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. Construire l&#39;Appareil de chirurgie REMARQUE: Pour assurer le poisson reste en vie pendant et après l&#39;opération, goutte à goutte anesthésie solution éthyle méthanesulfonate 3 benzoate (MS-222, ou Tricaine) à une demi-concentration (0,015%) avec une vitesse de 1 goutte / sec par la bouche du poisson en utilisant un système de goutte à goutte homebuilt représenté sur la figure 1. <ol><li> Faire une boîte comme indiqué sur la figure 1A (longueur 28 cm, largeur 10 cm, hauteur 5 cm), mettre une éponge (longueur de 6 cm, la largeur est de 9 cm, la hauteur est de 4 cm) dans le milieu et ouvrir un écart sur elle. </li><li> Faites une surface sphérique lisse à l&#39;extrémité de l&#39;aiguille avec liaison photopolymérisation (figure 1B) qui sera ensuite insérée dans la bouche du poisson. S&#39;assurer que la surface de l&#39;aiguille est lisse pour empêcher la bouche du poisson ne soit endommagée. </li><li> Ajouter la concentration de la moitié de la SP-222 (chambre à gauche, la longueur est de 6 cm, la largeur est de 10 cm, la hauteur est de 8 cm) et une solution saline (chambre à droite, la longueur est de 4 cm, la largeur est de 10 cm, la hauteur est de 8 cm) pour les réservoirs comme le montre la figure 1C. Note: frais MS-222 est recommandé. </li><li> Anesthésier les poissons dans 0,03% MS-222 pendant 2 min. </li><li> Placer le poisson dans l&#39;écart de l&#39;éponge et fixer le poisson avec deux paires de broches, insérées derrière la nageoire pectorale et à côté du pore du cloaque, respectivement 11. </li><li> Raccorder la bouche du poisson, MS-222, et de l&#39;eau d&#39;élevage avec un tube de type T, comme le montre la figure 1C. </li><li> Mettez le tube relié à la MS-222. </li></ol> 2. CGR Label rétrogrades de la droite Tectum REMARQUE: Avant de commencer, désinfecter tous les appareils avec 70-75% d&#39;éthanol, veiller à ce qu&#39;aucun éthanol reste. <ol><li> Rossing: enlever la peau légèrement sur ​​l&#39;ensemble du crâne avec sclerectome (vue dorsale du crâne est représenté sur les figures 2A et 2B </strong&gt;). </li><li> Chambre de compensation: laver le crâne avec une solution saline, puis sécher avec une ampoule de lavage de l&#39;oreille. </li><li> Corrosion initiale: corroder le crâne avec le décapant pendant 10 sec. </li><li> Chambre de compensation: essuyer complètement à l&#39;écart de la gravure avec un coton-tige, puis laver la zone avec du sérum physiologique et le sécher. </li><li> Protection: pour protéger le crâne dans d&#39;autres domaines, sauf le droit tectum de corrosion excessive, couvrir ces zones de liaison de photopolymérisation et guérir par l&#39;exposition à la lumière bleue avec illuminant portable. </li><li> Corrosion complète: mettre le décapant dans la zone centrale du droit tectum nouveau pour la gravure complète pendant 2 min. Remarque: Depuis crânes de poissons plus âgés sont calcifiées, la corrosion sera plus long. </li><li> Chambre de compensation: essuyer complètement à l&#39;écart de la gravure avec un coton-tige, puis laver la zone avec du sérum physiologique et sécher à nouveau. </li><li> Ouverture Crâne: retirez soigneusement le crâne Malacic (figure 2C) avec une pince pour exposer le tectum droite (figure 2D </strong&gt;). </li><li> Chambre de compensation: essuyer le mucus et effusing de sang du trou avec des morceaux de gelfoam et le laver avec une solution saline jusqu&#39;à ce que le saignement s&#39;est arrêté. </li><li> placement de Dye: mettre le colorant (pâte tissu-étiquetage) sur l&#39;ensemble du tectum droit et le couvrir avec gelfoam. </li><li> Séparer: couvrir le trou avec une échelle stérile du même animal. </li><li> Étanchéité: Bond Place de photopolymérisation sur la balance et guérir avec la lumière bleue de nouveau pendant 40 secondes. </li><li> Revival: laver la surface de la liaison et de relancer l&#39;animal avec une solution saline. </li><li> Soins infirmiers et de l&#39;élevage: Gardez le poisson dans 28,5 ° C moyenne de l&#39;embryon (ZFIN) pendant 5 jours et nourrir 2 fois / jour. Ne pas inclure les