1% (v / v) de Triton X-100 durante 20 min. </li><li> Añadir 3% (w / v) de albúmina de suero bovino (BSA) a las muestras durante 60 minutos para bloquear los sitios de unión de proteína sin reaccionar. Etiquetar las muestras durante la noche anti-βIII-tubulina (5 mg / ml en PBS suplementado con 1% de BSA) a 4 ° C. </li><li> Se incuban las células durante 90 min en la oscuridad con un anticuerpo secundario apropiado (ratón anti-anticuerpo conjugado con TRITC por ejemplo IgG) utilizando una concentración de 0,4-2 mg / ml en 1% de BSA en PBS. Etiquetar los núcleos celulares con DAPI (0,1 mg / ml en agua desionizada) durante 10 min. Nota: la conjugación de anticuerpos y MIG concentraciónht puede variar de acuerdo con el protocolo de empresa. Lávese las muestras con PBS dos veces durante 5 minutos después de cada etapa de tinción. <img alt="Figura 1" src="/files/ftp_upload/52566/52566fig1.jpg" /></li><li> Muestras Imagen de epifluorescencia o microscopía confocal utilizando al menos un 20 × objetivo. Elige el microscopio filtra en consecuencia para el anticuerpo secundario. Seleccionar un filtro DAPI (λ EX = 358 nm; EM λ = 488 nm) para visualizar los núcleos celulares. </li><li> Analizar las imágenes mediante la evaluación de: i) la longitud de las neuritas máximo (registro de la longitud desde la punta de la neurita hasta el comienzo del cuerpo celular), ii) el número de neuritas por célula neuronal (resumir todas las neuritas por célula) y iii) el porcentaje de células con neuritas (dividir el número total de células que expresan βIII-tubulina por el número total de células con un núcleo con tinción positiva) 19. </li></ol> Inducida por láser 4. intracelular de Ca2 + Imaging <ol&gt; <li> Preparar un 20% (w / v) solución madre de Pluronic F-127 por disolución de 2 g de soluto en 10 ml de sulfóxido de dimetilo anhidro (DMSO). Calentar la solución de Pluronic F-127 a 40 ° C durante aproximadamente 20 min para aumentar la solubilidad. Preparar con antelación si se almacena a temperatura ambiente. </li><li> En el día 3 después de la incubación NR, preparar una solución salina equilibrada (BSS; NaCl 135 mM, 4,5 mM KCl, 1,5 mM CaCl 2, 0,5 mM MgCl 2, glucosa 5,6 mM, y HEPES 10 mM, pH 7,4) y suplementado con 5 M de Fluo-4 a.m. en DMSO y 0,1% (w / v) de Pluronic F-127 solución. Nota: La solución de BSS puede ser preparado de antemano y se refrigeró durante un máximo de 1 mes. Añadir los suplementos (Fluo-4 de la mañana y Pluronic F-127) en el día del experimento. </li><li> Retire medio de diferenciación celular y reemplazarla con la solución BSS complementado. Para obtener los mejores resultados con un microscopio confocal invertido, incubar las células en un micro-pocillos del portaobjetos. </li><li> Cargar NG108-15 células con Fluo-04 a.m.durante 30 minutos en la oscuridad a 5% de CO 2/37 ° C. Tras el tiempo de incubación, lavar las muestras dos veces con BSS para eliminar cualquier fluoróforo descargada extracelular. </li><li> Pareja el láser con una fibra óptica monomodo y escindir la punta usando técnicas estándar. Observar la punta resultante bajo un microscopio óptico, para asegurar una superficie de alta calidad (es decir, la punta debe ser perpendicular al eje de la fibra y plana después de una inspección microscopía). <ol><li> Medir la potencia del láser de salida con un medidor de potencia estándar. Inserte la fibra de distribución de luz en un soporte de fibra óptica y pegarla en un microposicionador. Consulte Brown et al. 20 para más detalles sobre la preparación de la fibra óptica y posicionamiento. Precaución: Siga las normas generales de seguridad del láser durante la medición de la potencia del láser (por ejemplo, no mire directamente al rayo láser, Usar gafas de protección láser durante la manipulación, evitar la exposición a la luz parásita otros usuarios del laboratorio) &lt;./ Li&gt; </ol></li><li> Conectar un osciloscopio al láser portátil para controlar la modulación óptica. Utilice una señal binaria con frecuencias variables (0,5-2 Hz) y longitudes de pulso (20-100 ms). Conecte el láser utilizando la señal de modulación como la lógica transistor-transistor (TTL) de entrada para el microscopio, a raíz de la configuración se muestra en Paviolo et al. 13. <ol><li> Colocar las células bajo un microscopio confocal invertido y la posición de la fibra de suministro de luz 250 ± 50 m de distancia de la célula diana en el modo de iluminación de transmisión. A esta distancia, utilizar las ecuaciones presentadas en 3.3.1 para calcular el radio del haz en el blanco. </li></ol></li><li> Usa un láser de iones de argón (488 nm) para excitar el Fluo-4 a.m. colorante internalizado (λ EX = 494 nm; λ EM = 516 nm) y la LD sincronizado para la