Los autores desean agradecer al Dr. Louis Reichardt para los ratones TrkA taulacZ y Dr. Annette Markus para el debate profundo y sugerencias. Este trabajo fue apoyado por fondos de puesta en marcha de la Fundación Burke, así como la Fundación Whitehall beca de investigación 2010-08-61, una beca de investigación de Wings for Life Foundation (WFL-US-028/14), conceder ZB1-1102-1 del Christopher &amp; Dana Reeve Foundation, y subvenciones 1R01EY022409 y 3R01EY022409-01S1 del Instituto Nacional del Ojo, a JZ. KJO es un compañero de Goldsmith.

NOTA: Todos los procedimientos se ajusten a la Guía del NIH para el uso y cuidado de animales de laboratorio. El animal protocolo fue aprobado por el IACUC en el Weill Cornell Medical College. 1. Preparación del tejido <ol><li> La eutanasia hembras cronometrada del embarazo por dislocación cervical 15. Diseccionar embrionarias E16 - E18 embriones de las hembras cronometrada del embarazo y colocar los embriones individualmente en los pocillos de una placa de 6 pocillos, llenos de solución salina tamponada con fosfato fría (PBS). </li><li> Enjuague los embriones en PBS frío. </li><li> Retirar una pequeña porción de la membrana amniótica del embrión y el lugar en una solución de proteinasa K pre-preparado para la posterior determinación del genotipo. Alternativamente, si se pierde la membrana amniótica, retirar una pequeña porción de la punta de la cola del embrión y colocarlo en una solución de proteinasa K </li><li> Hacer agujeros en la piel en todo el embrión utilizando pasadores de insectos (por lo menos 100 por cada lado). </li><li> Retire PBS y poco a poco añadir nuevo máximo de 2% de paraformaldehído (PFA),pH 7,4, al pozo. </li><li> Agite suavemente durante 2 horas a 4 ° C. </li><li> Enjuague embriones dos veces en PBS y almacenar en PBS a 4 ° C hasta que la tinción. </li></ol> 2. Genotipado <ol><li> Sumergir membranas amnióticas o de las colas de embriones en proteinasa K mezcla de acuerdo con las instrucciones del fabricante. </li><li> Incubar a 56 ° C durante 10 min. </li><li> Extracto de ADN utilizando un kit de purificación de ADN disponible en el mercado. </li><li> Tomar 0,5 l de los totales de 200 l purificados solución de ADN genómico, combinarlo con 0,5 l de cada uno de los cebadores sentido y antisentido de genes específicos (10 mM), 2 l de dNTP Mix (2,5 mM de dATP, dCTP, dGTP y dTTP) , 0,2 Taq polimerasa l (5 U / l) y 2 l de tampón PCR (10x), añadir H 2 O hasta un volumen total de 20 l. NOTA: Las secuencias de cebador son los siguientes (Tabla 1): TrkA: TrkA_F: GCG GGC GCC GCC GCG ATG, TrkA_R: GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; Frag PCRción longitud: 400 pares de bases (pb). Bax: In5R: GTT GAC CAG AGT GGC GTA GG, Ex5F: TGA TCA GAA CCA TCA TG, NeoR: CCG CTT CCA TTG CTC AGC GG; PCR longitudes de los fragmentos: tipo salvaje, 304 pb; Nula Bax, 507 pb. TauLacZ: lacZup: ACA ACG TCG TGA GTG GGA AAA, lacZdown: ATC AAC ATT AAA TGT GAG CGA G; PCR longitud de los fragmentos: 360 pb. </li><li> Ejecutar una PCR con el siguiente programa para TrkA: paso 1: 1 min a 95 ° C, etapa 2: 10 segundos a 95 ° C, paso 3: 20 segundos a 68 ° C, etapa 4: 30 segundos a 72 ° C. Repita los pasos 2-4 para 40 ciclos. <ol><li> Para LacZ y Bax, ejecute una PCR con el siguiente programa: paso 1: 1 min a 95 ° C, etapa 2: 10 segundos a 95 ° C, paso 3: 1 min a 54 ° C, etapa 4: 1 min a 72 ° C, repita los pasos 2-4 para 35 ciclos. </li></ol></li><li> Run muestras de PCR en un gel de agarosa al 2% a 100 V en tampón 1x Tris-acetato-EDTA durante 20 minutos con un carril de escalera de ADN para evaluar longitudes de los fragmentos. </li><li> Analizar los embriones para detectar la presencia de tipo salvaje TrkA, TRKA-tauLacZ y Bax alelos nulos. Los genotipos de los embriones experimentales son TrkA taulacZ / WT: Bax - / - y TrkA taulacZ / taulacZ: Bax - / -, en función de las preguntas experimentales. Los embriones que carecen de la reportero tauLacZ serán negativos para la tinción axonal y por lo tanto pueden servir como controles negativos para calibrar el proceso de tinción. </li></ol> 3. La tinción de embriones <ol><li> Utilice un kit comercial para la tinción de lacZ con algunas modificaciones, como se describe a continuación en las secciones 3.2 a 3.8. En este caso, utilice el kit de Millipore. </li><li> Sumerja embriones en Tampón A durante al menos 1 hora, agitando suavemente a temperatura ambiente. </li><li> Directamente sumerja embriones en tampón B durante al menos 15 minutos, agitando suavemente a temperatura ambiente. </li><li> Mientras que los embriones son en Tampón A / B, solución Base de tejidos pre-caliente a 37 ° C. Usar 5 ml de la solución de base de tisú por embrión. </li><li> Mientras que los embriones son en Tampón A / B, preparar fresco 5-bromo- 4- cloro- 3- indolilo α-D-galactopiranósido (X-gal) a una concentración de 40 mg / ml en 100% de sulfóxido de dimetilo (DMSO). </li><li> Diluir X-gal en la solución de base de tisú pre-calentado a una concentración de 1 mg / ml y añadir 5 ml de X-gal / mezcla base de tejido a un vial de centelleo de vidrio. </li><li> Transferencia de embriones a un vial de centelleo de vidrio que contiene mezcla Base X-gal / Tissue. Sellar la tapa con Parafilm y se incuba a 37 ° C durante la noche, la comprobación de la presencia de la mancha azul. </li><li> Embriones Postfix con frescos PFA al 4%, pH 7,4. Agite suavemente durante 4 horas a 4 ° C. </li><li> Enjuague embriones dos veces en PBS frío. </li></ol> 4. Tejido Deshidratación y Compensación <ol><li> Coloque los embriones en 50% de metanol / 50% de mezcla de PBS durante 30 minutos a temperatura ambiente, agitando en los viales de vidrio. </li><li> Continuar para deshidratar en 75% de metanol / 25% PBS durante 30 min a temperatura ambiente, agitando en viales de vidrio. </li><li> Continuar para deshidratar en 90% de metanol / 10% de PBS durante 30 min a temperatura ambiente, agitando frascos de vidrio. </li><li> Continuar para deshidratar en 100% de metanol durante 30 min (2x) a temperatura ambiente, agitando en viales de vidrio. </li><li> Mientras que los embriones se deshidratante, preparar una 1: 1 (v: v) mezcla de alcohol de benzoilo y de benzoato de benzoilo. NOTA: alcohol de benzoilo y benzoilo benzoato es tóxico. </li><li> Sumergir los embriones en el 1: 1 (v: v) mezcla de 100% de metanol: 100% BABB durante 15 min. Sólo utilizar el vidrio porque el BABB se disolverá plástico. </li><li> Sumerja los embriones en el 100% BABB para borrar por completo el tejido y para visualizar la tinción con X-gal en el embrión despejado. NOTA: La imagen lo más pronto posible porque la mancha X-gal se desvanecerá con el tiempo. </li></ol> 5. todo el montaje de imágenes <ol><li> Estabilizar el embrión, inmerso en el 100% BABB, en los ángulos adecuados para la fotografía utilizando pinzas metálicas. Ilumine con una combinación de fuentes hasta la luz y bajo la luz de la luz. </li><li> Adquirir imágenes con un microscopio de disecciónequipado con una cámara digital. Tome varias exposiciones de campo claro en cinco planos focales sucesivas 0,5 mm de separación a lo largo del eje Z. Los tiempos de exposición y diafragmas pueden variar en función de la iluminación y la cámara especificaciones y necesitan ser optimizados para cada configuración. </li><li> Utilice software estándar de procesamiento de imágenes para procesar imágenes superpuestas y generar fotomontajes. </li></ol>

