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><tbody><tr><td>Spring scissors</td><td></td><td></td><td>straight tip, multiple suppliers</td></tr><tr><td>Surgical scissors</td><td></td><td></td><td>sharp and blunt end, multiple suppliers</td></tr><tr><td>Fine forceps</td><td></td><td></td><td>curved tips, Dumont #7, multiple suppliers</td></tr><tr><td>Razor blade</td><td></td><td></td><td>extra thin, multiple suppliers</td></tr><tr><td>Binocular with illumination</td><td></td><td></td><td>multiple suppliers, Stemi 2000-C, Zeiss</td></tr><tr><td>Petri dish</td><td></td><td></td><td>multiple suppliers</td></tr><tr><td>Liquid-blocker pen</td><td>Science Services</td><td>N71310</td><td></td></tr><tr><td>Polysine coated slides</td><td>Thermo Scientific</td><td>J2800AMNZ</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>TCS SPE</td><td></td></tr><tr><td>primary antibody Goat anti-Kirrel2</td><td>R&amp;amp;D Systems</td><td>AF2930</td><td>1:200</td></tr><tr><td>primary antibody Rabbit anti-mOR-EG</td><td>Baumgart et al., 2014</td><td></td><td>1:200</td></tr><tr><td>secondary antibodies</td><td>Life Technologies</td><td>A21206, A11057</td><td>1:500</td></tr><tr><td>Mounting medium, ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>441244</td><td>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

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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11.1K 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

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Semplice e assistita da computer Olfattivo Test per Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

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Comportamento

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imaging Pheromone Sensing in preparazione dei tessuti Vomeronasal acuta Slice mouse

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Neuroscienze

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

Le attività di imaging Odore-Evocati nel bulbo olfattivo del mouse utilizzando riflettanza ottica e segnali autofluorescenza

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

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 ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforato Patch-clamp registrazione di topo neuroni sensoriali olfattivi In neuroepithelium intatto: Analisi funzionale di neuroni Esprimendo un Identificato odorizzante Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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 ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Quadrupla Immunostaining della lampadina olfattiva per la visualizzazione di codici di identificazione molecolare Axon sensoriale olfattiva

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Una prova oggettiva e riproducibile di apprendimento olfattivo e discriminazione nei topi

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

Un efficace manuale disosso metodo per preparare intatto il mouse tessuto nasale con organizzazione anatomica Conservato

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

Biologia

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

<meta charset="utf8"></head><body>Indagine sui neuroni sensoriali olfattivi del topo attivati tramite immunocolorazione combinata e ibridazione in situ

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Indagine sui neuroni sensoriali olfattivi del topo attivati tramite immunocolorazione combinata e ibridazione in situ

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...