RESUMEN

The molecular basis for odor perception in humans remains enigmatic because of the difficulty in studying odorant receptors (ORs) outside their native environment. Efforts toward OR expression and functional profiling have been met with limited success because of the poor efficiency of their cell surface expression in vitro. Structures protruding from the surface of olfactory sensory neurons called cilia contain all of the components of the olfactory signal transduction machinery and can be placed in an ex vivo plate assay to rapidly measure odor-specific responses. Here, we describe an approach using cilia isolated from the olfactory sensory neurons of mice expressing two human ORs, OR1A1 and OR5AN1, that showed 10- to 100-fold more sensitivity to ligands as compared to previous assays. A single mouse can produce enough olfactory cilia for up to 4000 384-well assay wells, and isolated cilia can be stored frozen and thus preserved. This pipeline offers a sensitive and highly scalable ex vivo odor-screening platform that has the potential to decode human olfaction.

Asunto(s)

Neuronas Receptoras Olfatorias , Receptores Odorantes , Animales , Cilios/genética , Cilios/metabolismo , Humanos , Ratones , Odorantes , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Olfato/genética

RESUMEN

The olfactory system serves a critical function as a danger detection system to trigger defense responses essential for survival. The cellular and molecular mechanisms that drive such defenses in mammals are incompletely understood. Here, we have discovered an ultrasensitive olfactory sensor for the highly poisonous bacterial metabolite hydrogen sulfide (H2S) in mice. An atypical class of sensory neurons in the main olfactory epithelium, the type B cells, is activated by both H2S and low O2. These two stimuli trigger, respectively, Cnga2- and Trpc2-signaling pathways, which operate in separate subcellular compartments, the cilia and the dendritic knob. This activation drives essential defensive responses: elevation of the stress hormone ACTH, stress-related self-grooming behavior, and conditioned place avoidance. Our findings identify a previously unknown signaling paradigm in mammalian olfaction and define type B cells as chemosensory neurons that integrate distinct danger inputs from the external environment with appropriate defense outputs.

Asunto(s)

Reacción de Fuga/fisiología , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Olfato/fisiología , Animales , Sulfuro de Hidrógeno , Ratones , Mucosa Olfatoria/citología , Neuronas Receptoras Olfatorias/citología

RESUMEN

Sensing the level of oxygen in the external and internal environments is essential for survival. Organisms have evolved multiple mechanisms to sense oxygen. No function in oxygen sensing has been attributed to any mammalian olfactory system. Here, we demonstrate that low environmental oxygen directly activates a subpopulation of sensory neurons in the mouse main olfactory epithelium. These neurons express the soluble guanylate cyclase Gucy1b2 and the cation channel Trpc2. Low oxygen induces calcium influx in these neurons, and Gucy1b2 and Trpc2 are required for these responses. In vivo exposure of a mouse to low environmental oxygen causes Gucy1b2-dependent activation of olfactory bulb neurons in the vicinity of the glomeruli formed by axons of Gucy1b2+ sensory neurons. Low environmental oxygen also induces conditioned place aversion, for which Gucy1b2 and Trpc2 are required. We propose that this chemosensory function enables a mouse to rapidly assess the oxygen level in the external environment.

Asunto(s)

Células Quimiorreceptoras/metabolismo , Mucosa Olfatoria/metabolismo , Oxígeno/metabolismo , Guanilil Ciclasa Soluble/genética , Canales Catiónicos TRPC/genética , Animales , Ratones , Ratones Noqueados , Mutación , Mucosa Olfatoria/citología , Guanilil Ciclasa Soluble/metabolismo , Canales Catiónicos TRPC/metabolismo

