This work was funded by the Deutsche Forschungsgemeinschaft DFG (Exc257, SFB958).

HINWEIS: Alle Tierversuche wurden an der Charité Universitätsklinik Jena oder in Übereinstimmung mit dem deutschen Tierpflege Gesetze Vermeidung jeder unnötige Leiden der Tiere behandelt. 1. Vorbereitung Solutions und Dissection Arbeitsplatz <ol><li> Lösungen HINWEIS: die folgenden Lösungen vorbereiten, bevor Dissektion des Epithels. <ol><li> Lösungen für die Dissektion Verfahren: <ol><li> Bereiten Ringerlösung (pH 7,4) mit Konzentrationen von 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl 2, 1 mM MgCl 2 und 10 mM Glucose. </li><li> Vorbereiten einer PBS - / - Lösung (pH 7,4) in Konzentrationen von 2,68 mM KCl, 1,47 mM KH 2 PO 4, 136 mM NaCl und 8,1 mM Na 2 HPO 4. </li><li> Vorbereiten einer Fixierungslösung (pH 7,2) bei Konzentrationen von 1 × PBS - / -, 0,2 mM CaCl 2, 4% Saccharose und 4% Paraformaldehyd. Saccharose in der Fixierungslösung verbessert KryoSchneiden und Pick-up von Gefrierschnitten. Lagerung bei -20 ° C. </li></ol></li><li> Lösungen für die Färbeverfahren <ol><li> Bereiten Sie eine PBS + / + Lösung mit Konzentrationen von 1 × PBS - / -, 0,48 mM MgCl 2, 0,9 mM CaCl 2. </li><li> Vorbereiten eines PBST + / + Lösung mit Konzentrationen von 1x PBS + / + und 0,1% Triton X-100. </li><li> Bereiten Sie eine Blockierungslösung mit Konzentrationen von 1x PBST + / + und 1% Gelatine. </li></ol></li></ol></li><li> Arbeitsplatz <ol><li> Für die Präparation am Arbeitsplatz, mit einem Binokular mit heller Beleuchtung sowie einen flüssigkeits Blocker Stift. </li><li> Bereiten Sie eine Petrischale mit Ringerlösung und Klebeglasplättchen gefüllt. </li><li> Besorgen Sie sich die folgenden chirurgischen Instrumenten: ein Paar chirurgische Schere mit einem scharfen und einer stumpfen Spitze, einem Paar von Feder Schere mit gerader Spitze Form, zwei Pinzette mit feinen gebogenen Spitze Form und einer Rasierklinge ( <strong&gt; 1A). </li></ol></li></ol> 2. Herstellung der Nasenscheidewand <ol><li> Haus Tiere in zugelassenen Käfigen, die regelmäßig Zugang zu Nahrung und Wasser und geeignete Tag / Nacht-Zyklus. </li><li> Führen Anästhesie in einem geschlossenen Gefäß mit Gaze mit 100% Isofluran und Monitor Analgesie durch Testen hinteren Fuß Reflexe getränkt. Seit Genickbruch kann Blut im Nasenhöhlen führen, direkt zu enthaupten narkotisierten Mäusen. </li><li> Entfernen Sie die Haut, den Knochen des gesamten Schädel und Nasen aussetzen, und wischen Sie die restliche Blut und Gewebe gründlich mit einem Papiertuch. Entfernen Sie den Unterkiefer und die Vorderzähne. </li><li> Einzuschneiden die dorsale Knochennasen bilateral in 1-2 mm Abstand parallel zu der Nahtlinie an der einen Seite der Scheidewand (medial) von dem Oberkiefer (lateral) zu trennen. Teilen Sie die Nase mit einem einzigen Schnitt. Wenn Knochenreste und Nasenmuscheln sind immer noch auf das Septum angebracht sind, entfernen Sie diese sorgfältig, um das Septum vollständig auszusetzenohne es zu berühren. </li><li> Entfernen Sie die Rückennasenbein, indem Sie den feinen gebogenen Spitze einer Zange an der dorsalen Seite der Scheidewand zwischen Septum und Nasenbein. Mit leichtem Druck auf den Knochen, nach oben schieben und abnehmen. </li><li> Um den Zugang zu den beiden septal Gewebe, einer aus jeder Kavität bieten, vorsichtig den Oberkiefer von der anderen Seite des Kopfes. Bei der Herstellung von älteren Tieren (P&gt; 28), entfernen Sie die Spitze des Stirnbeins Abdeckung der Riechkolben. </li><li> Identifizieren Sie das Riechepithel durch seine leicht gelbe Farbe (1B). Die angrenzenden Atemwegsepithel ist weiß und zeigt die Bewegung des beweglichen Zilien, die unter dem Binokular gesehen werden kann. Entlang der Grenze zwischen Geruchs- und respiratorischen Epithel Schneiden mit einem Paar von schönen Frühlings Schere. </li><li> Erweitern Sie den Schnitt und trennen das Septum von der ventralen Verbindung zum Vomers. Dann entlang der Grenze zwischen dem Septum und Lamina cribrosa des Os ethmoidale