Este trabajo fue financiado por el Proyecto de Apertura de Hubei Key Laboratories (subvención no. 2021KFY055), la Fundación de Ciencias Naturales de la Provincia de Hubei (subvención no. 2020CFB240) y los Fondos de Investigación Fundamental para las Universidades Centrales (subvención no. 2042020kf0065).

Todos los pasos se realizan a temperatura ambiente a menos que se indique lo contrario. Todos los ratones C57BL / 6J utilizados se obtuvieron del Centro de Animales de Laboratorio de la Universidad de Wuhan, y todos los experimentos relacionados fueron aprobados por el Comité de Ética de los Experimentos con Animales de la Universidad de Wuhan. Se hicieron todos los esfuerzos para minimizar el sufrimiento de los ratones.
1. Enucleación y fijación de los ojos
<ol><li>Eutanasia a los ratones con asfixia de dióxido de carbono y enuclear el globo ocular suavemente usando pinzas sin dientes.</li>
	<li>Enjuague el globo ocular una vez en solución salina tamponada con fosfato (PBS).</li>
	<li>Perfore el globo ocular con una aguja de jeringa en la córnea bajo un microscopio (ocular WF: 10x/22; objetivo de aumento continuo: 0.67x-4.5x) para acelerar la fijación en el globo ocular y mejorar la eficiencia de fijación.</li>
	<li>Transfiera a una placa de 24 pocillos que contenga 4% de paraformaldehído (PFA).</li>
	<li>Fije el globo ocular en PFA al 4% durante 45 minutos y luego transfiéralo a 1x PBS para eliminar el exceso de PFA (lavar tres veces, 5 minutos / vez) inmediatamente.</li>
</ol>2. Aislamiento y aplanamiento de la retina
<ol><li>Transfiera el globo ocular a un portaobjetos de vidrio y colóquelo bajo un microscopio de disección (ocular WF: 10x/22; objetivo de aumento continuo: 0.67x-4.5x).</li>
	<li>Sostenga los fórceps en una mano y úselos para sujetar el margen de la córnea para evitar el movimiento del globo ocular. Use la otra mano para sostener las tijeras de disección y corte alrededor de la córnea a aproximadamente 1 mm de profundidad a través del limbo corneoescleral. Retire el cristalino y el cuerpo vítreo.</li>
	<li>Sostenga el disco óptico en el centro y, utilizando tijeras de disección, corte la retina radialmente a lo largo de los cuatro cuadrantes, alcanzando aproximadamente 2/3 del radio de la retina.</li>
	<li>Sujete uno de los bordes esclerales con pinzas dentadas. Con la otra mano, sostenga el borde de la esclerótica con pinzas sin dientes y muévase desde donde la esclerótica está fijada por las pinzas dentadas hacia la base de la esclerótica. Pelar cuidadosamente la retina.</li>
	<li>Extraiga PBS usando una pipeta (200 μL) para enjuagar y humedecer la retina, y luego retire cualquier exceso de PBS con un pequeño trozo de papel absorbente. Si es necesario, aplanar suavemente la retina con un cepillo de cerdas pequeñas.</li>
	<li>Pipetear lentamente metanol frío (preenfriado en un congelador de -20 °C) gota a gota sobre la superficie interna de la retina (el volumen de metanol no es fijo, siempre y cuando la retina pueda estar completamente cubierta por el metanol). La retina se vuelve blanca y desarrolla una mayor tenacidad.</li>
	<li>Aplanar suavemente la retina y eliminar las impurezas como las membranas pigmentarias residuales y el cuerpo vítreo con pequeños cepillos de cerdas. Invierta la retina con pequeños cepillos de cerdas y repita el proceso mencionado anteriormente.</li>
	<li>Transfiera la retina tratada con metanol a un pocillo de una placa de 24 pocillos y manténgala empapada en metanol.</li>
	<li>Guárdelo a -20 °C durante 1-2 h para la inmunotinción inmediata, o deje la retina en metanol y guárdelo a -20 °C para su almacenamiento a largo plazo.</li>
	<li>Retire el metanol de la placa de 24 pocillos y enjuague la retina con 1x PBS.</li>
</ol>3. Tinción inmunofluorescente de las CGR
<ol><li>Transfiera cuidadosamente la retina a una placa de hemaglutinación de 24 pocillos con una pipeta plástica de Pasteur y bloquee en un 5% de PBS-TX-BSA (5 g de polvo de albúmina de suero bovino disuelto en 100 ml de 1x solución de PBST; el PBST se forma agregando 10 ml de Triton X-100 a 2,000 ml de 1 x PBS) a temperatura ambiente durante 4 h o durante la noche a 4 °C. Mantenga el lado ganglionar hacia arriba.</li>
	<li>Extraer el PBS-TX-BSA e incubar la retina a 4 °C durante 48-96 h, añadiendo 100 μL del anticuerpo primario seleccionado (anticuerpo anti-RBPMS de cobaya) mediante una dilución 1:400.</li>
	<li>Retire el anticuerpo primario y enjuague la retina tres veces (20 minutos cada una) con 1x PBS, agitando a temperatura ambiente.</li>
	<li>Incubar la retina con agitación suave durante la noche a 4 °C con 100 μL del anticuerpo secundario correspondiente (ver Tabla de materiales), cianina Cy3 affiniPure burro anti-cobaya IgG (H+L), utilizando una dilución 1:400.</li>
	<li>Enjuague la retina con 1x PBS tres veces (20 minutos cada una).</li>
	<li>Coloque la retina plana sobre el portaobjetos con la capa de células ganglionares hacia arriba, deje caer una cantidad adecuada de agente antifluorescente alrededor de la retina con una pipeta y cubra el portaobjetos con un cubreobjetos (tenga cuidado de no tener burbujas). NOTA: Para determinar la cantidad adecuada de calmante antifluorescente, asegúrese de que cubra toda la retina. Después de eso, cubra el portaobjetos sin permitir que la retina flote y se mueva fácilmente.</li>
	<li>Selle la corredera alrededor con un agente de sellado para evitar que se mueva. Almacene las muestras a -20 ° C y protéjalas de la luz.</li>
	<li>Imagen de la retina con un microscopio fluorescente/confocal.</li>
</ol>

