Name: whole mount labeling of cilia in the main olfactory system of mice
Uploaded: 2014-12-27T14:00:00.000+00:00
Duration: 8 min 42 s
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).

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.

<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.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.1K 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

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.

Einfache und Computergestützte Riechtests für Mäuse

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 ...

Forschung

Verhalten

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 a Mouse Vomeronasal Akute Gewebeschnitt Vorbereitung

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. ...

Neurowissenschaften

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.

Imaging Gerüche hervorgerufene Aktivitäten in der Maus Riechhirn mit Optical Reflexions-und Autofluoreszenzsignale

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.

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 ...

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.

Perforated-Patch-Clamp-Aufzeichnung der Maus Riechzellen in Intact Neuroepithel: Funktionelle Analyse von Neuronen, die eine bestimmte Geruchsrezeptor

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.

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 ...

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.

Quadruple Immunfärbung der olfaktorischen Glühbirne zur Visualisierung von olfaktorischen sensorischen 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 ...

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.

Eine objektive und reproduzierbare Test der olfaktorischen Lern- und Diskriminierung bei Mäusen

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.

Eine effektive Methode, um Manuelle Zerlege Intact Maus Nasengewebe mit erhaltener Anatomische Organisation vorbereiten

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

Biologie

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.

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

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.

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

Ganze Berge Kennzeichnung von Cilia im Hauptriechsystem der Mäuse

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Perforated-Patch-Clamp-Aufzeichnung der Maus Riechzellen in Intact Neuroepithel: Funktionelle Analyse von Neuronen, die eine bestimmte Geruchsrezeptor

Ganze Berge Dissection und Immunfluoreszenz der erwachsenen Maus Cochlea

Quadruple Immunfärbung der olfaktorischen Glühbirne zur Visualisierung von olfaktorischen sensorischen Axon Molecular Identity Codes

Eine objektive und reproduzierbare Test der olfaktorischen Lern- und Diskriminierung bei Mäusen

Eine effektive Methode, um Manuelle Zerlege Intact Maus Nasengewebe mit erhaltener Anatomische Organisation vorbereiten

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