poissons dont la teinture ne parvient pas à rester dans le tectum pendant 5 jours dans l&#39;expérience. Remarque: moyen d&#39;embryons (EM) solution est vital pour la survie des poissons et de la température est le facteur clé pour l&#39;étiquetage Dil. </li></ol> 3. Wholemounting Retina et imagerie <ol><li> Placer le poisson dans un champ sombre pendant 2 heures avant rétine quilemount. </li><li> Anesthésier le poisson avec de l&#39;eau glacée. </li><li> Percer un trou dans la marge de la cornée et de faire une oeillère en supprimant la cornée avec des ciseaux venus. </li><li> Retirez la lentille, puis rincer le centre de la rétine avec de la glace-froid 1x PBS. </li><li> Rincer l&#39;écart entre la rétine et la sclérotique avec glacée 1x PBS jusqu&#39;à ce qu&#39;aucun pigment est à gauche. </li><li> Couper le nerf optique au niveau du disque. </li><li> Gardez la couche RGC vers le haut et maintenez la rétine avec une cuillère en verre self-made (figures 3A et 3B). Un compte-gouttes peut également être utilisé pour transférer la rétine. </li><li> Mettez la rétine dans une goutte de glycérine à 30% glacé sur une lame avec la couche RGC vers le haut (Figure 3C). </li><li> Retirez la glycérine puis aplatir la rétine sur la lame de verre avant de couper rétine en quatre quadrants (figure 3D). Garder la rétine déplié avec une couche RGC hausse sera utile pour aplatir la rétine. </li><li> Goutte à goutte glacée 50% de glycérine sur til rétine pour laver les fragments de pigment. </li><li> Retirez la glycérine puis déposez 20 pi de glace 75% de glycérine sur la rétine. </li><li> Couvrir l&#39;échantillon avec 1,8 cm x 1,8 cm de lamelle carré. </li><li> Sceller la lamelle avec du vernis à ongles. Immédiatement imagerie de la rétine est recommandée. </li><li> Image non-entrelacé la rétine entière sous une lentille d&#39;objectif 20X (NA = 0,70) avec DP72 CCD (chaque image est de 1360 x 1024 pixels). Chaque champ a trois axes différents. </li><li> Étendre la profondeur de chaque champ avec l&#39;outil de &quot;profondeur de champ étendue&quot; dans le logiciel. </li><li> Monter une rétine virtuel avec la fonction de Photomerge. </li><li> Sélectionner trois domaines de 680 x 512 pixels (surface réelle est de 350 um par 264 um, de 0,092 mm 2) dans chaque orientation de la rétine pour analyser la densité moyenne des CGR (Figure 4A). </li><li> Obtenir la zone avec la fonction de mesures - créer un polygone dans l&#39;imagelogiciel. </li><li> Calculer le nombre total de CGR par l&#39;équation «nombre CGR = x destin zone&quot; 9. </li></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=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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+number+and+distribution+of+retinal+ganglion+cells+after+optic+nerve+crush+in+zebrafish.">Changes in number and distribution of retinal ganglion cells after optic nerve crush in zebrafish.</a> Shi Yan Sheng Wu Xue Bao. 35, 159-162 (2002).</li><li>Sherpa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganglion+cell+regeneration+following+whole-retina+destruction+in+zebrafish.">Ganglion cell regeneration following whole-retina destruction in zebrafish.</a> Dev Neurobiol. 68, 166-181 (2008).</li><li>Zou, S., Tian, C., Ge, S., Hu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+of+retinal+ganglion+cells+is+not+essential+to+visual+functional+recovery+after+optic+nerve+injury+in+adult+zebrafish.">Neurogenesis of retinal ganglion cells is not essential to visual functional recovery after optic nerve injury in adult zebrafish.</a> PLoS One. 8, (2013).</li><li>Choi, D., Li, D., Raisman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+retrograde+neuronal+tracers+that+label+the+rat+facial+nucleus:+a+comparison+of+Fast+Blue,+Fluoro-ruby,+Fluoro-emerald,+Fluoro-Gold+and+DiI.">Fluorescent retrograde neuronal tracers that label the rat facial nucleus: a comparison of Fast Blue, Fluoro-ruby, Fluoro-emerald, Fluoro-Gold and DiI.</a> J Neurosci Methods. 117, 167-172 (2002).</li><li>Zou, S. 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We have nothing to disclose.