excitación de los NR endocitosis. Realice la imagen a RT de muestras y controles utilizando al menos un objetivo de 40 × recogiendo las exploraciones de series de tiempocon 256 x 256 píxeles / resolución de fotograma en el modo de ida y vuelta con una frecuencia de al menos 4 Hz. Realice una grabación sin ninguna excitación LD para identificar cualquier interferencia láser de argón-ion (ruido de línea de base). </li><li> Quitar el fondo de los datos utilizando algoritmos penalizados mínimos cuadrados ponderados de forma adaptativa 21. Representar gráficamente los inducidos Ca 2+ variaciones como una función del tiempo. Analizar los picos transitorios en la intensidad de fluorescencia con software de post-procesamiento por umbral en un nivel tres veces la desviación estándar del ruido de línea de base (3σ). </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subacute+and+chronic+toxicity+of+cetyltrimethylammonium+bromide+(CTAB),+a+cationic+surfactant,+in+the+rat.">The subacute and chronic toxicity of cetyltrimethylammonium bromide (CTAB), a cationic surfactant, in the rat.</a> Arch. Toxicol. 35 (2), 91-96 (1976).</li><li>Juste, J., Pastoriza-Santos, I., Liz-Marz&#225;n, L., Mulvaney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanorods:+synthesis,+characterization+and+applications.">Gold nanorods: synthesis, characterization and applications.</a> Coord. Chem. Rev. 249 (17-18), 1870-1901 (2005).</li><li>Dreaden, E. C., Alkilany, A. M., Huang, X., Murphy, C. J., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+golden+age:+gold+nanoparticles+for+biomedicine.">The golden age: gold nanoparticles for biomedicine.</a> Chem. Soc. Rev. 41 (7), 2740-2779 (2012).</li><li>Huang, X., Jain, P. K., El-Sayed, I. H., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmonic+photothermal+therapy+(PPTT)+using+gold+nanoparticles.">Plasmonic photothermal therapy (PPTT) using gold nanoparticles.</a> Lasers Med. Sci. 23 (3), 217-228 (2008).</li><li>Albert, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPV4+channels+mediate+the+infrared+laser-evoked+response+in+sensory+neurons.">TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.</a> J. Neurophysiol. 107 (12), 3227-3234 (2012).</li><li>Garcia-Elias, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4,5-biphosphate-dependent+rearrangement+of+TRPV4+cytosolic+tails+enables+channel+activation+by+physiological+stimuli.">Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (23), 9553-9558 (2013).</li><li>Shapiro, M. G., Homma, K., Villarreal, S., Richter, C. -. P., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+light+excites+cells+by+changing+their+electrical+capacitance.">Infrared light excites cells by changing their electrical capacitance.</a> Nat. Commun. 3 (736), (2012).</li><li>Roggan, A., Friebel, M., D&#246;rschel, K., Hahn, A., M&#252;ller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+circulating+human+blood+in+the+wavelength+range+400-2500+nm.">Optical properties of circulating human blood in the wavelength range 400-2500 nm.</a> J. Biomed. Opt. 4 (1), 36-46 (1999).</li><li>Byrnes, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+promotes+regeneration+and+functional+recovery+and+alters+the+immune+response+after+spinal+cord+injury.">Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury.</a> Lasers Surg. Med. 36 (3), 171-185 (2005).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=810+nm+wavelength+light:+an+effective+therapy+for+transected+or+contused+rat+spinal+cord.">810 nm wavelength light: an effective therapy for transected or contused rat spinal cord.</a> Lasers Surg. Med. 41 (1), 36-41 (2009).</li><li>Grossman, N., Schneid, N., Reuveni, H., Halevy, S., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=780+nm+low+power+diode+laser+irradiation+stimulates+proliferation+of+keratinocyte+cultures:+involvement+of+reactive+oxygen+species.">780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species.</a> Lasers Surg. Med. 22 (4), 212-218 (1998).</li><li>Wong-Riley, M. T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+directly+benefits+primary+neurons+functionally+inactivated+by+toxins+-+Role+of+cytochrome+c+oxidase.">Photobiomodulation directly benefits primary neurons functionally inactivated by toxins - Role of cytochrome c oxidase.</a> J. Biol. Chem. 280 (6), 4761-4771 (2005).</li><li>Beauvoit, B., Kitai, T., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+mitochondrial+compartment+to+the+optical+properties+pf+the+rat+liver:+a+theoretical+and+practical+approach.">Contribution of the mitochondrial compartment to the optical properties pf the rat liver: a theoretical and practical approach.</a> Biophys. J. 67 (6), 2501-2510 (1994).</li></ol>