geschnittene. Das Septum ist nun vollständig von allen Verbindungen mit dem Kopf getrennt. Verwenden Sie die in 1C gezeigt Schnittpositionen. </li></ol> 3. Isolierung des olfaktorischen Epithels für Immunfärbung <ol><li> Ein Klebeglasobjektträger in eine Petrischale mit Ringerlösung gefüllt ist. Ablegen Rand der Petrischale, so dass eine Hälfte des Glas liegt in der Ringerlösung (1A). </li><li> Verwenden Sie eine Zange, sorgfältig überweisen Sie den Siebbein mit dem Riechepithel in die Petrischale. Heben Sie es ohne packte sie mit den Pinzettenspitzen. </li><li> Besorgen Sie sich die senkrechten Platte des Siebbein mit einer Zange und mit einem zweiten, um vorsichtig die Epithel von einer Seite der Scheidewand. Schieben Sie die gebogene Spitze zwischen der senkrechten Platte und Epithel, das Epithel abziehen. </li><li> Seien Sie immer sicher, dass die ciliary Seite identifizieren, für den Fall, das Epithel blättert in der Ringer solutiauf. Wenn das Epithel dreht, ist es kaum möglich, die ciliary Seite später identifizieren. Meist rollt das Epithel mit der ciliary Fläche im Inneren. </li><li> Vergleichen Sie den eingeschrieben Gewebeform mit den in 1C gezeigt Schnittpositionen. Schnappen Sie sich das Epithel an einer Stelle an der Grenze und ziehen Sie sie auf den Objektträger. Mit der Zange nicht berühren ciliary Seite zu Verletzungen des Gewebes zu vermeiden. </li><li> Drehen Sie das Septum und wiederholen Sie den Vorgang mit dem Epithel von der anderen Seite. </li><li> Trocknen Sie die Objektträger um die beiden Stücke von Riechepithel mit einem Papiertuch und umgeben das Gewebe mit einer Flüssigkeit-Blocker Stift. </li><li> Befestigen Sie das Gewebe mit 150 ul Fixierungslösung für 10 min bei RT. Bezüglich der Zilien Stabilität und Integrität, kurze Fixierzeiten für eine Vielzahl der getesteten Antikörper. Innerhalb dieser 10 Minuten Zilien sind fest, aber wahrscheinlich nicht die ganze Epithelgewebe. Somit sofortige Visualisierung unter dem Mikroskop im Anschluß an die sTaining Verfahren. Allerdings könnte Fixierungszeiten für einzelne Antikörper variieren. </li></ol> 4. Färbeprotokoll HINWEIS: Gehen Sie mit dem Gewebe sehr sorgfältig, um ciliary Strukturen der Riechzellen zu bewahren. Lösungen entfernen durch Pipettieren. Keine Lösungen nicht fallen direkt auf das Epithel, wie mechanische Kräfte könnten die feinen Flimmerhärchen stören. Führen Sie alle Schritte bei Raumtemperatur, wenn nicht anders angegeben. <ol><li> Entfernen Sie die Fixierungslösung und waschen Sie 2 mal mit PBS + / +. </li><li> Inkubieren Sie die Epithel für mindestens 1 h mit Blockierlösung. </li><li> Planen primärer Antikörperlösung durch Lösen des Antikörpers in Blockierungslösung (1: 200 für die in dieser Studie verwendeten Antikörper) und Zentrifuge 5 min bei voller Geschwindigkeit, um irgendwelche Niederschläge zu entfernen. </li><li> Inkubieren Sie die Epithel mit Antikörperlösung in einer feuchten Kammer bei 4 ° CO / N. </li><li> Wasch Epithel mit PBS + / + 5 min bei mindestens 3 mal. </li><li>Bereiten sekundäre Antikörperlösung durch Lösen des Antikörpers in Blocking-Lösung (1: 500) und Zentrifuge 5 min bei voller Geschwindigkeit. </li><li> Inkubiere das Epithel mit sekundären Antikörperlösung für 1 Stunde in einem dunklen Raum. </li><li> Wasch Epithel mit PBS + / + 5 min bei mindestens 3 mal. </li><li> Entfernen Sie die Flüssigkeit Blocker mit einer Rasierklinge oder einem Papiertuch. 5 s Waschen Sie das Epithel mit destilliertem Wasser. </li><li> Erhalten Sie die Gewebe in Antifade Montage Reagenz. HINWEIS: Die Hintergrundfluoreszenz erhöht sich schnell innerhalb von Tagen; mikroskopische Analyse spätestens 2 Tage nach der Einbettung in Eindeckmedium wird daher empfohlen. Besondere Sorgfalt muss nicht getroffen werden, um das Gewebe zu quetschen bei der Suche der richtigen Brennebene, da es schwer beschädigt werden kann die Vorbereitung werden. Aufgrund out-of-focus-Fluoreszenz sind weitere Untersuchungen mit Konfokalmikroskopie empfohlen. </li></ol>