RESUMEN

Homeobox genes constitute a large family of genes widely studied because of their role in the establishment of the body pattern. However, they are also involved in many other events during development and adulthood. The main olfactory epithelium (MOE) is an excellent model to study neurogenesis in the adult nervous system. Analyses of homeobox genes during development show that some of these genes are involved in the formation and establishment of cell diversity in the MOE. Moreover, the mechanisms of expression of odorant receptors (ORs) constitute one of the biggest enigmas in the field. Analyses of OR promoters revealed the presence of homeodomain binding sites in their sequences. Here we characterize the expression patterns of a set of 49 homeobox genes in the MOE with in situ hybridization. We found that seven of them (Dlx3, Dlx5, Dlx6, Msx1, Meis1, Isl1, and Pitx1) are zonally expressed. The homeobox gene Emx1 is expressed in three guanylate cyclase(+) populations, two located in the MOE and the third one in an olfactory subsystem known as Grüneberg ganglion located at the entrance of the nasal cavity. The homeobox gene Tshz1 is expressed in a unique patchy pattern across the MOE. Our findings provide new insights to guide functional studies that aim to understand the complexity of transcription factor expression and gene regulation in the MOE. J. Comp. Neurol. 524:2713-2739, 2016. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Genes Homeobox/fisiología , Mucosa Olfatoria/metabolismo , Factores de Edad , Animales , Femenino , Expresión Génica , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucosa Olfatoria/química , Mucosa Olfatoria/citología , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética

RESUMEN

The mouse olfactory mucosa is a complex chemosensory tissue composed of multiple cell types, neuronal and non-neuronal. We have here applied RNA-seq hierarchically, in three steps of decreasing cellular heterogeneity: starting with crude tissue samples dissected from the nose, proceeding to flow-cytometrically sorted pools of mature olfactory sensory neurons (OSNs), and finally arriving at single mature OSNs. We show that 98.9% of intact olfactory receptor (OR) genes are expressed in mature OSNs. We uncover a hitherto unknown bipartition among mature OSNs. We find that 19 of 21 single mature OSNs each express a single intact OR gene abundantly, consistent with the one neuron-one receptor rule. For the 9 single OSNs where the two alleles of the abundantly expressed OR gene exhibit single-nucleotide polymorphisms, we demonstrate that monoallelic expression of the abundantly expressed OR gene is extremely tight. The remaining two single mature OSNs lack OR gene expression but express Trpc2 and Gucy1b2. We establish these two cells as a neuronal cell type that is fundamentally distinct from canonical, OR-expressing OSNs and that is defined by the differential, higher expression of 55 genes. We propose this tiered experimental approach as a paradigm to unravel gene expression in other cellularly heterogeneous systems.

Asunto(s)

Neuronas Receptoras Olfatorias/fisiología , Animales , Análisis por Conglomerados , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Ratones , Mucosa Olfatoria/citología , Mucosa Olfatoria/metabolismo , Receptores Odorantes/genética , Transcriptoma

RESUMEN

Chemoreception in the mouse olfactory system occurs primarily at two chemosensory epithelia in the nasal cavity: the main olfactory epithelium (MOE) and the vomeronasal epithelium. The canonical chemosensory neurons in the MOE, the olfactory sensory neurons (OSNs), express the odorant receptor (OR) gene repertoire, and depend on Adcy3 and Cnga2 for chemosensory signal transduction. The canonical chemosensory neurons in the vomeronasal epithelium, the vomeronasal sensory neurons (VSNs), express two unrelated vomeronasal receptor (VR) gene repertoires, and involve Trpc2 for chemosensory signal transduction. Recently we reported the discovery of two types of neurons in the mouse MOE that express Trcp2 in addition to Cnga2. These cell types can be distinguished at the single-cell level by expression of Adcy3: positive, type A and negative, type B. Some type A cells express OR genes. Thus far there is no specific gene or marker for type B cells, hampering further analyses such as physiological recordings. Here, we show that among MOE cells, type B cells are unique in their expression of the soluble guanylate cyclase Gucy1b2. We came across Gucy1b2 in an explorative approach based on Long Serial Analysis of Gene Expression (LongSAGE) that we applied to single red-fluorescent cells isolated from whole olfactory mucosa and vomeronasal organ of mice of a novel Trcp2-IRES-taumCherry gene-targeted strain. The generation of a novel Gucy1b2-IRES-tauGFP gene-targeted strain enabled us to visualize coalescence of axons of type B cells into glomeruli in the main olfactory bulb. Our molecular and anatomical analyses define Gucy1b2 as a marker for type B cells within the MOE. The Gucy1b2-IRES-tauGFP strain will be useful for physiological, molecular, cellular, and anatomical studies of this newly described chemosensory subsystem.

