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 los animales procedimientos fueron manejados en la Charité o Clínica Universitaria de Jena de acuerdo con las leyes de Cuidado Animal alemanes evitando cualquier sufrimiento innecesario de los animales. 1. Soluciones Preparación y Disección del lugar de trabajo <ol><li> Soluciones NOTA: Preparar las siguientes soluciones antes de comenzar la disección del epitelio. <ol><li> Soluciones para el procedimiento de disección: <ol><li> Preparar la solución de Ringer (pH 7,4) con concentraciones de NaCl 140 mM, KCl 5 mM, HEPES 10 mM, 2 mM CaCl 2, 1 mM de MgCl 2, y glucosa 10 mM. </li><li> Preparar una PBS - / - solución (pH 7,4) con concentraciones de 2.68 mM KCl, 1.47 mM KH 2 PO 4, NaCl 136 mM, y 8,1 mM Na 2 HPO 4. </li><li> Preparar una solución de fijación (pH 7,2) con concentraciones de 1x PBS - / -, 0,2 mM CaCl 2, 4% de sacarosa, y 4% de paraformaldehído. La sacarosa en la solución de fijación mejora crioseccionamiento y recoger de cryosections. Almacenar a -20 ° C. </li></ol></li><li> Soluciones para el procedimiento de tinción <ol><li> Preparar un PBS + / + solución con concentraciones de 1x PBS - / -, 0,48 mM MgCl2, 0,9 mM CaCl2. </li><li> Preparar una PBST + / + solución con concentraciones de 1x PBS + / + y 0,1% de Triton X-100. </li><li> Preparar una solución de bloqueo con concentraciones de 1x PBST + / + y 1% de gelatina. </li></ol></li></ol></li><li> Lugar de trabajo <ol><li> Para el lugar de trabajo de disección, usar un microscopio de disección con una iluminación brillante, así como una pluma líquido-bloqueante. </li><li> Preparar una placa de Petri, lleno de solución de Ringer, y portaobjetos de vidrio adhesivas. </li><li> Obtenga los siguientes instrumentos quirúrgicos: un par de tijeras quirúrgicas con un sostenido y una punta roma, un par de tijeras de primavera con forma de punta recta, dos pinzas con forma de punta curva fina y una hoja de afeitar ( <strong&gt; Figura 1A). </li></ol></li></ol> 2. Preparación del tabique nasal <ol><li> Casa animales en jaulas aprobados con acceso regular a alimentos y agua y el ciclo día / noche apropiada. </li><li> Realizar la anestesia en un recipiente que contiene una gasa empapada cerrado con 100% de isoflurano y el monitor analgesia probando los reflejos del pie trasero. Desde dislocación cervical puede causar que la sangre en las cavidades nasales, decapitar directamente ratones anestesiados. </li><li> Quitar la piel para exponer el hueso de todo el cráneo y la nariz, y limpiar la sangre y el tejido restante a fondo usando una toalla de papel. Retire la mandíbula inferior y los dientes delanteros. </li><li> Incisión en la médula dorsal de la nariz bilateralmente en 1-2 mm de distancia paralela a la línea de sutura para separar un lado del tabique (medial) de la maxilar (lateralmente). Dividir la nariz con un solo corte. Si los restos de hueso y cornetes todavía están unidos al tabique, eliminar estas cuidadosamente para exponer el tabique completamentesin tocarlo. </li><li> Quitar el hueso nasal dorsal deslizando la punta curvada multa de una pinza a lo largo de la cara dorsal del tabique, entre el septo y el hueso nasal. Aplique una ligera presión sobre el hueso, empuje hacia arriba y retírela. </li><li> Para proporcionar acceso a los dos tejidos septales, uno de cada cavidad, retirar cuidadosamente el maxilar superior del otro lado de la cabeza. En la preparación de los animales más viejos (P&gt; 28), retire la punta del hueso frontal que cubre los bulbos olfatorios. </li><li> Identificar el epitelio olfativo por su color ligeramente amarillo (Figura 1B). El epitelio respiratorio bordeando es blanco y muestra el movimiento de los cilios móviles que puede ser vista bajo el microscopio de disección. Corte a lo largo de la frontera entre el epitelio olfativo y respiratoria con un par de tijeras finas primavera. </li><li> Extienda el corte y separar el tabique de la conexión ventral al hueso vómer. A continuación, corte a lo largo de la frontera entre el tabique y la lámina cribosa del etmoides Ose. El tabique está completamente aislado de todas las conexiones a la cabeza. Use las posiciones de corte mostradas en la Figura 1C. </li></ol> 3. El aislamiento del epitelio olfativo para Inmunoticción <ol><li> Publicar un portaobjetos de vidrio adhesivo en la placa de Petri llena con solución de Ringer. Colocarlo sobre el borde de la placa de Petri, de manera que una mitad del vidrio se encuentra en solución de Ringer (Figura 1A). </li><li> Utilice unas pinzas para transferir cuidadosamente el hueso etmoidal con el epitelio olfativo a la placa de Petri. Levante sin agarrarlo con las puntas de las pinzas. </li><li> Coge la placa perpendicular del hueso etmoides con uno fórceps y utilizar un segundo uno para quitar cuidadosamente el epitelio de un lado del tabique. Deslice la punta curvada entre la lámina perpendicular y el epitelio de pelar el epitelio apagado. </li><li> Sé siempre asegúrese de identificar el lado ciliar, en caso de que el epitelio voltea en soluti de Ringeren. Si el epitelio voltea, no es posible identificar el lado ciliar después. Sobre todo, el epitelio se enrolla con la superficie ciliar dentro. </li><li> Comparar la forma de tejido inscrito con las posiciones de corte mostrado en la Figura 1C. Coge el epitelio en una posición en la frontera y tire de ella en el portaobjetos de vidrio. No toque el lado ciliar con las pinzas para evitar lesiones del tejido. </li><li> Girar el septum y repetir el procedimiento con el epitelio desde el otro lado. </li><li> Seque el portaobjetos de vidrio alrededor de las dos piezas de epitelio olfativo con una toalla de papel y rodear el tejido con un lápiz líquido bloqueador. </li><li> Fijar el tejido con la solución de fijación 150 l durante 10 min a RT. En cuanto a la estabilidad y la integridad de los cilios, usar tiempos de fijación cortos para una variedad de anticuerpos ensayados. Dentro de estos 10 min cilios son fijos, pero probablemente no todo el tejido epitelial. Por lo tanto, visualizar inmediata con el microscopio después de los scontienen procedimiento. Sin embargo, los tiempos pueden variar para la adherencia de anticuerpos individuales. </li></ol> 4. Protocolo de tinción NOTA: Maneje el tejido con mucho cuidado para preservar las estructuras ciliares de las neuronas sensoriales olfativas. Retire soluciones con la pipeta. No deje caer alguna solución directamente sobre el epitelio, como las fuerzas mecánicas podrían interrumpir la multa cilios. Realice todos los pasos a temperatura ambiente si no se indica lo contrario. <ol><li> Eliminar la solución de fijador y lavar 2 veces con PBS + / +. </li><li> Incubar el epitelio durante al menos 1 hora con solución de bloqueo. </li><li> Preparar la solución de anticuerpo primario mediante la disolución del anticuerpo en solución de bloqueo (1: 200 para los anticuerpos utilizados en este estudio) y centrifugar durante 5 min a velocidad máxima para eliminar cualquier precipitado. </li><li> Incubar el epitelio con una solución de anticuerpo en una cámara húmeda a 4 ° CO / N. </li><li> Epitelio lavado con PBS + / + durante 5 minutos al menos 3 veces. </li><li>Preparar la solución de anticuerpo secundario disolviendo el anticuerpo en solución de bloqueo (1: 500) y centrifugar durante 5 min a toda velocidad. </li><li> Incubar el epitelio con una solución de anticuerpo secundario durante 1 hr en una cámara oscura. </li><li> Epitelio lavado con PBS + / + durante 5 minutos al menos 3 veces. </li><li> Retire el líquido bloqueador con una hoja de afeitar o una toalla de papel. Lave el epitelio con agua destilada durante 5 s. </li><li> Preservar el tejido en antifade reactivo montaje. NOTA: La fluorescencia de fondo se incrementa rápidamente en cuestión de días; Por lo tanto, se recomienda el análisis microscópico, a más de 2 días después de la incorporación en medio de montaje. Especial cuidado se debe tener de no apretar el tejido cuando se busca el plano focal correcta, ya que puede dañar seriamente la preparación. Debido a la fluorescencia fuera de foco, se recomienda investigación adicional con microscopía 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...

