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. Konstruieren Sie das Gerät Chirurgie HINWEIS: Um sicherzustellen, dass während und nach der Operation, Tropfanästhesie-Lösung Ethyl-3-Aminobenzoat methanesulfonate (MS-222 oder Tricaine) bei einem halben Konzentration (0,015%) mit einer Geschwindigkeit von 1 Tropfen / s durch den Mund des Fisches die Fische am Leben bleibt mit einem in Abbildung 1 dargestellt selbstgebauten Tropfsystem. <ol><li> Machen Sie ein Feld wie in 1A gezeigt (Länge 28 cm, Breite 10 cm, Höhe 5 cm), legte ein Schwamm (Länge 6 cm, Breite 9 cm, Höhe 4 cm) in der Mitte, und öffnen Sie ein Lücke auf sie. </li><li> Machen Sie eine glatte Fläche auf der Kugelstern Ende der Nadel mit lichthärtenden Bindung (Abbildung 1B), die dann in das Fischmaul eingefügt werden. Sicherzustellen, dass die Oberfläche der Nadel glatt an den Mund des Fisches vor Beschädigung zu schützen ist. </li><li> Fügen Sie die Hälfte Konzentration von MS-222 (linke Kammer, Länge 6 cm, Breite 10 cm, Höhe 8 cm) und Salzlösung (rechte Kammer, Länge 4 cm, Breite 10 cm, Höhe 8 cm) mit den Reservoirs, wie in 1C gezeigt. Hinweis: Frische MS-222 wird empfohlen. </li><li> Anesthetize Fisch in 0,03% MS-222 für 2 min. </li><li> Den Fisch in die Lücke der Schwamm und fixieren Sie die Fische mit zwei Paaren von Stiften, hinter der Brustflosse und neben der Kloakenporen bzw. 11 eingesetzt. </li><li> Verbinden Sie den Fisch in den Mund, MS-222 und Aufzucht Wasser mit einem T-Typ Rohr wie in 1C gezeigt. </li><li> Schalter auf dem Rohr zu dem MS-222 verbunden. </li></ol> 2. Retrograde Label-RGZ von rechts Tectum HINWEIS: Vor Beginn Desinfektion aller Geräte, die mit 70-75% Ethanol, so dass kein Ethanol bleibt. <ol><li> Rossing: die Haut über den ganzen Schädel mit sclerectome leicht entfernen (dorsale Ansicht des Schädels in den 2A und 2B gezeigt </strong&gt;). </li><li> Clearing: Waschen Sie den Schädel mit Kochsalzlösung und dann trocknen Sie es mit Ohrwasch Glühbirne. </li><li> Initial Korrosion: Korrosion der Schädel mit dem Ätzmittel für 10 Sekunden. </li><li> Clearing: vollständig abwischen das Ätzmittel mit einem Wattestäbchen und dann waschen Sie den Bereich mit Kochsalzlösung und trocknen. </li><li> Schutz: Um den Schädel in anderen Bereichen außer dem Recht Tectum von überschüssigem Korrosion zu schützen, decken diese Bereiche mit lichthärtenden Bindung und dann heilen sie durch die Einwirkung von blauem Licht mit tragbaren Leucht. </li><li> Komplette Korrosion: Legen Sie das Ätzmittel im zentralen Bereich des rechten Tectum wieder für die komplette Ätzen für 2 min. Hinweis: Da Schädel von älteren Fische sind verkalkt, wird Korrosion länger dauern. </li><li> Clearing: vollständig abwischen das Ätzmittel mit einem Wattestäbchen und dann waschen Sie den Bereich mit Kochsalzlösung und trocknen Sie es noch einmal. </li><li> Schädel Eröffnung: vorsichtig die malacic Schädel (2C) mit einer Pinzette, um die richtige Tectum (2D aussetzen </strong&gt;). </li><li> Clearing: Wischen Sie den Schleim und Blut aus dem Loch ausströmende mit Stücken von Gelschaum und waschen Sie es mit Kochsalzlösung, bis die Blutung aufgehört hat. </li><li> Dye Platzierung: setzen Sie den Farbstoff (Gewebe-Markierungspaste) auf der gesamten rechten Tectum und bedecken Sie es mit Gelschaum. </li><li> Trennung: decken Sie das Loch mit einer sterilen Skala von demselben Tier. </li><li> Dichtung: Ort, Lichthärte Bindung auf die Waage und dann heilen sie mit Blaulicht wieder für 40 Sekunden. </li><li> Revival: Waschen Sie die Oberfläche der Anleihe und beleben das Tier mit Kochsalzlösung. </li><li> Pflege und Zucht: Halten Sie den Fisch in 28,5 ° C Embryo Medium (ZFIN) für 5 Tage und Zulauf 