Aislamiento de retina de ratón y tratamiento con metanol para el estudio de las células ganglionares de la retina

<ol>
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M., 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=Brn3a+as+a+marker+of+retinal+ganglion+cells:+qualitative+and+quantitative+time+course+studies+in+naive+and+optic+nerve-injured+retinas.">Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.</a> Investigative Ophthalmology &amp; Visual Science. 50 (8), 3860-3868 (2009).</li><li>Kwong, J. M., Caprioli, J., Piri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+binding+protein+with+multiple+splicing:+A+new+marker+for+retinal+ganglion+cells.">RNA binding protein with multiple splicing: A new marker for retinal ganglion cells.</a> Investigative Ophthalmology &amp; Visual Science. 51 (2), 1052-1058 (2010).</li><li>Stradleigh, T. W., Ishida, A. 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H., Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fixation+protocols+for+adherent+cultured+cells+applied+to+a+GFP+fusion+protein+of+the+epidermal+growth+factor+receptor.">Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor.</a> Cytometry. 35 (4), 353-362 (1999).</li><li>Baykal, B., Korkmaz, C., Kocabiyik, N., Ceylan, O. 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+influence+of+post-fixation+on+visualising+vimentin+in+the+retina+using+immunofluorescence+method.">The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method.</a> Folia Morphologica. 77 (2), 246-252 (2018).</li><li>Powner, M. B., 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=Visualization+of+gene+expression+in+whole+mouse+retina+by+in+situ+hybridization.">Visualization of gene expression in whole mouse retina by in situ hybridization.</a> Nature Protocols. 7 (6), 1086-1096 (2012).</li><li>Zhang, N., Cao, W., He, X., Xing, Y., Yang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+methanol+to+preserve+retinas+for+immunostaining.">Using methanol to preserve retinas for immunostaining.</a> Clinical and Experimental Ophthalmology. 50 (3), 325-333 (2022).</li><li>Kalesnykas, 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=Retinal+ganglion+cell+morphology+after+optic+nerve+crush+and+experimental+glaucoma.">Retinal ganglion cell morphology after optic nerve crush and experimental glaucoma.</a> Investigative Ophthalmology &amp; Visual Science. 53 (7), 3847-3857 (2012).</li><li>Parrilla-Reverter, 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=Time-course+of+the+retinal+nerve+fibre+layer+degeneration+after+complete+intra-orbital+optic+nerve+transection+or+crush:+a+comparative+study.">Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: a comparative study.</a> Vision Research. 49 (23), 2808-2825 (2009).</li></ol>