<ol>
	<li>Verze, L., 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=Cutaneous+innervation+in+hereditary+sensory+and+autonomic+neuropathy+type+IV.">Cutaneous innervation in hereditary sensory and autonomic neuropathy type IV.</a> Neurology. 55, 126-128 (2000).</li><li>Sethna, N. F., Meier, P. M., Zurakowski, D., Berde, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+sensory+abnormalities+in+children+and+adolescents+with+complex+regional+pain+syndromes.">Cutaneous sensory abnormalities in children and adolescents with complex regional pain syndromes.</a> Pain. 131, 153-161 (2007).</li><li>Uceyler, N., 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=Small+fibers+in+Fabry+disease:+baseline+and+follow-up+data+under+enzyme+replacement+therapy.">Small fibers in Fabry disease: baseline and follow-up data under enzyme replacement therapy.</a> J Peripher Nerv Syst. 16, 304-314 (2011).</li><li>Reichardt, L. F., Mobley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+the+distance,+or+not,+with+neurotrophin+signals.">Going the distance, or not, with neurotrophin signals.</a> Cell. 118, 141-143 (2004).</li><li>White, F. 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=Synchronous+onset+of+NGF+and+TrkA+survival+dependence+in+developing+dorsal+root+ganglia.">Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia.</a> J Neurosci. 16, 4662-4672 (1996).</li><li>Farinas, I., Wilkinson, G. A., Backus, C., Reichardt, L. F., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neurotrophin+and+Trk+receptor+functions+in+developing+sensory+ganglia:+direct+NT-3+activation+of+TrkB+neurons+in+vivo.">Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo.</a> Neuron. 21, 325-334 (1998).</li><li>Markus, A., Zhong, J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf+and+akt+mediate+distinct+aspects+of+sensory+axon+growth.">Raf and akt mediate distinct aspects of sensory axon growth.</a> Neuron. 35, 65-76 (2002).</li><li>Kuruvilla, 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=A+neurotrophin+signaling+cascade+coordinates+sympathetic+neuron+development+through+differential+control+of+TrkA+trafficking+and+retrograde+signaling.">A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling.</a> Cell. 118, 243-255 (2004).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf+kinase+signaling+functions+in+sensory+neuron+differentiation+and+axon+growth+in+vivo.">Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo.</a> Nature. 10, 598-607 (2007).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+olfactory+sensory+map+develops+in+the+absence+of+normal+projection+neurons+or+GABAergic+interneurons.">An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons.</a> Neuron. 21, 1273-1282 (1998).</li><li>Moqrich, 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=Expressing+TrkC+from+the+TrkA+locus+causes+a+subset+of+dorsal+root+ganglia+neurons+to+switch+fate.">Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate.</a> Nature. 7, 812-818 (2004).</li><li>Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax-deficient+mice+with+lymphoid+hyperplasia+and+male+germ+cell+death.">Bax-deficient mice with lymphoid hyperplasia and male germ cell death.</a> Science. 270 (5233), 96-99 (1995).</li><li>Lentz, S. I., Knudson, C. M., Korsmeyer, S. J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+support+the+development+of+diverse+sensory+axon+morphologies.">Neurotrophins support the development of diverse sensory axon morphologies.</a> J. Neurosci. 19, 1038-1048 (1999).</li><li>Patel, T. D., Jackman, A., Rice, F. L., Kucera, J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sensory+neurons+in+the+absence+of+NGF/TrkA+signaling+in+vivo.">Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.</a> Neuron. 25, 345-357 (2000).</li><li>Donovan, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-RAF+kinase+drives+developmental+axon+growth+and+promotes+axon+regeneration+in+the+injured+mature+CNS.">B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS.</a> The Journal of experimental medicine. 211, 801-814 (2014).</li><li>Mercer, K., 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=Expression+of+endogenous+oncogenic+V600EB-raf+induces+proliferation+and+developmental+defects+in+mice+and+transformation+of+primary+fibroblasts.">Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts.</a> Cancer research. 65, 11493-11500 (2005).</li><li>Tronche, F., 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=Disruption+of+the+glucocorticoid+receptor+gene+in+the+nervous+system+results+in+reduced+anxiety.">Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.</a> Nature genetics. 23, 99-103 (1999).</li><li>Madisen, L., 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=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nat Neurosci. 15, 793-802 (2012).</li><li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Schmidt, H., Rathjen, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiI-labeling+of+DRG+neurons+to+study+axonal+branching+in+a+whole+mount+preparation+of+mouse+embryonic+spinal+cord.">DiI-labeling of DRG neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord.</a> J Vis Exp. , (2011).</li></ol>

