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).

NOTA: Todos os procedimentos com animais foram manipulados no Charité University Clinic Jena ou de acordo com as leis Animal Care alemães evitando qualquer sofrimento desnecessário dos animais. 1. Soluções preparação e Dissection Workplace <ol><li> Soluções NOTA: Prepare as soluções a seguir antes de iniciar a dissecção do epitélio. <ol><li> Soluções para o procedimento de dissecção: <ol><li> Preparar a solução de Ringer (pH 7,4) com concentrações de 140 mM de NaCl, 5 mM de KCl, 10 mM de HEPES, 2 mM de CaCl2, 1 mM de MgCl2, e 10 mM de glucose. </li><li> Preparar uma solução de PBS - / - solução (pH 7,4) com concentrações de 2,68 mM de KCl, 1,47 mM de Na KH 2 PO 4, 136 mM de NaCl, 8,1 mM e 2 HPO 4. </li><li> Prepara-se uma solução fixadora (pH 7,2) com concentrações de 1x PBS - / -, 0,2 mM de CaCl2, 4% de sacarose, e 4% de paraformaldeído. Sacarose na solução fixadora melhora crioseccionamento e pick-up de criosecções. Armazenar a -20 ° C. </li></ol></li><li> Soluções para o procedimento de coloração <ol><li> Preparar uma solução de PBS + / + solução com concentrações de 1x PBS - / -, 0,48 mM de MgCl2, 0,9 mM de CaCl2. </li><li> Prepara-se uma PBST + / + solução com concentrações de 1x PBS + / + e 0,1% de Triton X-100. </li><li> Prepara-se uma solução de bloqueio com concentrações de 1x PBST + / + e 1% de gelatina. </li></ol></li></ol></li><li> Local de trabalho <ol><li> Para o local de trabalho dissecção, usar um microscópio de dissecação com iluminação brilhante, bem como uma caneta líquido-bloqueador. </li><li> Prepara-se uma placa de Petri, cheia com solução de Ringer, e lâminas de vidro adesivas. </li><li> Obter os seguintes instrumentos cirúrgicos: um par de tesouras cirúrgicas com uma afiada e uma ponta romba, um par de tesouras de Primavera com forma de ponta de linha reta, duas pinças com ponta curvada forma fina e uma lâmina de barbear ( <strong&gt; Figura 1A). </li></ol></li></ol> 2. Preparação do Septo nasal <ol><li> Casa animais em gaiolas aprovados, com acesso regular a alimentos e água e do ciclo de dia / noite adequado. </li><li> Realizar anestesia em um recipiente contendo gaze embebida fechado com 100% de isoflurano e monitor de analgesia, testando reflexos nos pés traseiros. Desde deslocamento cervical pode causar sangue nas cavidades nasais, decapitar diretamente ratos anestesiados. </li><li> Retire a pele para expor o osso de todo o crânio e nariz, e limpar o sangue restante e tecido, cuidadosamente, com uma toalha de papel. Remover o maxilar inferior e os dentes da frente. </li><li> Incisão no osso dorsal do nariz bilateralmente em 1-2 mm de distância paralela à linha de sutura se separar um lado do septo (medial) da maxila (lateralmente). Dividir o nariz com um único corte. Se restos de ossos e conchas ainda estão ligados ao septo, retire-os cuidadosamente para expor o septo completamentesem tocá-lo. </li><li> Remover o osso nasal dorsal deslizando a ponta curvada de uma pinça fina ao longo do lado dorsal do septo, entre o septo nasal e osso. Aplique uma leve pressão sobre o osso, empurre-o para cima e remova-o. </li><li> Para proporcionar o acesso aos dois tecidos do septo, um de cada cavidade, remover cuidadosamente a maxila do outro lado da cabeça. Ao preparar os animais mais velhos (P&gt; 28), remova a ponta do osso frontal que cobre os bulbos olfatórios. </li><li> Identificar o epitélio olfactivo pela sua cor ligeiramente amarela (Figura 1B). O epitélio respiratório na fronteira é branco e mostra o movimento dos cílios móveis que podem ser vistas sob o microscópio de dissecação. Corte ao longo da fronteira entre o epitélio olfativo e respiratória com um par de tesouras de primavera. </li><li> Estender o corte e separar o septo a partir da conexão ventral ao osso vômer. Em seguida, corte ao longo da fronteira entre o septo e lâmina cribrosa do etmoidal Ose. O septo é agora completamente isolada de todas as conexões com a cabeça. Use as posições de corte mostrado na Figura 1C. </li></ol> 3. O isolamento do Olfativo Epithelium para Immunostaining <ol><li> Coloque uma lâmina de vidro adesiva na placa de petri cheia com solução de Ringer. Colocá-lo sobre o bordo da placa de petri, de modo que uma metade do vidro encontra-se em solução (Figura 1A) da campainha. </li><li> Use uma pinça para transferir cuidadosamente o etmóide com o epitélio olfativo à placa de Petri. Levantá-lo sem agarrando-o com as pontas fórceps. </li><li> Agarrar a placa perpendicular do osso etmóide com uma pinça e usar uma segunda para remover cuidadosamente o epitélio de um lado do septo. Deslize a ponta curvada entre a placa perpendicular e epitélio de descascar o epitélio off. </li><li> Seja sempre a certeza de identificar o lado ciliar, no caso de o epitélio vira em soluti do Ringerno. Se o epitélio vira, dificilmente é possível identificar o lado ciliar depois. Principalmente, o epitélio enrola com a superfície do ciliar dentro. </li><li> Comparar a forma de tecido inscritos com as posições de corte mostrado na Figura 1C. Agarre o epitélio em uma posição na fronteira e puxe-o para a lâmina de vidro. Não toque no lado do ciliar com a pinça para evitar lesões do tecido. </li><li> Ligue o septo e repetir o procedimento com o epitélio do outro lado. </li><li> Seca-se a lâmina de vidro em torno de ambas as peças de epitélio olfactivo com uma toalha de papel e cercam o tecido com uma caneta líquido-bloqueador. </li><li> Fixar o tecido com solução fixadora 150 uL durante 10 min à TA. No que diz respeito a estabilidade e integridade dos cílios, use tempos de fixação curtos para uma variedade de anticorpos testados. Dentro destes 10 min cílios são fixos, mas provavelmente não o todo tecido epitelial. Assim, visualizar imediato com o microscópio subsequente para os stenção procedimento. No entanto, os tempos de fixador pode variar para anticorpos individuais. </li></ol> 4. Coloração Protocol NOTA: Lidar com o tecido com muito cuidado para preservar as estruturas ciliares dos neurônios olfatórios. Remover soluções por pipetagem. Não deixe cair quaisquer soluções diretamente no epitélio, como forças mecânicas poderia interromper os cílios bem. Execute todas as etapas na RT se não for indicado o contrário. <ol><li> Remover a solução fixadora e lavar 2 vezes com PBS + / +. </li><li> Incubar o epitélio durante pelo menos 1 hora com solução de bloqueio. </li><li> Preparar a solução de anticorpo primário por dissolução do anticorpo em solução (1: 200 para os anticorpos utilizados neste estudo) de bloqueio e centrifugar durante 5 minutos a toda a velocidade para remover quaisquer precipitados. </li><li> Incubar o epitélio com solução de anticorpo numa câmara húmida a 4 ° CO / N. </li><li> Lavar epitélio com PBS + / + durante 5 minutos, pelo menos, 3 vezes. </li><li>Preparar a solução de anticorpo secundário por dissolução do anticorpo em solução (1: 500) de bloqueio e centrifuga-se durante 5 minutos à velocidade máxima. </li><li> Incubar o epitélio com solução de anticorpo secundário durante 1 h numa câmara escura. </li><li> Lavar epitélio com PBS + / + durante 5 minutos, pelo menos, 3 vezes. </li><li> Retirar o líquido-bloqueador com uma lâmina de barbear ou toalha de papel. Lava-se a epitélio com água destilada durante 5 s. </li><li> Preserve o tecido em antifade reagente de montagem. NOTA: fluorescência de fundo aumenta rapidamente dentro de dias; análise microscópica, o mais tardar dois dias após a incorporação no meio de montagem é, portanto, recomendada. Cuidados especiais devem ser tomados para não apertar o tecido ao pesquisar o plano focal correto, uma vez que pode causar sérios danos à preparação. Devido a fluorescência fora de foco, é recomendável uma investigação mais aprofundada com a microscopia confocal. </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...