El epitelio olfatorio del ratón en la cavidad nasal comprende 6-10000000 bipolares neuronas sensoriales olfativas 1. Cada neurona olfativa elige una de 1.200 genes de receptores odoríferos para la expresión. Detección de los odorantes comienza por la unión a un receptor olfativo 2, que entonces activa la adenilato ciclasa de tipo III (ACIII) 3 a través de la proteína G específica olfativa Ga olf 4 odorante. El consiguiente aumento de la adenosina monofosfato cíclico (cAMP) abre un nucleótido cíclico-cerrada (GNC), canal catiónico no selectivo que conduce a afluencia de Ca 2+ y Na +, y, posteriormente, Ca 2 + afluencia conduce a la apertura de un Cl Ca 2+ activado - canal de 5,6. El resultante hacia afuera Cl - flujo es facilitado por un alto Cl intracelular - concentración constante mantenida por Cl - absorción, probablemente a través de la Na + / K + / Cl - NKCC1 cotransportador, laCl - / HCO 3 - SLC4A1 intercambiador, y tal vez los transportistas adicionales aún por identificar 6-8. Neuronas olfativas bipolares tienen axones individuales, no ramificados que se proyectan directamente al bulbo olfatorio, y una dendrita que se extiende a la superficie del epitelio y termina como un compartimento especializado, el mando dendríticas. De este mando, 10-30 cilios, que puede alcanzar una longitud de hasta 50-60 micras, emanar en el moco que cubre la superficie epitelial 9. Las proteínas de la cascada de transducción de señal canónica se localizan principalmente en la membrana de estas cilios. El aumento de la superficie del epitelio sensorial amplifica la capacidad de detectar olores. Debido a la densidad de las neuronas sensoriales, cilios que se extiende desde las perillas dendríticas vecinos se entremezclan. Esta interrelación resultados en una mezcla aleatoria de los cilios de diferentes neuronas, que expresan diferentes tipos de receptores olfativos, en la superficie del epitelio. La detección y la célulaular asignación de las proteínas ciliares que sólo están presentes en un subconjunto de neuronas sensoriales tanto, es difícil en criosecciones. Además, la localización precisa de dichas proteínas a lo largo de los cilios es apenas posible, ya que cryosections son típicamente más delgada que la longitud media de los cilios. Para permitir la investigación de ciliar localización de las proteínas de membrana hasta ahora no caracterizados en las neuronas olfativas, hemos optimizado una técnica de preparación de la cara en la que permite el análisis detallado de la localización de proteínas en los cilios. En pocas palabras, se sacrifica el ratón y la cabeza dividió cerca de la línea media. Los cornetes, nasal, y los huesos frontales se retiran para exponer el tabique. El tabique con la parte olfativa del epitelio de revestimiento se afloja mediante la reducción de todas las conexiones a la cavidad nasal. Después de poner el tabique en una placa de Petri llena con solución de Ringer, el epitelio se despega und transferido a un portaobjetos de vidrio recubierto. Tras una breve fixatpaso de iones, procedimientos inmunotinción se puede realizar si la manipulación es tan suave como sea posible para evitar el daño del tejido frágil. Se demuestra la resolución alcanzable mediante la comparación de la tinción de dos proteínas de membrana diferentes en cilios olfatorios en cryosections clásicos y en la preparación cara es 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.