Los autores no tienen conocimiento de ninguna afiliación, membresía, financiamiento o tenencia financiera que pueda afectar la objetividad de este estudio.

La fijación es un paso esencial para preservar la retina, lo que puede afectar cualquier investigación posterior de RGC basada en la morfología. La fijación exitosa captura rápidamente la estructura y el estado de las retinas en el momento de exponerlas al medio de fijación, lo cual es crítico para un análisis posterior. Aunque el formaldehído ha sido considerado como uno de los agentes fijadores más comunes para la fijación y preservación de tejidos y células, el formaldehído por sí solo no siempre funcio...

Las células ganglionares de la retina (CGR) son las únicas neuronas de proyección en la retina, e integran y transmiten información visual externa al cerebro1. Muchas enfermedades neurodegenerativas como el glaucoma y la neuropatía óptica traumática se caracterizan por daño irreversible y pérdida de CGR 2,3. El análisis de los cambios morfológicos y cuantitativos de las CGR es un paso crucial para determinar cómo se desarrollan y avanzan las enfermedades neurodegenerativas 4,5.
El ensayo de inmunofluorescencia indirecta es un método ampliamente aceptado para monitorear la distribución de proteínas y el recuento celular. En el laboratorio, la inmunotinción retiniana de montaje completo se utiliza comúnmente en estudios experimentales sobre CGR para evaluar las condiciones fisiológicas y patológicas de la retina6. Los marcadores más comunes utilizados para la cuantificación de RGC en toda la retina incluyen Brn3a, proteína de unión a ARN con empalme múltiple (RBPMS), y así sucesivamente 7,8. La caracterización de la cantidad y distribución de las CGR requiere inmunotinción retiniana de montaje completo de alta calidad. Generalmente, en los protocolos de inmunotinción, la retina se sumerge en fijadores químicos antes de ser incubada en anticuerpos. Los fijadores ideales no deben cambiar la forma de las células, la accesibilidad o afinidad de los epítopos por los anticuerpos, o las dimensiones lineales del tejido 9,10.
Debido a la compleja estructura de la retina, problemas como la fragilidad retiniana y el plegamiento, así como algunas dificultades comunes, como la contracción celular y los núcleos poco claros, son propensos a ocurrir cuando se hace un parche retiniano de montaje completo, lo que tiene un impacto negativo en la investigación experimental. Además, no todas las retinas se inmunotiñen inmediatamente, especialmente cuando se trata de las retinas de ratones transgénicos con precios caros que son de origen precioso, lo que requiere la preservación de las muestras de retina adicionales para su uso posterior.
La solución fijadora adecuada puede fijar el tejido rápidamente, evitar la autólisis tisular, preservar la morfología y estructura normales de las células tisulares y mantener la antigenicidad de las proteínas y otras sustancias10. En la actualidad, la fijación a base de formaldehído se ha utilizado ampliamente en diversos tejidos, incluyendo retinas separadas, oculares hemisecados y globos oculares enteros10. La contracción del tejido y la alteración morfológica de las células son los dos desafíos críticos encontrados después de la inmersión en formaldehído11. Además, las formulaciones de fijación modificada están emergiendo cada vez más para maximizar la retención de las propiedades originales de la retina y las células diana 9,10. Diferentes tratamientos de fijación retiniana pueden afectar la estructura retiniana, la inmunogenicidad de proteínas, la excitación fluorescente y el ciclo de enfriamiento de atenuación de manera diferente12,13. Las retinas fijadas con la solución de Davidson están más morfológicamente intactas en comparación con las fijadas con formalina, pero la solución de Davidson es menos compatible con algunos anticuerpos, como la molécula adaptadora de unión al calcio ionizada del marcador microglial 112. Teniendo en cuenta la naturaleza frágil de las retinas, los investigadores naturalmente se preguntarían si la integridad de la retina, así como las propiedades y la morfología de las células diana, cambiarán después del almacenamiento a largo plazo. Sin embargo, rara vez se han informado los posibles efectos de la solución de fijación sobre la morfología de las células retinianas y RGC después del almacenamiento durante varios meses. La optimización de la fijación retiniana es crítica para la evaluación y preservación de las CGR.
