Name: whole mount labeling of cilia in the main olfactory system of mice
Uploaded: 2014-12-27T14:00:00
Description: Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

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

NOTA: Tutte le procedure di animali sono stati trattati presso la Charité o Clinica Universitaria di Jena in accordo con le leggi per la cura degli animali tedesca evitando ogni sofferenza indebita di animali. 1. Preparazione Soluzioni e Dissection Workplace <ol><li> Soluzioni NOTA: Preparare le seguenti soluzioni prima di dissezione dell&#39;epitelio. <ol><li> Soluzioni per la procedura di dissezione: <ol><li> Preparare la soluzione di Ringer (pH 7,4) con concentrazioni di NaCl 14...

<ol>
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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...

L&#39;epitelio olfattivo mouse nella cavità nasale comprende 6-10.000.000 bipolari neuroni sensoriali olfattivi 1. Ogni neurone olfattivo sceglie uno dei 1.200 geni recettore olfattivo per l&#39;espressione. Rilevazione di odoranti inizia legandosi ad un recettore olfattivo 2, che attiva ciclasi di tipo III (ACIII) 3 tramite l&#39;olfatto specifica proteina G Gα olf 4 odorizzante. L&#39;aumento conseguente adenosina monofosfato ciclico (cAMP) apre una ciclica nucleotide-gated (CNG), canale cationico non selettivo che porta a afflusso di Ca 2+ e Na +, e successivamente Ca 2+ afflusso porta all&#39;apertura di un Cl Ca 2+ attivato - Canale 5,6. La risultante partenze Cl - flusso è facilitata da un elevato intracellulare Cl - concentrazione mantenuta costante Cl - captazione, probabilmente attraverso il Na + / K + / Cl - cotransporter NKCC1, laCl - / HCO3 - scambiatore SLC4A1, e forse altri trasportatori ancora essere identificate 6-8. Neuroni olfattivi bipolari presentano singoli, assoni non ramificati che proiettano direttamente al bulbo olfattivo, e un dendrite che si estende alla superficie dell&#39;epitelio e termina in un vano specializzata, la manopola dendritica. Da questa manopola, 10-30 cilia, che può raggiungere una lunghezza di fino a 50-60 micron, emanare nel muco che copre la superficie epiteliale 9. Proteine ​​della canonica cascata di trasduzione del segnale sono localizzati prevalentemente nella membrana di questi cilia. La superficie sensoriale maggiore dell&#39;epitelio amplifica la capacità di rilevare odoranti. A causa della densità di neuroni sensoriali, cilia estendentesi da manopole dendritiche vicini mescolano. Questa compenetrazione risultati in una miscela casuale di cilia da diversi neuroni, che esprimono diversi tipi di recettori olfattivi, sulla superficie dell&#39;epitelio. La rilevazione e la cellaassegnazione lare di proteine ​​ciliari che sono presenti solo in un sottogruppo di neuroni sensoriali è quindi difficile in criosezioni. Inoltre, la localizzazione precisa di tali proteine ​​lungo la cilia è appena possibile, poiché criosezioni sono tipicamente più sottile rispetto alla lunghezza media delle ciglia. Per attivare la ricerca di localizzazione ciliare di proteine ​​di membrana finora non caratterizzate in neuroni olfattivi, abbiamo ottimizzato una tecnica di preparazione volto en, che consente l&#39;analisi dettagliata di localizzazione delle proteine ​​in cilia. In breve, il mouse è sacrificato e la testa spaccata in prossimità della linea mediana. Turbinati, nasali e ossa frontali vengono rimossi per esporre il setto. Il setto con la parte olfattiva dell&#39;epitelio rivestimento viene allentata tagliando tutte le connessioni alla cavità nasale. Dopo aver messo il setto in una capsula di Petri riempita con la soluzione di Ringer, l&#39;epitelio è staccata und trasferito ad un vetrino rivestito. A seguito di una breve fixation passo, le procedure di colorazione può essere eseguita se la gestione è più delicata possibile per evitare danni del tessuto fragile. Dimostriamo la risoluzione ottenibile confrontando la colorazione di due diverse proteine ​​di membrana in cilia olfattiva in criosezioni classici e nella preparazione en face descritto.

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			<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.

Epitelio olfattivo en preparazioni faccia può essere usato per studiare la localizzazione delle proteine ​​nella cilia di neuroni sensoriali, consentendo l&#39;indagine dettagliata di proteine ​​la cui localizzazione è chiaro dopo l&#39;analisi di criosezioni. Questo problema può essere esemplificato nel caso della colorazione per Kin di proteine ​​IRRE-like 2 (Kirrel2). Kirrel2 (chiamato anche Neph3) è un membro della immunoglobuline (Ig) superfamiglia di proteine ​​di membrana e f...

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

Tutta Monte Etichettatura del Cilia nel Main olfattivo Sistema di 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.

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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Neuroscience

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

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Visualizzazione del sistema nervoso embrionale in Whole-mount Drosophila Embrioni

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

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

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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 ...

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Etichettatura di singole cellule nel sistema nervoso centrale di<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 ...

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Tutta Monte immunofluorescenza del mouse Retina neonatale per Indagare Angiogenesi<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. ...

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

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

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

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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 ...

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