The authors have no competing interests to disclose.

Los protocolos descritos en esta presentación se describe cómo la cultura, diferenciar y ópticamente a estimular las células neuronales utilizando absorbentes extrínsecos. Las características NR (por ejemplo, dimensiones, forma, longitud de onda de resonancia de plasmón y química de la superficie) y los parámetros de estimulación láser (tales como longitud de onda, longitud de pulso, frecuencia de repetición, etc.) se pueden variar para que coincida con diferentes necesidades experimentales...

Estudios recientes han demostrado que el calentamiento transitorio asociado con la absorción de luz infrarroja por el agua (longitud de onda&gt; 1400 nm) se puede utilizar para inducir potenciales de acción en el tejido nervioso 1 y transitorios de calcio intracelular en cardiomiocitos 2. El uso de luz infrarroja ha planteado un gran interés para aplicaciones en prótesis neurales, debido al potencial de resolución espacial más fina, la falta de contacto directo con el tejido, la reducción al mínimo de artefactos de estimulación, y la eliminación de la necesidad de modificar genéticamente las células antes de la estimulación ( como se requiere en la optogenética) 1. A pesar de todos estos beneficios, modelos térmicos desarrollados recientemente sugirieron que los tejidos / células diana pueden verse afectados por efectos de calentamiento y acumulativos, cuando se utilizan múltiples sitios de estímulo y / o altas tasas de repetición 3,4. En respuesta a estos desafíos, los investigadores han reconocido el potencial de utilizar ABSOR extrínsecabros de la estimulación del nervio para producir efectos de calentamiento más localizadas en el tejido. Huang et al. Demostró este principio mediante el uso de nanopartículas de ferrita superparamagnéticas para activar remotamente los canales TRPV1 sensibles a la temperatura en las células HEK 293 con una radio-frecuencia del campo magnético 5. Aunque esta técnica puede permitir una penetración más profunda (campos magnéticos interactúan relativamente débilmente con el tejido), las respuestas sólo se registraron durante períodos de segundos, en lugar de las duraciones de milisegundos requeridos en los dispositivos biónicos 5. Del mismo modo, Farah et al. Estimulación eléctrica demostrada de neuronas corticales de rata con micro-partículas negras en vitro. Mostraron precisión a nivel celular en la estimulación utilizando duraciones de pulso del orden de cientos de microsegundo y energías en el rango de mu J, permitiendo potencialmente las tasas de repetición más rápidas 6. El uso de absorbentes de extrínsecos también se ha aplicado para inducircambios morfológicos in vitro. Ciofani et al. Mostraron un aumento de ~ 40% en el crecimiento externo celular neuronal utilizando piezoeléctricos nitruro de boro nanotubos excitados por ultrasonido 7. Del mismo modo, se ha informado de nanopartículas de óxido de hierro endocitosis en las células PC12 para mejorar la diferenciación de neuritas en una manera dependiente de la dosis, debido a la activación de moléculas de adhesión celular con el óxido de hierro 8. Recientemente, el interés en absorbedores extrínsecos para ayudar a la estimulación neural también se ha centrado en el uso de nanopartículas de oro (Au NPs). Au NPs tienen la capacidad de absorber de manera eficiente la luz láser en el pico plasmónica y para disipar en el ambiente circundante en forma de calor 9. Entre todas las formas de las partículas disponibles, la absorción óptica de nanorods oro (Au NRS) convenientemente coincide con la ventana terapéutica de tejidos biológicos (infrarrojo cercano - NIR, longitud de onda entre 750-1,400 nm) 10. Además, en el context de estimulación neural, el uso de Au NR proporciona biocompatibilidad relativamente favorable y una amplia gama de opciones de funcionalización de la superficie 11. Estudios recientes han demostrado que un efecto estimulador en la diferenciación puede ser inducida después de exposiciones láser continuo de Au NR en NG108-15 células neuronales 12. Del mismo modo, los transitorios de calcio intracelulares se registraron en las células neuronales cultivadas con Au NR después de la irradiación láser modulada con frecuencias variables y longitudes de pulso 13. Despolarización de la membrana celular también se registró después de NIR láser de iluminación de Au NR en cultivos primarios de neuronas del ganglio espiral 14. La primera aplicación in vivo con irradiado Au NR se ha demostrado recientemente. Eom y compañeros de trabajo expuestos Au rupias en su pico plasmónica y registraron un aumento de seis veces en la amplitud de los potenciales de acción del nervio compuesto (CNaPS) y una disminución de tres veces en el umbral de estimulación en ratas nervios ciáticos. Los cuartosrespuesta hanced se atribuyó a los efectos de calentamiento locales que resultan de la excitación del pico plasmónica NR 15. En el presente trabajo, se especifican los protocolos para la investigación de los efectos de la estimulación con láser en NG108-15 células neuronales cultivadas con Au rupias. Estos métodos proporcionan una simple, pero potente, forma de irradiar poblaciones celulares in vitro utilizando técnicas y materiales biológicos estándar. El protocolo se basa en un diodo láser de fibra acoplada (LD) que permite un funcionamiento seguro y la alineación repetible. La preparación de la muestra y los métodos de irradiación láser Au NR se pueden ampliar a diferentes formas de partículas y cultivos de células neuronales, siempre que los protocolos de síntesis y cultura específicas son conocidas, respectivamente.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Au NR</td>
			<td>Sigma Aldrich</td>
			<td>716812</td>
			<td></td>
		</tr>
		<tr>
			<td>NG108-15</td>
			<td>Sigma Aldrich</td>
			<td>8811230</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6546</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Life Technologies</td>
			<td>10100147</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Life Technologies</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BDH</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;III-tubulin</td>
			<td>Promega</td>
			<td>G7121</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC-conjugated anti-mouse IgG antibody</td>
			<td>Sigma Aldrich</td>
			<td>T5393</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P6867</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Varian Medical Systems Inc.</td>
			<td>Cary 50 Bio</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Eppendorf</td>
			<td>Mini Spin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic bath</td>
			<td>Unisonics Australia</td>
			<td>FPX 10D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Kendro</td>
			<td>Hera Cell 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td>Hettich</td>
			<td>Rotofix 32A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diode</td>
			<td>Optotech</td>
			<td>780 nm single mode fibre - coupled LD</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Thorlabs</td>
			<td>780 HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Power meter</td>
			<td>Coherent</td>
			<td>Laser Check</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>http://rsb.info.nih.gov/ij/index.html</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent microscope</td>
			<td>Axon Instruments</td>
			<td>ImageX-press 5000A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;-slide well</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Carl Zeiss Microscopy Ltd.</td>
			<td>LSM 510 meta-confocal microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS210</td>
			<td></td>
		</tr>
	</tbody>
</table>