2 mal / Tag. Sie sind nicht Fisch, dessen Farbstoff nicht in der Tectum für 5 Tage in dem Experiment bleiben. Hinweis: Embryo-Medium (EM)-Lösung ist für Fische Überleben und Temperatur ist der Schlüsselfaktor für Dil-Kennzeichnung. </li></ol> 3. Wholemounting Retina und Imaging <ol><li> Legen Sie den Fisch in einem Dunkelfeld für 2 Stunden, bevor die Netzhautlemount. </li><li> Anesthetize die Fische mit Eiswasser. </li><li> Stechen Sie ein Loch in den Rand der Hornhaut und eine Augenmuschel durch Entfernen der Hornhaut mit venus Schere. </li><li> Nehmen Sie die Linse und dann spülen Sie das Zentrum der Netzhaut mit eiskaltem 1x PBS. </li><li> Spülen Sie die Lücke zwischen Netzhaut und Lederhaut mit eiskaltem 1x PBS, bis kein Pigment übrig ist. </li><li> Schneiden Sie den Sehnerv an der Scheibe. </li><li> Halten Sie die RGC-Schicht nach oben und halten Sie die Netzhaut mit einem selbstgefertigten Glaslöffel (3A und 3B). Eine Pipette kann auch verwendet werden, um die Netzhaut übertragen werden. </li><li> Setzen Sie die Netzhaut in einem Tropfen eisigen 30% Glycerin auf einem Objektträger mit der RGC-Schicht nach oben (3C). </li><li> Entfernen Sie das Glycerin und dann flach auf die Netzhaut auf dem Glasträger vor dem Schneiden Netzhaut in vier Quadranten (Abbildung 3D). Halten Sie die Netzhaut entfaltet mit RGC Schicht nach oben hilfreich für die Abflachung der Netzhaut sein. </li><li> Drip eisigen 50% Glycerin auf Ter Netzhaut weg zu waschen die Fragmente der Pigment. </li><li> Entfernen Sie das Glycerin und dann fallen 20 ul eisigen 75% Glycerin auf der Netzhaut. </li><li> Decken Sie die Probe mit einem 1,8 cm x 1,8 cm im Quadrat Deckglas. </li><li> Deckglas mit Nagellack. Unmittelbare Abbildung der Netzhaut empfohlen. </li><li> Bild ohne Zeilensprung die ganze Netzhaut unter einem 20fach Objektiv (NA = 0,70) mit DP72 CCD (jedes Bild ist 1360 x 1024 Pixel). Jedes Feld hat drei unterschiedliche Schwerpunkte. </li><li> Verlängern Sie die Tiefe jedes Feld mit dem Instrument der &quot;erweiterten Schärfentiefe&quot; in der Software. </li><li> Montieren Sie einen virtuellen Netzhaut mit der Funktion der Photomerge. </li><li> Wählen drei Felder von 680 x 512 Pixel (tatsächliche Fläche beträgt 350 &amp;mgr; m 264 &amp;mgr; m, 0,092 mm 2) in jeder Ausrichtung der Retina durchschnittlichen Dichte von RGCs (4A) zu analysieren. </li><li> Erhalten Sie den Bereich mit der Funktion der Messungen - erstellen Sie ein Polygon-Feature im BildSoftware. </li><li> Berechnen Sie die Gesamtzahl der RGZ durch die Gleichung &quot;RGZ Nummer = Schicksal x-Bereich&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. 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. Q., 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=Using+the+optokinetic+response+to+study+visual+function+of+zebrafish.">Using the optokinetic response to study visual function of zebrafish.</a> J Vis Exp. , (2010).</li><li>Tokuoka, H., Yoshida, T., Matsuda, N., Mishina, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+by+glycogen+synthase+kinase-3beta+of+the+arborization+field+and+maturation+of+retinotectal+projection+in+zebrafish.">Regulation by glycogen synthase kinase-3beta of the arborization field and maturation of retinotectal projection in zebrafish.</a> J Neurosci. 22, 10324-10332 (2002).</li><li>Xiao, T., Roeser, T., Staub, W., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GFP-based+genetic+screen+reveals+mutations+that+disrupt+the+architecture+of+the+zebrafish+retinotectal+projection.">A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection.</a> Development. 132, 2955-2967 (2005).</li><li>Li, L., Dowling, J. 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.

