Olfactory systems have evolved the extraordinary capability to detect and discriminate volatile odorous molecules (odorants) in the environment. Fundamentally, this process relies on the interaction of odorants and their cognate olfactory receptors (ORs) encoded in the genome. Here, we conducted a cell-based screen using over 800 mouse ORs against seven odorants, resulting in the identification of a set of high-affinity and/or broadly-tuned ORs. We then test whether heterologously expressed ORs respond to odors presented in vapor phase by individually expressing 31 ORs to measure cAMP responses against vapor phase odor stimulation. Comparison of response profiles demonstrates this platform is capable of discriminating between structural analogs. Lastly, co-expression of carboxyl esterase Ces1d expressed in olfactory mucosa resulted in marked changes in activation of specific odorant-OR combinations. Altogether, these results establish a cell-based volatile odor detection and discrimination platform and form the basis for an OR-based volatile odor sensor.
Understanding how genes and experiences work in concert to generate phenotypic variability will provide a better understanding of individuality. Here, we considered this in the main olfactory epithelium, a chemosensory structure with over a thousand distinct cell types in mice. We identified a subpopulation of olfactory sensory neurons, defined by receptor expression, whose abundances were sexually dimorphic. This subpopulation of olfactory sensory neurons was over-represented in sex-separated mice and robustly responsive to sex-specific semiochemicals. Sex-combined housing led to an attenuation of the dimorphic representations. Single-cell sequencing analysis revealed an axis of activity-dependent gene expression amongst a subset of the dimorphic OSN populations. Finally, the pro-apoptotic gene Bax is necessary to generate the dimorphic representations. Altogether, our results suggest a role of experience and activity in influencing homeostatic mechanisms to generate a robust sexually dimorphic phenotype in the main olfactory epithelium.
A fundamental challenge in studying principles of organization used by the olfactory system to encode odor concentration information has been identifying comprehensive sets of activated odorant receptors (ORs) across a broad concentration range inside freely behaving animals. In mammals, this has recently become feasible with high-throughput sequencing-based methods that identify populations of activated ORs in vivo. In this study, we characterized the mouse OR repertoires activated by the two odorants, acetophenone and 2,5-dihydro-2,4,5trimethylthiazoline, from 0.01% to 100% (v/v) as starting concentrations using phosphorylated ribosomal protein S6 capture followed by RNA-Seq. We found Olfr923 to be one of the most sensitive ORs that is enriched by acetophenone. Using a mouse line that genetically labels Olfr923-positive axons, we provided evidence that acetophenone activates the Olfr923 glomeruli in the olfactory bulb. Through molecular dynamics stimulations, we identified amino acid residues in the Olfr923 binding cavity that facilitate acetophenone binding. This study sheds light on the active process by which unique OR repertoires may collectively facilitate the discrimination of odorant concentrations. 4 Significance Statement The ability of animals to discriminate odors over a range of odor concentrations while recognizing concentration-invariant odor identity presents an encoding challenge for the olfactory system. To further our understanding on how animals sense odors at different concentrations, it is important to describe how odor concentration information is represented at the receptor level. Here, we establish a sensitive in vivo approach to screen populations of odorant receptors enriched in the odor-activated sensory neurons in mice. We identified comprehensive lists of enriched odorant receptors against a 10,000-fold concentration range for two odorants. Describing the concentration-dependent activation for unique populations of odorant receptors is fundamental for future studies in determining how individual odorant receptors contribute to olfactory sensitivity and odor intensity coding.
24Understanding how genes and experiences work in concert to generate phenotypic variability will 25 provide a better understanding of individuality. Here, we considered this in the context of the main 26 olfactory epithelium, a chemosensory structure with over a thousand distinct cell-types, in mice. We 27 identified a subpopulation of at least three types of olfactory sensory neurons, defined by receptor 28 expression, whose abundances were sexually dimorphic. This subpopulation of olfactory sensory 29 neurons was over-represented in sex-separated female mice and responded robustly to the male-30 specific semiochemicals 2-sec-butyl-4,5-dihydrothaizole and (methylthio)methanethiol. housing led to a robust attenuation of the female over-representation. Testing of Bax null mice 32 revealed a Bax-dependence in generating the sexual dimorphism in sex-separated mice. Altogether, 33 our results suggest a profound role of experience in influencing homeostatic neural lifespan 34 mechanisms to generate a robust sexually dimorphic phenotype in the main olfactory epithelium. a subset of ORs that exhibit sexually dimorphic expression under sex-separated conditions. In situ 61 mRNA hybridization probing for the expression of these ORs demonstrated the proportions of OSNs 62 expressing these ORs to be over-represented in female mice. Activity-dependent labeling experiments 63 further identified this subpopulation of OSNs as selective responders to odor cues generated by 64 mature male mice. Targeted screening of previously identified sex-specific and sex-enriched volatiles 65 demonstrated that this subpopulation of OSNs responded robustly to the reproductive-behavior and 66 physiology modifying semiochemicals 2-sec-butyl-4,5-dihydrothaizole (SBT) and 67 (methylthio)methanethiol (MTMT) in vivo. Finally, to test the role of experience in generating this 68 sexual dimorphism, we switched male and female mice from sex-separated conditions to sex-69 combined conditions and learned the sexual dimorphism had severely attenuated. Examination of sex-70 separated mutant mice null for the BCL2-associated X protein (Bax -/-) revealed a failure to generate 71 robust sexual dimorphisms within the whole olfactory mucosa. During the course of our investigations, 72 a report, van der Linden et al. 2018, was also published with some overlapping findings. Altogether, 73 these results suggest a link between specific olfactory experiences and OSN lifespan as a means to 74 influence sensory cell-level odor representations in the olfactory system. 75 76Results 77 Identification of sexually dimorphic ORs in the MOE 78We first performed RNA-Seq on the whole olfactory mucosa of male and female mice at various ages 79 housed under sex-separated conditions ( Figure 1A). Differential expression analysis of ORs revealed no 80 obvious sexually dimorphic OR expression at 3 weeks (weaning) age. In contrast, progressive 81 differential expression analysis of ORs at 9, 26, and 43 weeks age revealed at least three OR genes: 82Olfr910, Olfr912, and Olfr1295, to...
Olfactory receptors (ORs) constitute the largest multi-gene family in the mammalian genome, with hundreds to thousands of loci in humans and mice respectively. The rapid expansion of this massive family of genes has been generated by numerous duplication and diversification events throughout evolutionary history. This size, similarity, and diversity has made it challenging to define the principles by which ORs encode olfactory stimuli. Here, we performed a broad surveying of OR responses, using an in vivo strategy, against a diverse panel of odorants. We then used the resulting interaction profiles to uncover relationships between OR responses, odorants, odor molecular properties, and OR sequences. Our data and analyses revealed that ORs generally exhibited sparse tuning towards odorants and their molecular properties. Odor molecular property similarity between pairs of odorants was informative of odor response similarity. Finally, ORs sharing response to an odorant possessed amino acids at poorly conserved sites that exhibited both, predictive power towards odorant selectivity and convergent evolution. The localization of these residues occurred primarily at the interface of the upper halves of the transmembrane domains, implying that canonical positions govern odor selectivity across ORs. Altogether, our results provide a basis for translating odorants into receptor neuron responses for the unraveling of mammalian odor coding.
Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to "de-orphanize" these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.
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