gold nanorod-assisted optical stimulation of neuronal cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

Mediante el uso de los Protocolos 1, 2 y 3 descritos aquí, se observó un efecto estimulador en la diferenciación en células NG108-15 neuronales cultivadas con Au NP (Au rupias, poli (estirenosulfonato) presenta el revestimiento Au rupias y revestido con sílice Au NR) después de láser exposiciones entre 1,25 y 7,5 W · cm de -2. Las imágenes confocales de marcado con rhodamineB Au NR demostraron que las partículas se interiorizaron de 1 día de incubación 12. La localización se observó p...

Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Oro nanorod asistida estimulación óptica de las células neuronales

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

gold-nanorod-assisted-optical-stimulation-of-neuronal-cells

Research

JoVE Journal

Neuroscience

8.9K vistas.  Swinburne University of Technology. This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Video: Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

La combinación de Estimulación Magnética Transcraneal y fMRI para examinar la red en modo por defecto

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Investigación

Neurociencias

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Uso del protocolo Theta-Burst para explorar Mechasnism de plasticidad en los individuos con el síndrome de X frágil y el autismo

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

El dispositivo de TMS NeuroStar: Realización de la FDA aprobó el Protocolo para el tratamiento de la depresión

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Células vivas imágenes de células de los órganos sensoriales Precursor en Intacto Drosophila Las pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Control óptico de una proteína neuronal utilizando un ácido Unnatural Amino genéticamente codificados en las neuronas

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Incremento dependiente del tiempo en la respuesta de la red a la estimulación de cultivos de células neuronales en matrices de microelectrodos

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Tomografía de coherencia óptica: Proyección de imagen de ratón ganglionares de la retina las células In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Estimulación eléctrica transcraneal crónica y grabación Intracortical en ratas

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Diferenciación neuronal de las células madre embrionarias del ratón in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...