Asunto(s)

Guanilato Ciclasa/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Canales Catiónicos TRPC/metabolismo , Secuencia de Aminoácidos , Animales , Guanilato Ciclasa/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/enzimología , Receptores Citoplasmáticos y Nucleares/genética , Guanilil Ciclasa Soluble , Canales Catiónicos TRPC/genética

RESUMEN

The mouse olfactory system contains two distinct chemosensory epithelia, the main olfactory epithelium (MOE) and the vomeronasal epithelium (VNE). Their sensory neurons express odorant receptor genes and vomeronasal receptor genes, respectively, and differ fundamentally in their signal transduction pathways. Genes required for chemosensory transduction are the cyclic nucleotide-gated channel subunit Cnga2 and the transient receptor potential cation channel Trpc2, respectively. Here, we document two previously unrecognized types of Trpc2+ neurons in the MOE of mice of various ages, including adults. These cell types express Cnga2 and can be distinguished by expression of adenylate cyclase Adcy3 (positive: type A; negative: type B). A third of MOE neurons that express the odorant receptor genes Olfr68/Olfr69 coexpress Trpc2 and are type A cells. In Trpc2-IRES-taulacZ gene-targeted mice, some labeled axons coalesce into glomeruli in the main olfactory bulb. Our findings have implications for the conventional VNE-centric interpretation of the behavioral phenotypes of Trpc2 knockout mice.

Asunto(s)

Células Quimiorreceptoras/metabolismo , Mucosa Olfatoria/metabolismo , Canales Catiónicos TRPC/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Células Quimiorreceptoras/clasificación , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mucosa Olfatoria/citología , Mucosa Olfatoria/crecimiento & desarrollo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Canales Catiónicos TRPC/genética

RESUMEN

In the mouse, mature olfactory sensory neurons (OSNs) express one allele of one of the ~1200 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express the same OR coalesce into homogeneous glomeruli at conserved positions in the olfactory bulb. ORs are involved in OR gene choice and OSN axonal wiring, but the mechanisms remain poorly understood. One approach is to substitute an OR genetically with another GPCR, and to determine in which aspects this GPCR can serve as a surrogate OR under experimental conditions. Here, we characterize a novel gene-targeted mouse strain in which the mouse ß2-adrenergic receptor (ß2AR) is coexpressed with tauGFP in OSNs that choose the OR locus M71 for expression (ß2AR→M71-GFP). By crossing these mice with ß2AR→M71-lacZ gene-targeted mice, we find that differentially tagged ß2AR→M71 alleles are expressed monoallelically. The OR coding sequence is thus not required for monoallelic expression - the expression of one of the two alleles of a given OR gene in an OSN. We detect strong ß2AR immunoreactivity in dendritic cilia of ß2AR→M71-GFP OSNs. These OSNs respond to the ß2AR agonist isoproterenol in a dose-dependent manner. Axons of ß2AR→M71-GFP OSNs coalesce into homogeneous glomeruli, and ß2AR immunoreactivity is detectable within these glomeruli. We do not find evidence for expression of endogenous ß2AR in OSNs of wild-type mice, also not in M71-expressing OSNs, and we do not observe overt differences in the olfactory system of ß2AR and ß1AR knockout mice. Our findings corroborate the experimental value of the ß2AR as a surrogate OR, including for the study of the mechanisms of monoallelic expression.