O epitélio olfativo do mouse na cavidade nasal compreende 6-10.000.000 neurônios olfatórios bipolares 1. Cada neurônio olfativo escolhe um dos 1.200 genes de receptores olfativos para expressão. Detecção de odorantes inicia por ligação a um receptor olfactivo 2, que, em seguida, activa adenilil ciclase do tipo III (ACIII) 3 através da proteína G específica olfactiva Gu olf 4 odorante. O aumento resultando em monofosfato de adenosina cíclico (cAMP) abre uma cíclico (CNG), canal de cátions não seletivos levando a influxo de Ca 2+ e Na +, e, posteriormente, Ca 2+ fechado de nucleotídeo influxo leva à abertura de um Cl Ca 2+ ativado - canal 5,6. O resultante exterior Cl - fluxo é facilitada por uma alta Cl intracelular - concentração mantida constante por Cl - absorção, provavelmente através de Na + / K + / Cl - NKCC1 co-transportador, oCl - / HCO3 - trocador SLC4A1, e transportadores ainda a ser identificados talvez adicionais 6-8. Neurónios olfactivos bipolares têm axónios, não ramificados individuais que se projectam directamente para o bolbo olfactivo e um dendrito que se estende para a superfície do epitélio e termina como um compartimento especializado, o botão dendrítica. A partir deste botão, 10-30 cílios, que pode atingir um comprimento de até 50-60 mm, emanam no muco que cobre a superfície epitelial 9. As proteínas da cascata de transdução de sinal canónico estão principalmente localizados na membrana destas cílios. O aumento da superfície do epitélio sensorial amplifica a capacidade de detectar odorantes. Devido à densidade de neurônios sensoriais, os cílios se estende desde maçanetas dendríticas vizinhos se misturam. Este entrelaçamento resulta numa mistura aleatória dos cílios de diferentes neurónios, que expressam diferentes tipos de receptores olfactivos, na superfície do epitélio. A célula de detecção ealocação ular de proteínas ciliares que estão presentes em apenas um subconjunto de neurônios sensoriais, portanto, difícil de criosecções. Além disso, a localização precisa de tais proteínas ao longo dos cílios é quase impossível, visto que criocortes são tipicamente mais fina do que o comprimento médio dos cílios. Para ativar a investigação da ciliar localização de proteínas de membrana até agora descaracterizados em neurônios olfativos, otimizamos um en face preparação técnica que permite a análise detalhada de localização de proteínas em cílios. Resumidamente, o rato é sacrificado ea cabeça dividida perto da linha média. Cornetos nasais, e os ossos frontais são removidos para expor o septo. O septo com a parte olfativa do epitélio de revestimento é solto por cortar todas as conexões para a cavidade nasal. Depois de colocar o septo numa placa de petri cheia com solução de Ringer, o epitélio é descascada und transferido para uma lâmina de vidro revestido. Após uma breve FIXATion passo, os procedimentos imunocoloração pode ser realizada se a manipulação é o mais suave possível para evitar danos do tecido frágil. Nós demonstramos a resolução alcançável através da comparação da coloração de duas proteínas de membrana em diferentes cílios olfactiva em criocortes clássicos e na preparação cara en descrito.

