The beta 2-adrenergic receptor as a surrogate odorant receptor in mouse olfactory sensory neurons

In the mouse, mature olfactory sensory neurons (OSNs) express one allele of one of the similar to 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 beta 2-adrenergic receptor (beta 2AR) is coexpressed with tauGFP in OSNs that choose the OR locus M71 for expression (beta 2AR -> M71-GFP). By crossing these mice with beta 2AR -> M71-lacZ gene-targeted mice, we find that differentially tagged beta 2AR -> 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 (beta 2AR immunoreactivity in dendritic cilia of (beta 2AR -> M71-GFP OSNs. These OSNs respond to the beta 2AR agonist isoproterenol in a dose-dependent manner. Axons of beta 2AR -> M71-GFP OSNs coalesce into homogeneous glomeruli, and beta 2AR immunoreactivity is detectable within these glomeruli. We do not find evidence for expression of endogenous beta 2AR in OSNs of wild-type mice, also not in M71-expressing OSNs, and we do not observe overt differences in the olfactory system of beta 2AR and beta 1AR knockout mice. Our findings corroborate the experimental value of the beta 2AR as a surrogate OR, including for the study of the mechanisms of monoallelic expression. (C) 2013 Elsevier Inc All rights reserved

Effets de l'environnement sur la plasticité des neurones olfactifs durant le développement (étude anatomique, moléculaire, physiologique et comportementale)

Dans le but d évaluer l impact d une exposition postnatale sur une population spécifique de neurones olfactifs, nous avons exposé au Lyral des souris transgéniques dont les neurones récepteurs olfactifs MOR23 expriment la protéine fluorescente, GFP. Nous avons évalué l impact d une exposition précoce sur la population des neurones MOR23, en utilisant plusieurs méthodes. Par des observations anatomiques, nous montrons que le nombre de neurones olfactifs exprimant MOR23 diminue chez les sujets traités. Cette baisse concerne principalement les neurones matures. Ensuite, des analyses en qPCR quantitatives sur de petits échantillons de cellules isolées indiquent que les neurones MOR23 des souris exposées expriment davantage de récepteurs, de canaux couplés aux nucléotides cycliques (CNGA2) et de phosphodiestérase (PDE1C). La surexpression du récepteur MOR23 par neurone est transitoire. Enfin, cette surexpression est corrélée avec les propriétés électriques des neurones en réponse au ligand du récepteur MOR23 étudiées en patch-clamp. Afin d écarter tout effet aspécifique inhérent à une éventuelle toxicité du Lyral, nous avons réalisé des enregistrements d électroolfactogramme (EOG), une étude transcriptomique (41 gènes) et une étude d immunohistochimie sur la muqueuse olfactive montrant que l EO reste intact après exposition de trois semaines au Lyral. Nous montrons également que l effet est dépendant du couple ligand-récepteur. Enfin, par des observations comportementales, nous montrons que les souris exposées développent une préférence pour le Lyral et passent plus de temps à l explorer.Ensemble, ces résultats suggèrent que les neurones olfactifs font preuve d une certaine plasticité et sont capables de s adapter à l environnementIn order to investigate the consequences of postnatal odorant exposure on a specific population of olfactory sensory neurons (OSNs), we have taken the following experimental approach. MOR23-GFP mice were daily exposed to Lyral for 21 days starting at birth and three lines of investigations were carried out. Using anatomical analysis we observe that the density of OSNs expressing MOR23 decreases after odorant exposure. This decrease concerns primarily matures OSN (MOR23-OMP+). In order to study molecular changes within individual OSNs, mRNA levels for olfactory signaling pathway components were quantitatively analyzed using qPCR on GFP-labeled neurons (7 per mouse). mRNAs for CNGA2, PDE1C and MOR23 olfactory receptor were up-regulated in exposed mice, whereas ACIII transcript levels remained stable. This effect is not permanent: we observed an anatomical and a molecular recovery. Patch-clamp recordings on MOR23 dendritic knobs correlate with qPCR datas. To exclude any aspecific effect due to a possible Lyral toxicity we performed EOG, immunohistochemistry and qPCR on total olfactory epithelium (OE). These experiments show that 3 weeks of Lyral exposure does not damage the OE. Then qPCR on isolated cells reveals that the effect is couple ligand-receptor dependant: M71 neurons are not affected by acetophenone exposure. Finally, we performed behavioral experiments on mice from both groups. Exposed mice favored their exposure odor in olfactory preference test and spend more time exploring Lyral than non-exposed mice. These observations suggest that the environment can induce plasticity in olfactory sensory neuronsDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Postnatal odorant exposure induces peripheral olfactory plasticity at the cellular level