Asunto(s)

Neuronas Receptoras Olfatorias/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Odorantes/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Isoproterenol/farmacología , Ratones , Ratones Endogámicos C57BL , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/efectos de los fármacos , Receptores Adrenérgicos beta 2/genética , Receptores Odorantes/genética

RESUMEN

From the approximately 1,200 odorant receptor (OR) genes in the mouse genome, an olfactory sensory neuron is thought to express only one gene. The mechanisms of OR gene choice are not understood. A 2.1 kilobase region (the H element) adjacent to a cluster of seven OR genes has been proposed as a trans- and pan-enhancer for OR gene expression. Here, we deleted the H element by gene targeting in mice. The deletion abolishes expression of a family of three OR genes proximal to H, and H operates in cis on these genes. Deletion of H has a graded effect on expression of a distal group of four OR genes, commensurate with genomic distance. There is no demonstrable effect on expression of OR genes located outside the cluster. Our findings are not consistent with the hypothesis of H as an essential trans-acting enhancer for genome-wide regulation of OR gene expression.

Asunto(s)

Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Receptores Odorantes/genética , Animales , Axones/metabolismo , Secuencia de Bases , Cromosomas de los Mamíferos/metabolismo , Marcación de Gen , Genoma/genética , Ratones , Datos de Secuencia Molecular , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/metabolismo , Eliminación de Secuencia

RESUMEN

Odorant receptor (OR) genes can be classified into two types: fish-like class I OR genes and mammalian-specific class II OR genes. We have previously shown that Lhx2, a LIM-homeodomain protein, binds to the homeodomain site in the promoter region of mouse M71, a class II OR, and that a knockout mutation in Lhx2 precludes expression of all tested class II OR genes including M71. Here, we report that most class I OR genes, which are expressed in a dorsal region of the olfactory epithelium, are still expressed in Lhx2-deficient embryos. There are two exceptions: two class I OR genes, which are normally expressed in a more ventral region, are no longer expressed in Lhx2 mutant mice. Lhx2 is transcribed in olfactory sensory neurons irrespective of expression of class I or class II OR genes. Thus, a deficiency of Lhx2 has a differential impact on class I and class II OR gene expression.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Neuronas Receptoras Olfatorias/embriología , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/genética , Factores de Transcripción/deficiencia , Animales , Embrión de Mamíferos , Expresión Génica , Proteínas de Homeodominio/genética , Inmunohistoquímica , Hibridación in Situ , Proteínas con Homeodominio LIM , Ratones , Mucosa Olfatoria/embriología , Mucosa Olfatoria/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética

RESUMEN

Odorant identity is represented in the olfactory bulb (OB) by the glomerular activity pattern, which reflects a combination of activated odorant receptors (ORs) in the olfactory epithelium. To elucidate this neuronal circuit at the molecular level, we established a functional OR identification strategy based on glomerular activity by combining in vivo Ca(2+) imaging, retrograde dye labeling, and single-cell RT-PCR. Spatial and functional mapping of OR-defined glomeruli revealed that the glomerular positional relationship varied considerably between individual animals, resulting in different OR maps in the OB. Notably, OR-defined glomeruli exhibited different ligand spectra and far higher sensitivity compared to the in vitro pharmacological properties of corresponding ORs. Moreover, we found that the olfactory mucus was an important factor in the regulation of in vivo odorant responsiveness. Our results provide a methodology to examine in vivo glomerular responses at the receptor level and further help address the long-standing issues of olfactory sensitivity and specificity under physiological conditions.

