This is supported by 973 MOST grant (Grant No. 2011CB504402, 2012CB947602), National Natural Science Foundation of China (Grant No. 91132724, U1332136) and the 'Hundred Talents Project' of Chinese Academy of Science. The protocol was approved by the Committee on the Ethics of Animal Experiments of the USTC (Permit Number: USTCACUC1103013).

1. Construya la Cirugía del Aparato NOTA: Para asegurar el pescado se mantiene vivo durante y después de la operación, la anestesia por goteo solución de acetato de metanosulfonato de 3-aminobenzoato (MS-222, o tricaína) a una media de concentración (0,015%) con una velocidad de 1 gota / segundo a través de la boca del pez usando un sistema de goteo de construcción casera se muestra en la Figura 1. <ol><li> Haga una caja, como se muestra en la Figura 1A (longitud es de 28 cm, el ancho es de 10 cm, la altura es de 5 cm), coloque una esponja (la longitud es de 6 cm, la anchura es de 9 cm, la altura es de 4 cm) en el centro y abrir un brecha en él. </li><li> Hacer una superficie globular liso en el extremo de la aguja con el bono de curación de luz (Figura 1B) que luego se inserta en la boca de los pescados. Asegúrese de que la superficie de la aguja es suave para evitar que la boca del pez de ser dañado. </li><li> Añadir la concentración media de MS-222 (cámara de la izquierda, la longitud es de 6 cm, el ancho es de 10 cm, la altura es de 8 cm) y solución salina (cámara derecha, la longitud es de 4 cm, el ancho es de 10 cm, la altura es de 8 cm) para los depósitos como se muestra en la Figura 1C. Nota: Se recomienda Fresh MS-222. </li><li> Anestesie peces en 0,03% de MS-222 durante 2 min. </li><li> Coloque el pescado en la brecha de la esponja y fijar los peces con dos pares de pasadores, insertado detrás de la aleta pectoral y al lado del poro cloacal, respectivamente 11. </li><li> Conectar la boca del pez, MS-222, y agua de cría con un tubo de tipo T como se muestra en la figura 1C. </li><li> Conectar el tubo conectado a la MS-222. </li></ol> 2. CGR etiqueta retrógrados de la derecha Tectum NOTA: Antes de comenzar, desinfecte todos los aparatos con etanol al 70-75%, lo que garantiza que ningún etanol permanece. <ol><li> Rossing, alejar un poco la piel sobre todo el cráneo con sclerectome (vista dorsal del cráneo se muestra en las figuras 2A y 2B </strong&gt;). </li><li> Compensación: lavar el cráneo con solución salina y luego séquela con oreja bombilla lavado. </li><li> Corrosión inicial: corroer el cráneo con el reactivo de ataque durante 10 s. </li><li> Compensación: completamente limpie el grabador con un hisopo de algodón y luego lave el área con solución salina y se seca. </li><li> Protección: con el fin de proteger el cráneo en otras áreas, excepto el tectum derecha desde el exceso de corrosión, cubrir estas áreas con vínculo lámpara de polimerización y después se cura por exposición a la luz azul con iluminador portátil. </li><li> Corrosión completa: poner el grabador en la zona central de la tectum la derecha de nuevo para el grabado completo para 2 min. Nota: Dado que los cráneos de peces de más edad están calcificados, la corrosión se necesitará más tiempo. </li><li> Compensación: completamente limpie el grabador con un hisopo de algodón y luego lave el área con solución salina y se seca de nuevo. </li><li> Apertura Cráneo: retirar con cuidado el cráneo malacicas (Figura 2C) con unas pinzas para exponer el tectum derecha (Figura 2D </strong&gt;). </li><li> Compensación: limpiar la mucosidad y effusing sangre del agujero con piezas de espuma de gel y se lava con solución salina hasta que el sangrado se ha detenido. </li><li> Colocación tinte: poner el tinte (pasta de tejido-etiquetado) en todo el tectum derecha y se cubre con espuma de gel. </li><li> Segregar: cubra el orificio con una escala estéril del mismo animal. </li><li> Sellado: Bond Place lámpara de polimerización a la báscula y luego curarla con la luz azul de nuevo durante 40 segundos. </li><li> Revival: lavar la superficie de la fianza y revivir el animal con solución salina. </li><li> Enfermería y cría: Mantener el pescado en 28.5 ° C medio de embrión (ZFIN) durante 5 días y alimentar a 2 veces / día. No incluya pez cuyo tinte no logra permanecer en el tectum durante 5 días en el experimento. Nota: media de Embriones (EM) solución es vital para la supervivencia de los peces y de la temperatura es el factor clave para el marcaje con Dil. </li></ol> 3. Wholemounting Retina e Imagen <ol><li> Coloque el pescado en un campo oscuro durante 2 horas antes de la retina quelemount. </li><li> Se anestesia el pescado con agua helada. </li><li> Perforar un agujero en el margen de la córnea y hacer una taza de ojo mediante la eliminación de la córnea con tijeras venus. </li><li> Retire el objetivo y luego vaciar el centro de la retina con helado PBS 1x. </li><li> Enjuague la brecha entre la retina y la esclerótica con helado 1x PBS hasta que no quede ningún pigmento. </li><li> Cortar el nervio óptico en el disco. </li><li> Mantener la capa de CGR hacia arriba y mantenga la retina con una cuchara de cristal hecho a sí mismo (Figuras 3A y 3B). Un gotero también se puede utilizar para transferir la retina. </li><li> Ponga la retina en una gota de hielo 30% de glicerina en una diapositiva con la capa de CGR hacia arriba (Figura 3C). </li><li> Retire la glicerina y luego aplanar la retina en el portaobjetos de vidrio antes de cortar retina en cuatro cuadrantes (Figura 3D). Mantener la retina desarrollado con capa RGC alza será de utilidad para aplanar la retina. </li><li> Por goteo helada 50% de glicerina en tél retina para lavar los fragmentos de pigmento. </li><li> Retire la glicerina y luego dejar caer 20 l de helado 75% de glicerina en la retina. </li><li> Cubra la muestra con 1,8 cm x 1,8 cm cubreobjetos cuadrado. </li><li> Sellar el cubreobjetos con esmalte de uñas. Se recomienda de formación de imágenes inmediata de la retina. </li><li> Imagen no entrelazada toda la retina bajo un lente objetivo de 20X (NA = 0,70) con DP72 CCD (cada imagen es 1360 x 1024 píxeles). Cada campo tiene tres enfoques diferentes. </li><li> Extender la profundidad de cada campo con la herramienta de &quot;profundidad de campo&quot; en el software. </li><li> Montar una retina virtual con la función de photomerge. </li><li> Seleccionar tres campos de 680 x 512 píxeles (área real es de 350 micras por 264 micras, 0,092 mm 2) en cada orientación de la retina para analizar la densidad promedio de CGR (Figura 4A). </li><li> Obtener el área con la función de las mediciones de - crear una entidad poligonal en la imagende software. </li><li> Calcular el número total de CGR mediante la ecuación &quot;número CGR = área destino x&quot; 9. </li></ol>

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A., Noack, C., Boehringer, D., Biermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+of+fluorogold-prelabeled+retinal+ganglion+cells+with+calcein-AM:+A+new+method+for+assessing+cell+vitality.">Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality.</a> J Neurosci Methods. 192, 233-239 (2010).</li><li>Schwalb, J. 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=Two+factors+secreted+by+the+goldfish+optic+nerve+induce+retinal+ganglion+cells+to+regenerate+axons+in+culture.">Two factors secreted by the goldfish optic nerve induce retinal ganglion cells to regenerate axons in culture.</a> J Neurosci. 15, 5514-5525 (1995).</li><li>Watanabe, M., Inukai, N., Fukuda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+retinal+ganglion+cells+after+transection+of+the+optic+nerve+in+adult+cats:+a+quantitative+study+within+two+weeks.">Survival of retinal ganglion cells after transection of the optic nerve in adult cats: a quantitative study within two weeks.</a> Vis Neurosci. 18, 137-145 (2001).</li><li>Chiu, K., Lau, W. M., Yeung, S. C., Chang, R. C., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+labeling+of+retinal+ganglion+cells+by+application+of+fluoro-gold+on+the+surface+of+superior+colliculus.">Retrograde labeling of retinal ganglion cells by application of fluoro-gold on the surface of superior colliculus.</a> J Vis Exp. , (2008).</li><li>Bakken, T. E., Stevens, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+system+scaling+in+teleost+fish.">Visual system scaling in teleost fish.</a> J Comp Neurol. 520, 142-153 (2012).</li><li>Zhou, L. X., Wang, Z. 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E. <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+olfactoretinal+centrifugal+pathway+may+relate+to+the+visual+system+defect+in+night+blindness+b+mutant+zebrafish.">Disruption of the olfactoretinal centrifugal pathway may relate to the visual system defect in night blindness b mutant zebrafish.</a> Journal of Neuroscience. 20, 1883-1892 (2000).</li><li>Kassing, V., Engelmann, J., Kurtz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Single-Cell+Responses+in+the+Optic+Tectum+of+Adult+Zebrafish+with+Dextran-Coupled+Calcium+Dyes+Delivered+via+Local+Electroporation.">Monitoring of Single-Cell Responses in the Optic Tectum of Adult Zebrafish with Dextran-Coupled Calcium Dyes Delivered via Local Electroporation.</a> PLoS One. 8, (2013).</li></ol>

We have nothing to disclose.

Marcado retrógrado de CGR es importante investigar la supervivencia de CGR en animales mamíferos, pero no se ha usado en el pez cebra. Los métodos alternativos, tinción HE 7 y 8 tinción de anticuerpos, no son normas de oro para contar el número de CGR, y las líneas transgénicas con todas las CGR marcadas aún no se ha construido el 12, 13. En este video, se introduce un método para retrogradar CGR etiqueta en la retina del pez cebra adulto. Aunque aproximadamente el 1% de los ax...

