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 140 mM, KCl 5 mM, HEPES 10 mM, 2 mM CaCl 2, 1 mM MgCl 2, e glucosio 10 mM. </li><li> Preparare un PBS - / - Soluzione (pH 7.4) con concentrazioni di 2.68 mM KCl, 1.47 mM Na KH 2 PO 4, 136 mM NaCl, e 8,1 mm 2 HPO 4. </li><li> Preparare una soluzione di fissativo (pH 7,2) con concentrazioni di 1x PBS - / -, 0.2 mM CaCl 2, 4% di saccarosio, e 4% paraformaldeide. Saccarosio nella soluzione fissativo migliora cryosezionamento e pick-up di criosezioni. Conservare a -20 ° C. </li></ol></li><li> Soluzioni per la procedura di colorazione <ol><li> Preparare un + / + soluzione PBS con concentrazioni di 1x PBS - / -, 0.48 mM MgCl 2, 0.9 mM CaCl 2. </li><li> Preparare una PBST + / + soluzione con concentrazioni di PBS 1x + / + e 0,1% Triton X-100. </li><li> Preparare una soluzione di saturazione con concentrazioni di 1x PBST + / + e 1% di gelatina. </li></ol></li></ol></li><li> Workplace <ol><li> Per il lavoro dissezione, utilizzare un microscopio da dissezione con illuminazione brillante, così come una penna liquida-bloccante. </li><li> Preparare una capsula di Petri, riempito con soluzione di Ringer, e vetrini adesivi. </li><li> Ottenere i seguenti strumenti chirurgici: un paio di forbici chirurgiche con una forte e una punta smussata, un paio di forbici a molla di forma punta dritto, due pinze di finissima perfetta forma ricurva e una lama di rasoio ( <strong&gt; Figura 1A). </li></ol></li></ol> 2. Preparazione del setto nasale <ol><li> Animali domestici in gabbia approvati regolare accesso al cibo e all&#39;acqua e appropriato ciclo giorno / notte. </li><li> Eseguire l&#39;anestesia in un recipiente contenente una garza imbevuta chiuso con il 100% isoflurano e il monitor analgesia testando i riflessi del piede posteriore. Dal dislocazione cervicale può causare sangue nelle cavità nasali, decapitare direttamente topi anestetizzati. </li><li> Togliere la pelle per esporre l&#39;osso dell&#39;intero cranio e il naso, e spazzare via il sangue e tessuto rimanente accuratamente con un tovagliolo di carta. Rimuovere la mandibola e denti anteriori. </li><li> Incidere l&#39;osso dorsale del naso bilateralmente in 1-2 mm distanza parallela alla linea di sutura per separare una parte del setto (medialmente) dal mascellare (lateralmente). Dividere il naso con un unico taglio. Se resti ossei e turbinati sono ancora attaccati al setto, rimuoverli con cura per esporre completamente il settosenza toccarlo. </li><li> Rimuovere la dorsale osso nasale facendo scorrere la punta ricurva multa di una pinza lungo il lato dorsale del setto, tra setto e osso nasale. Applicare una leggera pressione sull&#39;osso, spingere verso l&#39;alto e rimuoverlo. </li><li> Per fornire l&#39;accesso ai due tessuti settali, uno da ciascuna cavità, rimuovere accuratamente mascella dell&#39;altro lato della testa. Quando si prepara animali più vecchi (P&gt; 28), rimuovere la punta dell&#39;osso frontale che copre i bulbi olfattivi. </li><li> Identificare l&#39;epitelio olfattivo dal suo colore leggermente giallo (Figura 1B). L&#39;epitelio respiratorio confinante è bianca e mostra il movimento delle ciglia mobili che può essere visto sotto il microscopio da dissezione. Tagliare lungo il confine tra epitelio olfattivo e respiratorio, con un paio di forbici di primavera belle. </li><li> Estendere il taglio e separare il setto dalla connessione ventrale all&#39;osso vomere. Poi tagliare lungo il confine tra il setto e la lamina cribrosa del ethmoidal Ose. Il setto è ora completamente isolato da tutti i collegamenti alla testa. Utilizzare le posizioni di taglio illustrati nella Figura 1C. </li></ol> 3. Isolamento del olfattivo Epithelium per Immunostaining <ol><li> Inserire un vetrino adesivo nella capsula di Petri riempita con soluzione di Ringer. Posizionarlo sul bordo della capsula di Petri, in modo che la metà del vetro risiede nella soluzione di Ringer (Figura 1A). </li><li> Usare una pinza per trasferire accuratamente l&#39;osso etmoide con l&#39;epitelio olfattivo alla capsula di Petri. Sollevare senza afferrare con le punte pinze. </li><li> Afferrare la piastra perpendicolare dell&#39;osso etmoide con una pinza e utilizzare un secondo per rimuovere con attenzione l&#39;epitelio da un lato del setto. Far scorrere la punta ricurva tra la piastra perpendicolari e epitelio a sbucciare la epitelio off. </li><li> Assicurarsi sempre di identificare il lato ciliare, nel caso in cui l&#39;epitelio lanci in soluti del Ringeron. Se l&#39;epitelio ribalta, è difficilmente possibile identificare il lato ciliare seguito. Per lo più, l&#39;epitelio arrotola con la superficie interna ciliare. </li><li> Confrontare la forma del tessuto iscritti con le posizioni di taglio illustrati nella Figura 1C. Prendete l&#39;epitelio in una posizione al confine e tirarlo sul vetrino. Non toccare il