<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.

Epitélio olfactivo en preparações cara pode ser utilizado para investigar a localização de proteínas nos cílios dos neurónios sensoriais, o que permite a investigação aprofundada das proteínas cuja localização é claro após análise de criocortes. Este problema pode ser exemplificado no caso da coloração de proteína para Kin IRRE-like 2 (Kirrel2). Kirrel2 (também chamado Neph3) é um membro da superfamília de imunoglobulina (Ig) de proteínas e funções da membrana como uma proteín...

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.

Monte todo Rotulagem de Cilia na Olfativo Sistema de Ratos Principal

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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Research

JoVE Journal

Neuroscience

11.1K visualizações.  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.

Vídeo: 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.

Simples e Testes Olfativo para Ratos assistido por computador

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 ...

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.

Imagem Pheromone Sensing em um Preparação Slice rato Vomeronasal aguda do tecido

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. ...

Neurociência

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.

Atividades de imagem Odor-Evocados no bulbo olfatório do rato usando reflectância óptica e Sinais de autofluorescência

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.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

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.

Perfurado gravação patch-clamp de rato Olfactory neurônios sensoriais na Intact neuroepitélio: Análise Funcional do Neurônios Expressando uma Odorant Receptor Identificados

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.

Monte todo Dissecção e Imunofluorescência do mouse cóclea Adulto

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.

Imunossinção Quadruple da Lâmpada Olfativa para Visualização de Códigos de Identidade Molecular de Axon Sensorial Olfativo

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.

Uma objetiva e reproduzível teste de aprendizagem olfactiva e discriminação em ratos

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.

Um método eficaz para Desossa manual Prepare Intacto Rato tecido nasal com organização anatômica preservada

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>Investigação de neurônios sensoriais olfativos de camundongos ativados por meio de imunocoloração combinada e hibridização 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.

Investigação de neurônios sensoriais olfativos de camundongos ativados por meio de imunocoloração combinada e hibridização in situ

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