Epitelio olfatorio en los preparativos de la cara puede ser utilizado para investigar la localización de las proteínas en los cilios de las neuronas sensoriales, lo que permite la investigación detallada de las proteínas cuya localización está claro después de un análisis de cryosections. Este problema puede ser ejemplificado en el caso de la tinción de la proteína para Kin-IRRE como 2 (Kirrel2). Kirrel2 (también llamado Neph3) es un miembro de la superfamilia de proteínas y funciones de la...

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.

Todo el montaje de etiquetado de los cilios en el sistema olfativo principal de ratones

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 vistas.  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.

Simple y asistida por ordenador de pruebas olfativas para Ratones

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 ...

Investigación

Comportamiento

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.

Imágenes de detección de feromonas en una preparación de ratón Vomeronasal aguda lámina de tejido

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. ...

Neurociencias

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.

Actividades de imagen de olores provocados en el bulbo olfatorio de ratón utilizando reflexión óptica y señales de autofluorescencia

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.

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<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.

Perforado grabación de patch-clamp de ratón neuronas sensoriales olfativas en neuroepitelio Intacto: Análisis funcional de las neuronas que expresan un odorante del receptor Identificado

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.

Todo el montaje La disección y de inmunofluorescencia del Adulto ratón cóclea

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.

Imunocoloración Cuádruple del Bulbo Olfativo para la Visualización de Códigos de Identidad Molecular del Axón 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.

Una prueba objetiva y Reproducible de aprendizaje olfativo y discriminación en ratones

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 método eficaz para Deshuesado Manual Preparar Intact ratón tejido nasal con la organización anatómica Conservas

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

Biología

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 ...

Todo el montaje de etiquetado de los cilios en el sistema olfativo principal de ratones

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