Retrograde Markierung von RGCs ist wichtig, RGCs Überleben in Säugetieren zu erforschen, aber es war nicht in Zebrafisch verwendet. Die alternativen Methoden, HE-Färbung 7 und 8 Antikörper-Färbung, sind nicht Goldstandards zum Zählen RGZ Nummer und transgenen Linien mit allen RGZ markiert wurde noch nicht gebaut 12, 13 ist. In diesem Video stellen wir eine Methode, um Label RGZ in der Netzhaut des erwachsenen Zebrafisch rückläufig. Obwohl etwa 1% der Axone zu Riechkolben oder and...

Wie erwachsenen Zebrafisch haben eine starke Fähigkeit zur Axone nach Verletzung des Sehnervs 1 regenerieren, eine geeignete Methode zu zählen, die ganze RGZ ist wichtig, um das Überleben und die Regeneration RGZ 2 auszuwerten. Basierend auf den Methoden der retrograde Markierung RGZ in Säugetier-und Goldfisch 3-5 konstruierten wir die Methode, um von der RGZ Tectum in erwachsenen Zebrafisch zu beschriften. Bei erwachsenen Zebrafisch, sollten zwei kritischen technischen Belange beachtet werden: der Schädel des erwachsenen Zebrafisch ist sehr klein, 6; sie leben in einer Wasserumgebung. Hier behandeln wir den Schädel mit Ätzmittel, die die Gefahren, die mit Bohrungen 5 zugeordnet minimiert. Dann haben wir das Loch versiegeln mit lichthärtenden Bindung, die das Überleben der Tiere nach der Operation verbessert. Bisher wurden mehrere andere Techniken angenommen, um RGZ Nummer auf indirekte Weise zu zählen. HE-Färbung in der Retina Abschnitte Etiketten aller Arten von Zellen in der RGCL 7. Antikörpermarkierung in der ganzen retina wie Insel-1, können auch beschriften Amacrinzellen 8. Obwohl retrograde Label aus der Sehnerv Stumpf können alle RGZ in der Netzhaut zu kennzeichnen, kann er nicht in das Gedränge Modell übernommen werden, da sie bewirkt, dass zusätzliche Schädigung des Sehnervs. Unter Ausnutzung der retrograden Etikett von der Tectum haben wir RGZ Überleben und die Regeneration im Sehnerv verknallt erforscht. Die Ergebnisse zeigen, dass fast alle RGZ überlebt und über 90% der RGZ regeneriert, um der Tectum in der ersten Woche im Gedränge Modell 9. Um alle RGZ erfolgreich zu kennzeichnen, wurde Dil Paste nach Vergleich mit mehreren anderen Handelsfarbstoffen 10 gewählt. Erstens ist es insbesondere für in vivo Gewebemarkierungs gestalten. Zweitens ist es ein lipophiler Farbstoff, der nicht in Wasser diffundieren kann. Zusätzlich kann diese Fluoreszenz für eine lange Zeit, die es zu einem hervorragenden Kandidaten für RGCs Überleben Forschung macht anhalten.

<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

Als retrograde Markierung retinalen Ganglienzellen (RGC) kann RGZ Somata von sterbenden Websites zu isolieren, hat sich der Goldstandard für die Zählung in RGZ RGZ Überleben und Regenerationsversuche. Viele Studien haben in Säugetieren durchgeführt, um RGZ Überleben nach Verletzung des Sehnervs zu erforschen. Allerdings hat retrograde Markierung von RGC in erwachsenen Zebrafisch noch nicht berichtet worden, obwohl einige alternative Methoden können Zellzahlen in retinalen Ganglienzellschichten (RGCL) zählen. In Anbetracht der geringen Größe des erwachsenen Zebrafisch Schädel und das hohe Risiko des Todes nach dem Bohren auf dem Schädel, öffnen wir den Schädel mit Hilfe von Säure-Ätz-und versiegeln das Loch mit einem lichthärtenden Bindung, die die Überlebensrate deutlich verbessern konnte. Nach Absorption der Farbstoffe für 5 Tage, sind fast alle markierten RGCs. Da diese Methode muss nicht den Sehnerv durchschneiden, ist es in der Forschung der RGZ Überleben unersetzlich nach Sehnerv verknallt in erwachsenen Zebrafisch. Hier stellen wirdieses Verfahren Schritt für Schritt und repräsentative Ergebnisse liefern.

Wie 4B-D-Show, ist die Anzahl der DiI +-Zellen zwei Drittel der DAPI +-Zellen in RGCL. In einer normalen Netzhaut, ein Montagebild der gesamten Netzhaut (Fig. 4E) zeigt Dil + RGCs werden über die gesamte Netzhaut, sondern in einem regenerierten Netzhaut (Fig. 4F), wie RGCs im Zentralbereich nicht zu ihrem Ziel zu regenerieren verteilten In der ersten Woche, konnten sie nicht markiert werden. <p class="jove_content" fo:keep-together....

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.

Retrograde Markierung von retinalen Ganglienzellen im erwachsenen Zebrafisch mit Fluoreszenzfarbstoffen

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

Serie: 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.

Labeling und Imaging Cells in der Zebrafisch Hinterhirn

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

Forschung

Neurowissenschaften

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.

Eine isolierte Retinal Vorbereitung auf leichte Ansprache aus genetisch markierten retinalen Ganglienzellen Rekord

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.

Optical Imaging von Neuronen im Crab stomatogastrischen Ganglion mit spannungsabhängigen Farbstoffen

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.

Die Transfektion von Maus retinalen Ganglienzellen durch In vivo Elektroporation

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.

Retrograde Fluoreszenzmarkierung Ermöglicht Gezielte Extracellular Einzel-Einheit Aufnahme von identifizierten Neuronen<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.

Retrograde Loading von Nerven, Tracts, und Spinalwurzeln mit Fluoreszenzfarbstoffen

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.

Genetic Study of Axonregeneration mit kultivierten adulten Spinalganglienneuronen

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.

Implementieren Dynamische Clamp mit Synaptic und künstliche Leitfähigkeiten in Maus retinalen Ganglienzellen

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 leichte Schäden Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafisch

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.

Einzelzell-RNA-Seq von definierten Subsets von Retinal-Ganglion-Zellen

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