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The authors have nothing to disclose.

The en face preparation technique described in this protocol provides a powerful tool for the detailed analysis of the olfactory system. So far, most studies characterizing the localization of signaling proteins use immunostainings of cryosections. They present a good overview of the olfactory epithelium, and protein expression in distinct cell types or regions can be easily identified. However, expression in olfactory cilia is sometimes hard to detect. Even if ciliary localization is obvious, cryosections offer...

Die Maus Riechepithel in der Nasenhöhle umfasst 6-10.000.000 bipolaren Riechzellen 1. Jedes olfaktorischen Neuronen wählt eine der 1200 Geruchsrezeptorgene zum Ausdruck. Der Nachweis von Geruchsstoffen beginnt mit der Geruchsstoff Bindung an einen Geruchsrezeptor 2, die dann aktiviert Adenylylcyclase Typ-III (ACIII) 3 über den Riech spezifischen G-Protein G &amp; agr olf 4. Der resultierende Anstieg des zyklischen Adenosinmonophosphat (cAMP) geöffnet ein zyklisches-Nukleotid-gesteuerten (CNG), nicht selektiven Kationenkanal nach Einströmen von Ca 2+ und Na +, und anschließend Ca 2+ -Einstrom führenden führt zum Öffnen eines Ca 2+ aktivierten Cl - Kanal 5,6. Die resultierende äußere Cl - Fluß wird durch eine hohe intrazelluläre Cl erleichtert - Aufnahme, wahrscheinlich über die Na + / K + / Cl - - ​​Cotransporter NKCC1, die Konzentration von stationären Cl beibehaltenCl - / HCO 3 - Tauscher SLC4A1, und vielleicht weitere noch zu identifizierenden Transporter 6-8. Bipolare olfaktorischen Neuronen einzigen, unverzweigte Axone, die direkt an den Riechkolben ragen und Dendriten, die an die Oberfläche des Epithels erstreckt und endet als spezialisierten Kompartiment, dendritischen Knopf. Von diesem Knopf, 10-30 Zilien, die eine Länge von bis zu 50 bis 60 &amp; mgr; m erreichen kann, ausgehen in den Schleim für den epithelialen Oberfläche 9. Proteine ​​der kanonischen Signaltransduktionskaskade werden hauptsächlich in der Membran dieser Zilien lokalisiert. Die erhöhte sensorische Oberfläche des Epithels verstärkt die Fähigkeit, Geruchsstoffe. Aufgrund der Dichte der sensorischen Neuronen, die sich von Zilien benachbarten dendritischen Noppen vermischen. Diese Vermischung führt zu einer zufälligen Mischung von Härchen verschiedener Neuronen exprimieren verschiedene Geruchsrezeptoren, die auf der Oberfläche des Epithels. Der Nachweis und die Zelledere Zuteilung Zilienproteine ​​die nur in einer Teilmenge von sensorischen Neuronen vorhanden sind, ist daher schwierig in Kryoschnitten. Darüber hinaus ist die genaue Lokalisierung solcher Proteine ​​entlang der Zilien kaum möglich, da Kryoschnitte sind typischerweise dünner als die durchschnittliche Länge der Wimpern. Zur Untersuchung der ciliary Lokalisierung von bisher nicht charakterisierte Membranproteine ​​in Riechzellen aktivieren optimierten wir ein eigenes Gesicht Präparationstechnik, die die detaillierte Analyse der Proteinlokalisierung in Zilien ermöglicht. Kurz gesagt wird die Maus getötet und der Kopf aufgeteilt in der Nähe der Mittellinie. Nasenmuscheln, nasal, und frontalen Knochen entfernt werden, um das Septum freizulegen. Das Septum der olfaktorischen Teil des Epithel wird durch Schneiden alle Verbindungen in der Nasenhöhle gelöst. Nachdem an der Scheidewand in eine Petrischale mit Ringerlösung gefüllt ist, wird das Epithel ab und auf eine beschichtete Glasobjektträger übertragen geschält. Nach einer kurzen FIXATIonenschritt kann Immunofärbung Verfahren durchgeführt werden, wenn die Behandlung ist so schonend wie möglich, um eine Beschädigung der zerbrechlichen Gewebe zu vermeiden. Wir zeigen die erzielbare Auflösung durch den Vergleich der Färbung von zwei verschiedenen Membranproteinen in olfaktorischen Zilien in der klassischen Gefrierschnitten und in der beschriebenen en face Vorbereitung.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spring scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">straight tip, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Surgical scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">sharp and blunt end, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fine forceps</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">curved tips, Dumont #7, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Razor blade</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">extra thin, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Binocular with illumination</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers, Stemi 2000-C, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid-blocker pen</td>
			<td style="width:127px;">Science Services</td>
			<td style="width:119px;">N71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polysine coated slides</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:119px;">J2800AMNZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Confocal microscope</td>
			<td style="width:127px;">Leica Microsystems</td>
			<td style="width:119px;">TCS SPE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Goat anti-Kirrel2</td>
			<td style="width:127px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2930</td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Rabbit anti-mOR-EG</td>
			<td style="width:127px;">Baumgart et al., 2014</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">secondary antibodies</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">A21206, A11057</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mounting medium, ProLong Gold antifade reagent</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">P36930</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma</td>
			<td align="right" style="width:119px;">441244</td>
			<td style="width:167px;">toxic, work under fume hood</td>
		</tr>
	</tbody>
</table>