Asunto(s)

Bulbo Olfatorio/fisiología , Vías Olfatorias/fisiología , Neuronas Receptoras Olfatorias/fisiología , Animales , Calcio/metabolismo , Línea Celular , Clonación Molecular , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Eugenol/análogos & derivados , Eugenol/farmacología , Hemiterpenos , Humanos , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucosa Nasal/fisiología , Odorantes , Bulbo Olfatorio/efectos de los fármacos , Mucosa Olfatoria/efectos de los fármacos , Mucosa Olfatoria/fisiología , Vías Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/efectos de los fármacos , Ácidos Pentanoicos/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

In mammals, conventional odorants are detected by OSNs located in the main olfactory epithelium of the nose. These neurons project their axons to glomeruli, which are specialized structures of neuropil in the olfactory bulb. Within glomeruli, axons synapse onto dendrites of projection neurons, the mitral and tufted (M/T) cells. Genetic approaches to visualize axons of OSNs expressing a given odorant receptor have proven very useful in elucidating the organization of these projections to the olfactory bulb. Much less is known about the development and connectivity of the lateral olfactory tract (LOT), which is formed by axons of M/T cells connecting the olfactory bulb to central neural regions. Here, we have extended our genetic approach to mark M/T cells of the main olfactory bulb and their axons in the mouse, by targeted insertion of IRES-tauGFP in the neurotensin locus. In NT-GFP mice, we find that M/T cells of the main olfactory bulb mature and project axons as early as embryonic day 11.5. Final innervation of central areas is accomplished before the end of the second postnatal week. M/T cell axons that originate from small defined areas within the main olfactory bulb, as visualized by localized injections of fluorescent tracers in wild-type mice at postnatal days 1 to 3, follow a dual trajectory: a branch of tightly packed axons along the dorsal aspect of the LOT, and a more diffuse branch along the ventral aspect. The dorsal, but not the ventral, subdivision of the LOT exhibits a topographical segregation of axons coming from the dorsal versus ventral main olfactory bulb. The NT-GFP mouse strain should prove useful in further studies of development and topography of the LOT, from E11.5 until 2 weeks after birth.

Asunto(s)

Proteínas Fluorescentes Verdes/metabolismo , Neurotensina/metabolismo , Bulbo Olfatorio/embriología , Bulbo Olfatorio/crecimiento & desarrollo , Vías Olfatorias/embriología , Vías Olfatorias/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismo , Neurotensina/genética , Bulbo Olfatorio/citología , Vías Olfatorias/citología , Transgenes/genética

RESUMEN

First described in 1973, the Grueneberg ganglion (GG) is an arrow-shaped neuronal structure at the anterior end of the nasal cavity. It lines both sides of the nasal septum, within the nasal vestibule, close to the opening of the naris. The functions of the GG and the pattern of projections to the brain are not known. Here, we report that neurons of the mouse GG express olfactory marker protein, which is normally expressed in mature olfactory or vomeronasal sensory neurons. The approx. 500 cells in each GG are arranged in several densely packed cell clusters. Individual cells give rise to single axons, which fasciculate to form a nerve bundle that projects caudally. The axons terminate in glomeruli of the olfactory bulb, one or two large glomeruli associated with a semicircle of up to 10 smaller, somewhat diffusely organized glomeruli that surround the most anterior part of the accessory olfactory bulb. Development of the GG starts around embryonic day 16 and appears to be completed at birth; cell numbers then undergo a minor decrease during postnatal development. The strategic location of the GG, expression of olfactory marker protein, axonal projections to glomeruli at particular locations in the olfactory bulb and early development suggest that this neuronal structure performs specific chemosensory functions at neonatal stages.

Asunto(s)

Axones/fisiología , Ganglios Sensoriales/fisiología , Bulbo Olfatorio/fisiología , Vías Olfatorias/fisiología , Animales , Carbocianinas , Recuento de Células , Células Quimiorreceptoras/fisiología , Femenino , Ganglios Sensoriales/citología , Ganglios Sensoriales/crecimiento & desarrollo , Genes Reporteros , Operón Lac , Ratones , Bulbo Olfatorio/citología , Proteína Marcadora Olfativa/metabolismo , Vías Olfatorias/citología , Vías Olfatorias/crecimiento & desarrollo , Embarazo