Proporcionamos una descripción detallada de un método confiable y técnicamente sencillo que utilizamos para la tinción retiniana murina de montaje completo. Nuestro método enfatiza la preparación y almacenamiento adecuados de las retinas para la investigación de RGC, teniendo en cuenta la necesidad de almacenamiento a largo plazo del tejido retiniano, así como aspectos específicos de la formación o degradación de fluoróforos.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well cell culture cluster</td>
			<td>Costar</td>
			<td></td>
			<td>Eyeball fixation</td>
		</tr>
		<tr>
			<td>24-well hemagglutination plate</td>
			<td>Labedit Company</td>
			<td></td>
			<td>Incubation antibody</td>
		</tr>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>Citotest</td>
			<td></td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Anti-fluorescent quenching mountant</td>
			<td>Servicebio</td>
			<td>G1401</td>
			<td>Slow down fluorescence quenching</td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumin)</td>
			<td>Servicebio</td>
			<td>GC305010</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>OLYMPUS</td>
			<td></td>
			<td>Apply 40x objective lens</td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>RWD Life science Co.,LTD&#160;</td>
			<td>77001S</td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>20210624</td>
			<td>GC&#8805;99.5%</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>SecheVite</td>
			<td></td>
			<td>Sealing agent</td>
		</tr>
		<tr>
			<td>Needles&#160;</td>
			<td>Shanghai Kindly Enterprises Development Group Co., Ltd.</td>
			<td></td>
			<td>Accelerate the fixation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution</td>
			<td>Servicebio</td>
			<td>G1101</td>
			<td>Eyeball fixation</td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline pH 7.4)</td>
			<td>Servicebio</td>
			<td>G0002</td>
			<td>Rinse the eyeball&#160;</td>
		</tr>
		<tr>
			<td>Primary antibody: guinea pig anti-RNA-binding protein with multiple splicing (RBPMS)</td>
			<td>PhosphoSolutions</td>
			<td>Cat. #1832-RBPMS</td>
			<td>For immunofluorescence. Used at 1:400</td>
		</tr>
		<tr>
			<td>Secondary antibody: Cy3 affiniPure donkey anti-guinea pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>706-165-148</td>
			<td>For immunofluorescence. Used at 1:400</td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
	</tbody>
</table>

methanol-based whole-mount preparation for the investigation of retinal ganglion cells

Las células ganglionares de la retina (CGR), que son las neuronas de proyección de la retina, propagan información visual externa al cerebro. Los cambios patológicos en las CGR tienen una estrecha relación con numerosas enfermedades degenerativas de la retina. La inmunotinción retiniana de montaje completo se utiliza con frecuencia en estudios experimentales sobre CGR para evaluar las condiciones de desarrollo y patológicas de la retina. En algunas circunstancias, algunas muestras valiosas de retina, como las de ratones transgénicos, pueden necesitar ser retenidas durante un largo período sin afectar la morfología o el número de CGR. Para obtener resultados experimentales creíbles y reproducibles, es esencial utilizar un medio de conservación eficaz. Aquí, describimos el efecto del metanol como un medio fijo auxiliar para preparaciones retinianas de montaje completo y almacenamiento a largo plazo. En resumen, durante el proceso de aislamiento, el metanol frío (-20 ° C) se pipetea sobre la superficie de la retina para ayudar a fijar los tejidos y facilitar su permeabilidad, y luego las retinas se pueden almacenar en metanol frío (-20 ° C) antes de ser inmunoteñidas. Este protocolo describe el flujo de trabajo de aislamiento de retina y el protocolo de almacenamiento de muestras de tejido, que es útil y práctico para la investigación de CGR.