whole mount labeling of cilia in the main olfactory system of mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Riechepithel en face Zubereitungen können verwendet werden, um die Lokalisierung von Proteinen in den Zilien der Sinneszellen zu untersuchen, so dass die detaillierte Untersuchung von Proteinen, deren Lokalisierung ist unklar, nach Analyse von Gefrierschnitten werden. Dieses Problem kann in dem Fall der Färbung für Kin IRRE-like protein 2 (Kirrel2) beispielhaft dargestellt werden. Kirrel2 (auch genannt Neph3) ist ein Mitglied der Immunoglobulin (Ig) Superfamilie von Membranproteinen und fungiert als homophil...

Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Ganze Berge Kennzeichnung von Cilia im Hauptriechsystem der Mäuse

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

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JoVE Journal

Neuroscience

11.0K Aufrufe.  Universitaetsklinikum Jena. Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Serie: Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualisierung des embryonalen Nervensystems in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

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Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Flash-Photolyse von Caged Compounds in den Zilien der Riechzellen

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DiI-Markierung von DRG-Neuronen zu Study Axonal Branching in einer Whole Mount Vorbereitung von embryonalen Maus-Spinal Cord

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Beschriften einzelner Zellen im zentralen Nervensystem<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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

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

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

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

Ganze Berge Immunfluoreszenzfärbung der Neonatal Maus Retina zu untersuchen Angiogenese<em&gt; In vivo</em

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

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

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

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

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

Zerlegung des Transversus Abdominis Muskels für die neuromuskuläre Junction-Analyse

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

Ganze Berge Immunmarkierung der olfaktorischen Rezeptorneuronen in der<em&gt; Drosophila</em&gt; Antennen

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

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Live-Imaging des Ependymale Cilia in der Seitenventrikel des Maus-Gehirn-

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

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

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

Ganze Berge Dissection und Immunfluoreszenz der erwachsenen Maus Cochlea

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

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

HSV-Mediated Transgene Expression von chimären Konstrukten Behavioral Funktion von GPCR Heteromeren bei Mäusen zu studieren

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...