The authors declare that there are no competing interests.

El procedimiento de tinción X-gal-descrita anteriormente de ratones embrionarios TrkA taulacZ permite la visualización rápida y detallada de las proyecciones de los axones de larga distancia en el embrión intacto fijo. Debido a la nula fondo Bax estos ratones permiten el sondaje de los mecanismos que pueden contribuir tanto al crecimiento del axón neuronal y la supervivencia de señalización. El apareamiento con transgénicos o knockout ratones de interés permite una evaluación compl...

Establecimiento de redes neuronales precisos es un complejo proceso de desarrollo esencial para la funcionalidad del sistema nervioso. Perturbación en este proceso conduce a la disfunción neuronal que ha sido implicado en enfermedades neurológicas humanas 1-3. Para estudiar los mecanismos moleculares subyacentes de crecimiento de los axones y la inervación diana en mamíferos, hemos desarrollado un protocolo para visualizar las trayectorias axonales de TrkA-expresión de las neuronas sensoriales usando una combinación de dos líneas de ratones modificados genéticamente. TrkA es un receptor para el factor de crecimiento nervioso NGF y es un marcador funcional de las neuronas sensoriales nociceptivas 4. TrkA es altamente expresado en las neuronas nociceptivas durante el desarrollo temprano y media la supervivencia de neuronas dependientes de NGF, el crecimiento axonal, la arborización y el objetivo inervación 5-9. En los ratones TrkA taulacZ, el gen TrkA de tipo salvaje se sustituye por una expresión taulacZ cassette 10, de tal manera que la morfología axonal de las neuronas TrkA-positivos putativos se puede visualizar por β-gal (X-gal) tinción 11. Mediante una línea de TrkA taulacZ / WT heterocigóticos, podemos examinar los factores que pueden regular o interferir con el desarrollo de proyecciones aferentes sensoriales en vivo. Por otra parte, la expresión de TrkA está ausente en ratones homocigotos TrkA taulacZ / taulacZ, que por lo tanto pueden ser utilizados para evaluar el crecimiento del axón promoción de mecanismos en la ausencia de la señalización de NGF / TrkA. Dado que las neuronas nociceptivas dependen de NGF / TrkA señalización no sólo para el crecimiento axonal, sino también para la supervivencia, empleamos otra línea de ratones, que carecen del gen Bax pro-apoptóticos, para inhibir la apoptosis en las neuronas DRG embrionarias, rescatar de la muerte celular que es lo contrario observado en ausencia de la señalización de TrkA. El Bax - / - fondo 12 por lo tanto permite la molecudisección lar de las vías de señalización que afectan específicamente axón 7-9,13-15 crecimiento. En TrkA - / -: Bax - / - ratones, DRG neuronas sobreviven, pero inervación aferente sensorial en la piel es la supresión total de 14,15. Podemos activar selectivamente las vías de señalización para determinar sus respectivas contribuciones al desarrollo de las proyecciones de los axones. La utilidad de este método es que permite la evaluación de los cambios en los fenotipos de crecimiento axonal cuando diferentes modificaciones genéticas se crían en el TrkA taulacZ / taulacZ: Bax - / - o TrkA taulacZ / WT: Bax - / - fondos.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

whole-mount imaging of mouse embryo sensory axon projections

La visualización de proyecciones neuronales de larga duración en los embriones es esencial para obtener una comprensión de cómo los mamíferos desarrollan redes neuronales. Aquí se describe un método para etiquetar in situ un subconjunto de ganglios de la raíz dorsal (GRD) proyecciones axón para evaluar sus características fenotípicas utilizando varias líneas de ratones manipulados genéticamente. Las neuronas TrkA-positivo son las neuronas nociceptivas, dedicada a la transmisión de señales de dolor. Utilizamos una línea de ratones TrkA taulacZ para etiquetar las trayectorias de todos los axones periféricos TrkA-positivo en el embrión de ratón intacto. Criamos aún más la línea TrkA taulacZ sobre un fondo nulo Bax, que esencialmente suprime la apoptosis neuronal, a fin de evaluar las preguntas relacionadas con el crecimiento independientemente de los posibles efectos de las manipulaciones genéticas sobre la supervivencia neuronal. Posteriormente, los ratones modificados genéticamente de interés son criados con el TrkA TaullacZ / Bax línea nula y son entonces listo para su estudio usando las técnicas descritas en el presente documento. Esta presentación incluye información detallada sobre los planes de cría de ratón, genotipificación en el momento de la disección, la preparación del tejido, la coloración y el desmonte para permitir la visualización de larga duración trayectorias axonal en la preparación de todo el montaje.

Los genotipos de TrkA WT / taulacZ: Bax - / - y TrkA taulacZ / taulacZ: Bax - / - embriones se pueden determinar de forma inequívoca por genotipo PCR estándar (Figura 1). Tinción pantallas X-gal detallan cenadores axonales periféricos por vía subcutánea en embriones convencionalmente manchadas (Figuras 2, 3a), y en todo el embrión después de la compensación de tejido (Figuras 3b, 4). Hemos...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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 ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Research

JoVE Journal

Neuroscience

10.0K vistas.  Weill Medical College of Cornell University. 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.

Video: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Investigación

Neurociencias

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Disección de un ojo del ratón de un monte entero del epitelio pigmentario de la retina

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

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 ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Todo el montaje de la tinción de inmunofluorescencia del Neonatal ratón Retina de investigar Angiogénesis<em&gt; En vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Flat Mount Imaging de ratón de la piel y su aplicación al análisis del folículo del pelo Patrones y sensorial Axon Morfología

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagen Ca<sup&gt; 2 +</sup&gt; Dinámica de Terminales cono fotorreceptor axón de la retina del ratón

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Todo el montaje La disección y de inmunofluorescencia del Adulto ratón cóclea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Inyección de raíz dorsal ganglio de raíz dorsal y Lesión por aplastamiento como modelo para la regeneración del axón sensorial

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

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 ...