Después de la disección, la retina debe verse como un trébol plano de cuatro hojas. En este estudio, mediante el uso del protocolo descrito anteriormente, la retina se volvió blanca después de que se agregó metanol (Figura 1). Mientras tanto, la retina cambió de suave a flexible y plana. A continuación, los RGC fueron etiquetados con anti-RBPMS8. Se tomaron cuatro campos de imagen en toda la retina de montaje (n = 3) utilizando un microscopio confocal (ocular:...

Watch this Scientific Journal Video about Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells at JoVE.com

Autor destacado: Preparación de montaje completo a base de metanol para la investigación de las células ganglionares de la retina  

El metanol se puede utilizar como medio fijo auxiliar para preparaciones retinianas de montaje completo y almacenamiento a largo plazo, lo que es útil para la investigación de las células ganglionares de la retina.

Preparación de montaje completo a base de metanol para la investigación de células ganglionares de la retina

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

La investigación tiene como objetivo proporcionar un flujo de trabajo de aislamiento de retina modificado y un protocolo de almacenamiento de muestras de tejido, que es útil y práctico para investigar las CGR. Las retinas son frágiles y se pliegan, lo que limita la investigación experimental al hacer un parche retiniano de montaje completo. Además, todas las retinas no requieren inmunotinción inmediata.A veces, las muestras de retina adicionales necesitan preservación para su uso posterior. Proporcionamos una descripción detallada de un método confiable y técnicamente sencillo de tinción de retina de montaje completo que enfatiza la preparación adecuada y la necesidad del almacenamiento a largo plazo de retinas para la investigación de RGC. Continuaremos trabajando en la investigación básica de las neuropatías ópticas degenerativas.Además, se explorará el mecanismo molecular de la lesión del nervio óptico y la regeneración de accidentes y los posibles objetivos clínicos para diagnosticar y tratar enfermedades neurodegenerativas.

methanol-based-whole-mount-preparation-for-investigation-retinal

Research

JoVE Journal

Neuroscience

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

2.9K vistas.  Renmin Hospital of Wuhan University. La investigación tiene como objetivo proporcionar un flujo de trabajo de aislamiento de retina modificado y un protocolo de almacenamiento de muestras de tejido, que es útil y práctico para investigar las CGR. Las retinas son frágiles y se pliegan, lo que limita la investigación experimental al hacer un parche retiniano de montaje completo. Además, todas las retinas no requieren inmunotinción inmediata.A veces, las muestras de retina adicionales necesitan preservación para su u...

Video: Autor destacado: Preparación de montaje completo a base de metanol para la investigación de las células ganglionares de la retina  

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Una preparación aislada de retina para registrar la respuesta de la luz a partir de semillas etiquetadas las células ganglionares retinianas

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

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 ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

La transfección de células de ratón del ganglio de la retina por In vivo La electroporación

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Preparación Todo el montaje del Adulto Drosophila Cordón nervioso ventral para inyección gigante tinte de fibra

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Implementar Clamp dinámico con conductancias sinápticas y artificiales en células de ratón ganglionares retinianas

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Disección Del Músculo Transverso Abdominis Para El Análisis De La Unión Neuromuscular De Montaje Completo

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Un simple de un solo paso para la disección Protocolo Todo el montaje Preparación de adulto<em&gt; Drosophila</emLos cerebros&gt;

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

RNA-Seq de una sola célula de subconjuntos definidos de células de ganglio retiniano

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

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 ...