RESUMEN

The detection of thousands of volatile odorants is mediated by several hundreds of different G protein-coupled olfactory receptors (ORs). The main strategy in encoding odorant identities is a combinatorial receptor code scheme in that different odorants are recognized by different sets of ORs. Despite increasing information on agonist-OR combinations, little is known about the antagonism of ORs in the mammalian olfactory system. Here we show that odorants inhibit odorant responses of OR(s), evidence of antagonism between odorants at the receptor level. The antagonism was demonstrated in a heterologous OR-expression system and in single olfactory neurons that expressed a given OR, and was also visualized at the level of the olfactory epithelium. Dual functions of odorants as an agonist and an antagonist to ORs indicate a new aspect in the receptor code determination for odorant mixtures that often give rise to novel perceptual qualities that are not present in each component. The current study also provides insight into strategies to modulate perceived odorant quality.

Asunto(s)

Receptores Odorantes/antagonistas & inhibidores , Receptores Odorantes/metabolismo , Olfato/fisiología , Animales , Animales Recién Nacidos , Anisoles/farmacología , Calcio/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Electrofisiología , Eugenol/farmacología , Colorantes Fluorescentes , Humanos , Isoproterenol , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Odorantes , Mucosa Olfatoria/citología , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/efectos de los fármacos , Receptores Odorantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Safrol/farmacología , Relación Estructura-Actividad

RESUMEN

The main and accessory olfactory epithelia of the mouse are composed of many cell populations. Each sensory neuron is thought to express one allele of one of the approximately 1000 odorant or approximately 300 vomeronasal receptor genes. Sensory neurons die and are replaced by new neurons that differentiate from precursor cells throughout the lifetime of the individual. Neuronal replacement is asynchronous, resulting in the co-existence of cells at various stages of differentiation. Receptor gene diversity and ongoing neuronal differentiation produce complex mosaics of gene expression within these epithelia. Accurate description of gene expression patterns will facilitate the understanding of mechanisms of gene choice and differentiation. Here we report a detailed protocol for two- and three-color fluorescent RNA in situ hybridization (ISH) and its combination with immunohistochemistry, or detection of bromodeoxyuridine (BrdU)-incorporated DNA after labeling. The protocol is applied to cryosections of the main and accessory olfactory epithelia in mouse.

Asunto(s)

Hibridación Fluorescente in Situ/métodos , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , ARN Mensajero/análisis , Receptores Odorantes/genética , Animales , Bromodesoxiuridina , ADN/análisis , ADN/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Inmunohistoquímica/métodos , Ratones , ARN Mensajero/genética , Olfato/genética

RESUMEN

An in situ Ca2+ -imaging technique was adopted to monitor odorant responses of more than several hundreds of neurons simultaneously in an intact coronal slice of the mouse olfactory epithelium. The sensitivity and resolution of the slice Ca2+ -imaging were high enough to distinguish between olfactory receptor neurons with threshold concentrations in a one-order difference for a particular odorant at the single-cell level. Increasing odorant concentrations resulted in increases in the numbers of odorant-responsive neurons, which were visualized in situ in the coronal slice. The methodology established in this study is a powerful tool to visualize spatial distributions of odorant responsive neurons at a cellular resolution, and to construct odor maps in a coronal view of the olfactory epithelium.

Asunto(s)

Calcio/fisiología , Ácido Egtácico/análogos & derivados , Fura-2/análogos & derivados , Mucosa Olfatoria/fisiología , Neuronas Receptoras Olfatorias/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Anestésicos Locales/farmacología , Animales , Animales Recién Nacidos , Alcohol Bencilo/farmacología , Colforsina/farmacología , Diagnóstico por Imagen/métodos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ácido Egtácico/metabolismo , Fura-2/metabolismo , Heptanol/farmacología , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Odorantes , Mucosa Olfatoria/efectos de los fármacos , Neuronas Receptoras Olfatorias/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Potasio/farmacología , Estimulación Química