Mammalian olfactory sensory neurons (OSNs) form the primary elements of the olfactory system. Inserted in the olfactory mucosa lining of the nasal cavity, they are exposed to the environment and their lifespan is brief. Several reports say that OSNs are regularly regenerated during the entire life and that odorant environment affects the olfactory epithelium. However, little is known about the impact of the odorant environment on OSNs at the cellular level and more precisely in the context of early postnatal olfactory exposure. Here we exposed MOR23-green fluorescent protein (GFP) and M71-GFP mice to lyral or acetophenone, ligands for MOR23 or M71, respectively. Daily postnatal exposure to lyral induces plasticity in the population of OSNs expressing MOR23. Their density decreases after odorant exposure, whereas the amount of MOR23 mRNA and protein remain stable in the whole epithelium. Meanwhile, quantitative PCR indicates that each MOR23 neuron has higher levels of olfactory receptor transcripts and also expresses more CNGA2 and phosphodiesterase 1C, fundamental olfactory transduction pathway proteins. Transcript levels return to baseline after 4 weeks recovery. Patch-clamp recordings reveal that exposed MOR23 neurons respond to lyral with higher sensitivity and broader dynamic range while the responses' kinetics were faster. These effects are specific to the odorant-receptor pair lyral-MOR23: there was no effect of acetophenone on MOR23 neurons and no effect of acetophenone and lyral on the M71 population. Together, our results clearly demonstrate that OSNs undergo specific anatomical, molecular, and functional adaptation when chronically exposed to odorants in the early stage of life

Identification of new binding partners of the chemosensory signaling protein Gγ13 expressed in taste and olfactory sensory cells

Tastant detection in the oral cavity involves selective receptors localized at the apical extremity of a subset of specialized taste bud cells called taste receptor cells (TRCs). The identification of the genes coding for the taste receptors involved in this process have greatly improved our understanding of the molecular mechanisms underlying detection. However, how these receptors signal in TRCs, and whether the components of the signaling cascades interact with each other or are organized in complexes is mostly unexplored. Here we report on the identification of three new binding partners for the mouse G protein gamma 13 subunit (Gγ13), a component of the bitter taste receptors signaling cascade. For two of these Gγ13 associated proteins, namely GOPC and MPDZ, we describe the expression in taste bud cells for the first time. Furthermore, we demonstrate by means of a yeast two-hybrid interaction assay that the C terminal PDZ binding motif of Gγ13 interacts with selected PDZ domains in these proteins. In the case of the PDZ domain-containing protein zona occludens-1 (ZO-1), a major component of the tight junction defining the boundary between the apical and baso-lateral region of TRCs, we identified the first PDZ domain as the site of strong interaction with Gγ13. This association was further confirmed by co-immunoprecipitation experiments in HEK 293 cells. In addition, we present immunohistological data supporting partial co-localization of GOPC, MPDZ, or ZO-1, and Gγ13 in taste buds cells. Finally, we extend this observation to olfactory sensory neurons (OSNs), another type of chemosensory cells known to express both ZO-1 and Gγ13. Taken together our results implicate these new interaction partners in the sub-cellular distribution of Gγ13 in olfactory and gustatory primary sensory cells

Postnatal odorant exposure induces peripheral olfactory plasticity

Olfactory sensory neurons (OSNs) form an interface between the environment and the brain, converting chemical information (odorants) into electrical signals and sending these signals to the brain. Little is known about the consequences of long term odorant exposure on OSNs. Our goal is to understand the anatomical, molecular and physiological effects of odorant exposure at the cellular level and more precisely in the context of an early postnatal olfactory exposure. We focus our work on specific populations of OSNs expressing particular ORs using gene-targeted mice. MOR23-GFP mice were exposed daily to Lyral and anatomical, molecular and physiological properties of these neurons were analyzed. The density of MOR23 neurons decreased after odorant exposure while the level of mRNA for the receptor remained stable at the entire mucosa level. To investigate molecular changes within individual OSNs, mRNA levels for olfactory signaling pathway components were quantitatively analyzed using qPCR on GFP-containing neurons (7 per mouse). The levels of mRNAs for CNGA2, PDE1C and MOR23 olfactory receptor were higher in exposed OSNs compared to control. Using patch-clamp recordings on the dendritic knobs of MOR23 neurons in an intact preparation we observed that exposed OSNs displayed a lower detection threshold compared to control OSNs while the dynamic range of the dose-response was broader. Responses of exposed neurons were also faster and shorter than the responses of control neurons. Postnatal odorant exposure induces molecular and physiological plasticity in individual MOR23 neurons. Taken together, our data suggest that the olfactory epithelium presents deep anatomical, molecular and functional changes when chronically exposed to odorant molecules in early stage of life. Acknowledgements: Funding was provided by CNRS (ATIP grant), by Conseil Régional de Bourgogne (FABER and PARI grants), and by Université de Bourgogne (BQR program)

Odor-induced electrical and calcium signals from olfactory sensory neurons in situ

International audienceElectrophysiological recording and optical imaging enable the characterization of membrane and odorant response properties of olfactory sensory neurons (OSNs) in the nasal neuroepithelium. Here we describe a method to record the responses of mammalian OSNs to odorant stimulations in an ex vivo preparation of intact olfactory epithelium. The responses of individual OSNs with defined odorant receptor types can be monitored via patch-clamp recording or calcium imaging

Odorant response properties of septal organ neurons: broad tuning and high sensitivity

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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 a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs' coding properties and their modulation at the membrane level