Marquage rétrograde de RGC est important de rechercher la survie CGR chez les animaux mammifères, mais il n&#39;a pas été utilisée dans le poisson-zèbre. Les méthodes alternatives, coloration HE 7 et la coloration des anticorps 8, ne sont pas des normes de référence pour compter le nombre CGR, et les lignées transgéniques avec tous les CGR marquées n&#39;a pas encore été construit 12, 13. Dans cette vidéo, nous introduisons une méthode de rétrograder CGR d&#39;étiquette...

Comme le poisson zèbre adulte a une forte capacité à régénérer les axones après une blessure du nerf optique 1, une méthode appropriée de compter l&#39;ensemble CGR est essentiel d&#39;évaluer la survie et la régénération CGR 2. Basé sur les méthodes de CGR en matière d&#39;étiquetage rétrogrades dans les mammifères et les poissons rouges 3 - 5, nous avons construit la méthode d&#39;étiqueter CGR du tectum chez le poisson zèbre adulte. Pour le poisson zèbre adulte, deux problèmes techniques critiques doivent être notées: le crâne de poisson zèbre adulte est très faible 6; ils vivent dans un environnement d&#39;eau. Ici, nous traitons le crâne avec gravure qui minimise les risques associés au forage 5. Ensuite, nous fermons le trou avec liaison photopolymérisation qui améliore la survie des animaux après la chirurgie. Auparavant, plusieurs autres techniques ont été adoptées à compter le nombre CGR de façon indirecte. Coloration HE dans les sections de la rétine étiquettes tous les types de cellules dans le RGCL 7. marquage de l&#39;anticorps dans l&#39;ensemble retina, comme îlot-1, peut aussi marquer les cellules amacrines 8. Bien que l&#39;étiquette rétrograde du nerf optique peut souche étiqueter tous les CGR de la rétine, il ne peut être adopté dans le modèle de l&#39;écrasement car elle cause un préjudice supplémentaire pour le nerf optique. Profitant de l&#39;étiquette rétrograde du tectum, nous avons déniché survie CGR et la régénération du nerf optique dans l&#39;écrasement. Les résultats montrent que presque tous les CGR survécu et plus de 90% des CGR régénérés au tectum lors de la première semaine dans le modèle d&#39;écrasement 9. Dans le but d&#39;étiqueter correctement les CGR, Dil pâte a été choisi après comparaison avec plusieurs autres colorants commerciaux 10. Tout d&#39;abord, il est spécialement conçu pour le marquage in vivo de tissus. D&#39;autre part, il s&#39;agit d&#39;un colorant lipophile qui ne peut pas se diffuser dans l&#39;eau. En outre, cette fluorescence peut persister pendant une longue période qui en fait un excellent candidat pour la recherche de la survie 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

Cellules rétrogrades en matière d&#39;étiquetage de la rétine ganglionnaires (CGR) peuvent isoler CGR soma de mourir sites, il est devenu l&#39;étalon-or pour compter CGR dans la survie CGR et des expériences de régénération. De nombreuses études ont été réalisées chez les animaux mammifères à la recherche survie CGR après une blessure du nerf optique. Cependant, marquage rétrograde de RGC chez le poisson zèbre adulte n&#39;a pas encore été signalé, bien que certaines méthodes peuvent compter le nombre de cellules en couches de cellules ganglionnaires de la rétine (RGCL). Compte tenu de la petite taille du crâne de poisson zèbre adulte et le risque élevé de décès après le forage sur le crâne, nous ouvrons le crâne à l&#39;aide de gravure à l&#39;acide et sceller le trou avec un lien de photopolymérisation, ce qui pourrait améliorer de manière significative le taux de survie. Après avoir absorbé les colorants pendant 5 jours, presque tous les CGR sont étiquetés. Comme cette méthode n&#39;a pas besoin de sectionner le nerf optique, il est irremplaçable dans la recherche de la survie CGR après écrasement du nerf optique chez le poisson zèbre adulte. Ici, nous introduisonsce procédé étape par étape et de fournir des résultats représentatifs.

Comme les figures 4B-D spectacle, le nombre de cellules DiI + est de deux tiers des DAPI + cellules RGCL. Dans une rétine normale, une image de montage de l&#39;ensemble de la rétine (figure 4E) montre que DiI + CGR sont répartis sur toute la rétine, mais dans une rétine régénérée (figure 4F), comme CGR dans la zone centrale n&#39;ont pas régénéré à leur cible à la première semaine, ils ne pouvaient pas être étiquetés....

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.

Marquage rétrograde des cellules ganglionnaires de la rétine à l&#39;âge adulte de poisson zèbre avec des colorants fluorescents

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

Vidéo: 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.

L&#39;étiquetage et l&#39;imagerie des cellules dans le cerveau postérieur Zebrafish

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

Recherche

Neurosciences

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.

Une préparation isolée rétinienne pour enregistrer la réponse de Lumière génétiquement marqués cellules ganglionnaires rétiniennes

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.

Imagerie optique des neurones dans le ganglion de crabe Stomatogastric avec tension colorants sensibles

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 transfection des cellules ganglionnaires de la rétine de la souris par In vivo L&#39;électroporation

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.

Marquage fluorescent rétrograde Permet ciblée extracellulaire seule unité d&#39;enregistrement de neurones identifiés<em&gt; In 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.

Chargement rétrograde des nerfs, des tracts, et les racines spinales avec des colorants fluorescents

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.

Étude génétique de la régénération des axones avec cultivées adultes dorsaux neurones ganglion de la racine

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.

La mise en œuvre Clamp dynamique avec conductances synaptiques et artificielle chez la souris cellules ganglionnaires rétiniennes

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.

A Novel lumière paradigme de dommages pour utilisation dans les études de régénération rétinienne chez le poisson zèbre adulte

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.

L&#39;ARN monocellulaire-séquentiel des sous-ensembles définis des cellules ganglionnaires rétiniennes

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