lato ciliare con le pinze per evitare lesioni del tessuto. </li><li> Spegnere il setto e ripetere la procedura con l&#39;epitelio dall&#39;altro lato. </li><li> Asciugare il vetrino attorno entrambi i pezzi di epitelio olfattivo con un tovagliolo di carta e circondare il tessuto con una penna liquido-bloccante. </li><li> Fissare il tessuto con 150 microlitri soluzione fissativa per 10 min a RT. Per quanto riguarda la stabilità e l&#39;integrità cilia, utilizzare tempi di fissazione brevi per una varietà di anticorpi testati. All&#39;interno di questi 10 min cilia sono fissi, ma probabilmente non l&#39;intero tessuto epiteliale. Così, immediata visualizzare con microscopio successivamente alle scontenenti procedura. Tuttavia, i tempi possono variare fissativo per gli anticorpi individuali. </li></ol> 4. Protocollo di colorazione NOTA: Maneggiare il tessuto con molta attenzione per preservare le strutture ciliari dei neuroni sensoriali olfattivi. Rimuovere soluzioni pipettando. Non far cadere alcuna soluzione direttamente sul epitelio, come forze meccaniche potrebbero comprometterne il bene cilia. Eseguire tutti i passi a RT, se non diversamente indicato. <ol><li> Rimuovere la soluzione di fissativo e lavare 2 volte con PBS + / +. </li><li> Incubare l&#39;epitelio per almeno 1 ora con soluzione bloccante. </li><li> Preparare la soluzione anticorpo primario sciogliendo l&#39;anticorpo in soluzione bloccante (1: 200 per gli anticorpi utilizzati in questo studio) e centrifugare per 5 min a piena velocità per rimuovere eventuali precipitati. </li><li> Incubare l&#39;epitelio con soluzione di anticorpi in una camera umida a 4 ° CO / N. </li><li> Lavare epitelio con PBS + / + per 5 minuti almeno 3 volte. </li><li>Preparare la soluzione di anticorpo secondario sciogliendo l&#39;anticorpo in soluzione bloccante (1: 500) e centrifugare per 5 min a piena velocità. </li><li> Incubare l&#39;epitelio con soluzione di anticorpo secondario per 1 ora in una camera oscura. </li><li> Lavare epitelio con PBS + / + per 5 minuti almeno 3 volte. </li><li> Rimuovere il liquido-bloccante con una lametta o un tovagliolo di carta. Lavare l&#39;epitelio con acqua distillata per 5 s. </li><li> Preservare il tessuto in antifade reattivo di montaggio. NOTA: aumenta rapidamente fluorescenza di fondo in pochi giorni; analisi microscopica non più tardi di due giorni dopo l&#39;incorporazione in mezzo di montaggio è quindi consigliato. Particolare cura deve essere presa non stringere il tessuto durante la ricerca sul piano focale corretto, in quanto può danneggiare gravemente il preparato. A causa di fluorescenza out-of-focus, si raccomanda ulteriori indagini al microscopio confocale. </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...

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.

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<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.

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...

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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.

Tutta Monte Etichettatura del Cilia nel Main olfattivo Sistema di Mice

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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11.0K Views.  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.

Video: 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.

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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Neuroscienze

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.

Fotolisi lampo di composti Caged nel Cilia di neuroni sensoriali olfattivi

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-Etichettatura di neuroni DRG per studiare Branching assonale in un monte intero Preparazione di Spinal Cord embrionali di topo

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.

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 ...

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.

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. ...

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.

Dissezione del muscolo Transversus Abdominis per l'analisi della giunzione neuromuscolare a montaggio intero

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.

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel<em&gt; Drosophila</em&gt; Antenna

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.

Immagini dal vivo del ependimale Cilia nei ventricoli laterali del cervello di topo

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.

Tutta Monte dissezione e immunofluorescenza del topo adulto Coclea

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-mediata Transgene Espressione di chimerici costrutti di studio comportamentale funzione di GPCR Heteromers nei topi

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