In VivoIdentification of Eugenol-Responsive and Muscone-Responsive Mouse Odorant Receptors

Abstract

Our understanding of mammalian olfactory coding has been impeded by the paucity of information about the odorant receptors (ORs) that respond to a given odorant ligand in awake, freely behaving animals. Identifying the ORs that respond in vivo to a given odorant ligand from among the ϳ1100 ORs in mice is intrinsically challenging but critical for our understanding of olfactory coding at the periphery. Here, we report an in vivo assay that is based on a novel gene-targeted mouse strain, S100a5-tauGFP, in which a fluorescent reporter selectively marks olfactory sensory neurons that have been activated recently in vivo. Because each olfactory sensory neuron expresses a single OR gene, multiple ORs responding to a given odorant ligand can be identified simultaneously by capturing the population of activated olfactory sensory neurons and using expression profiling methods to screen the repertoire of mouse OR genes. We used this in vivo assay to re-identify known eugenol-and muscone-responsive mouse ORs. We identified additional ORs responsive to eugenol or muscone. Heterologous expression assays confirmed nine eugenol-responsive ORs (Olfr73, Olfr178, Olfr432, Olfr610, Olfr958, Olfr960, Olfr961, Olfr913, and Olfr1234) and four muscone-responsive ORs (Olfr74, Olfr235, Olfr816, and Olfr1440). We found that the human ortholog of Olfr235 and Olfr1440 responds to macrocyclic ketone and lactone musk odorants but not to polycyclic musk odorants or a macrocyclic diester musk odorant. This novel assay, called the Kentucky in vivo odorant ligand-receptor assay, should facilitate the in vivo identification of mouse ORs for a given odorant ligand of interest.

Implausibility of the vibrational theory of olfaction

“…S3.3). This response is consistent with a recent report (27), in which OR5AN1 was identified as a human muscone OR, based on homology to the mouse OR MOR215-1, functionally cloned from muscone-responsive glomeruli; a second report on OR5AN1 as the only functional human homolog of mouse muscone ORs in vivo (54); and a third report that only a small number of receptors are thought to be involved in sensing musk odor (55). Our screening and the following confirmation experiments did not reveal any human OR that responded to only one, two, or three of the four isotopomers of 1.…”

“…; therefore, muscone may be recognized by only a small set of ORs, including OR5AN1 in humans, and genetic variation in these receptors may cause muscone anosmia" (27). McClintock et al (54) identified five highly related ORs, including MOR215-1, that are likely to be activated by muscone in freely behaving mice, supporting multiple muscone receptors. Importantly, however, the only functional human counterpart of these ORs is OR5AN1.…”

Hedgehog signaling regulates ciliary localization of mouse odorant receptors

“…To address whether the reduction in ciliary OR accumulation affected the function of OSNs, we analyzed the S100 calcium binding protein A5 (S100A5) that is transiently expressed to high levels in odorant-stimulated OSNs and that requires intact olfactory cilia to be expressed (22,23). In concordance with reduced olfactory transduction in vismodegib-treated mice we found that the number of S100A5-positive OSNs was reduced in vismodegib-treated mice compared with controls (Fig.…”

Monoallelic Expression of Olfactory Receptors

“…Both types of ORs are expressed by the olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE), which is the largest structure of the olfactory system. Although the odorant ligands recognized by most ORs remain unknown, in vitro and in vivo studies have recently been making rapid progress toward identification of pairings of odorants and ORs (McClintock et al 2014, Nara et al 2011). Odorant–OR pairing is combinatorial: each odorant activates multiple ORs, and higher concentrations of an odorant activate additional ORs (Malnic et al 1999, McClintock et al 2014, Nara et al 2011).…”

“…Although the odorant ligands recognized by most ORs remain unknown, in vitro and in vivo studies have recently been making rapid progress toward identification of pairings of odorants and ORs (McClintock et al 2014, Nara et al 2011). Odorant–OR pairing is combinatorial: each odorant activates multiple ORs, and higher concentrations of an odorant activate additional ORs (Malnic et al 1999, McClintock et al 2014, Nara et al 2011). Most ORs appear to be tuned toward specific odorants, or odorants with a specific physicochemical feature, but some ORs are broadly tuned and recognize many, structurally diverse odorants (Grosmaitre et al 2009, Nara et al 2011).…”