Como adultos de pez cebra tiene una fuerte capacidad de regenerar axones después de la lesión del nervio óptico 1, un método adecuado para contar toda la CGR es fundamental para evaluar la supervivencia y regeneración de 2 CGR. Sobre la base de los métodos de la CGR etiquetado retrógradas en mamíferos y peces de colores 3-5, se construyó el método para etiquetar CGR del tectum en adultos de pez cebra. Para adultos de pez cebra, dos preocupaciones técnicos críticos deben tenerse en cuenta: el cráneo del pez cebra adulto es muy pequeño 6; viven en un ambiente de agua. Aquí, tratamos el cráneo con mordiente lo que minimiza los riesgos asociados con la perforación de 5. Luego, sellamos el agujero con la luz de bonos de curado que mejora la supervivencia de los animales después de la cirugía. Anteriormente, se han adoptado varias otras técnicas para contar el número de CGR de manera indirecta. Tinción HE en las secciones de retina etiquetas de todos los tipos de células en el RGCL 7. Etiquetado de anticuerpos en toda la rEtina, tales como islote-1, también puede etiquetar células amacrinas 8. Aunque la etiqueta retrógrada del muñón del nervio óptico puede etiquetar todos los CGR en la retina, no se puede adoptar en el modelo de aplastamiento debido a que causa lesión adicional al nervio óptico. Aprovechando etiqueta retrógrada desde el tectum, hemos investigado la supervivencia y regeneración de la CGR en aplastamiento del nervio óptico. Los resultados muestran que casi todas las CGR y sobrevivieron más de 90% de CGR regeneran a la tectum en la primera semana en el modelo de aplastamiento 9. Con el fin de etiquetar con éxito todas las CGR, pasta de Dil fue elegido después de la comparación con varios otros colorantes comerciales 10. En primer lugar, está especialmente diseñado para el etiquetado in vivo de tejidos. En segundo lugar, es un colorante lipófilo que no puede difundirse en agua. Además, esta fluorescencia puede persistir durante un largo tiempo que le hace un excelente candidato para la investigación de la supervivencia de CGR.

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">MS222</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:152px;">E10521</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">DiI</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:152px;">N22880</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">lightcuring bond</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Durafill bond</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:152px;">Gluma Etch</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Gluma Etch 35 Gel</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:152px;">Blue LED</td>
			<td style="width:152px;">Shenruo Medical Equipment Co.</td>
			<td style="width:152px;">Power Blue Light Curing Unit</td>
			<td style="width:152px;">China</td>
		</tr>
	</tbody>
</table>

retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes

Etiquetado células ganglionares de la retina Como retrógradas (CGR) se pueden aislar CGR somata de sitios de morir, se ha convertido en el estándar de oro para el recuento de la CGR en la supervivencia de CGR y los experimentos de regeneración. Muchos estudios se han realizado en animales mamíferos para investigar la supervivencia de CGR después de la lesión del nervio óptico. Sin embargo, aún no se ha informado de marcado retrógrado de CGR en el pez cebra adultos, aunque algunos métodos alternativos pueden contar el número de células en capas de la retina de células ganglionares (RGCL). Teniendo en cuenta el pequeño tamaño del cráneo adultos de pez cebra y el alto riesgo de muerte después de la perforación en el cráneo, se abre el cráneo con la ayuda de grabado ácido y sellar el agujero con una fianza de curado de luz, lo que podría mejorar significativamente la tasa de supervivencia. Después de absorber los colorantes durante 5 días, casi todos los CGR están etiquetados. Como este método no es necesario seccionar el nervio óptico, que es insustituible en la investigación de la supervivencia de CGR después del aplastamiento del nervio óptico en adultos de pez cebra. Aquí, presentamoseste método paso a paso y ofrecer resultados representativos.

Como figuras 4B-D espectáculo, el número de células Dil + es de dos tercios de + células DAPI en RGCL. En una retina normal, una imagen de montaje de toda una retina (Figura 4E) muestra que con Dil + CGR se distribuyen en toda la retina, pero en una retina regenerada (Figura 4F), como CGR en la zona central no se han regenerado a su objetivo en la primera semana, que no podían ser etiquetados. <p class="jove_content" fo:keep-tog...

Watch this Scientific Journal Video about Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes at JoVE.com

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Etiquetado retrógrada de células ganglionares retinianas en adultos de pez cebra con tintes fluorescentes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

retrograde-labeling-retinal-ganglion-cells-adult-zebrafish-with

Research

JoVE Journal

Neuroscience

9.4K vistas.  University of Science and Technology of China. We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Video: Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Etiquetado y las Pilas de imágenes en la parte posterior del cerebro de pez cebra

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Investigación

Neurociencias

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 ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Imagen óptica de neuronas en el ganglio Cangrejo Stomatogastric con tintes sensibles al voltaje

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Marcaje fluorescente retrógrada Permite dirigida extracelular sola unidad de grabación de neuronas identificadas<em&gt; En vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Cargando retrógrada de los nervios, panfletos, y las raíces espinales con tintes fluorescentes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Estudio genético de la regeneración axonal en neuronas cultivadas de Adultos ganglio de la raíz dorsal

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

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 ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Una novela ligera Paradigm daños para su uso en estudios de regeneración retiniana en adultos de pez cebra

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

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 ...