The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odors. Here we used a combination of calcium imaging and single-cell RT-PCR to identify odorant receptors (ORs) for odorants with related structures but varied odors. We found that one OR recognizes multiple odorants and that one odorant is recognized by multiple ORs, but that different odorants are recognized by different combinations of ORs. Thus, the olfactory system uses a combinatorial receptor coding scheme to encode odor identities. Our studies also indicate that slight alterations in an odorant, or a change in its concentration, can change its "code," potentially explaining how such changes can alter perceived odor quality.

No abstract

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca 2+ -imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca 2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca 2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

1. Odor responses to two homologous series of n-fatty acids (nFA) and n-aliphatic alcohols (nAA) with a straight chain of three to nine carbons were examined by measuring odor-induced [Ca2+]i increase in mouse olfactory receptor neurons (ORNs) isolated by the tissue-printing method. 2. One-third of the ORNs responsive to nFA and/or nAA were alternately sensitive to either type of odorant. Their sensitivities were usually near maximal for one or two odorants and decreased with differences in the carbon chain length from the tuned odorants. 3. Two-thirds of the ORNs responsive to nFA and/or nAA were sensitive to both types of odorants. Most of them were also tuned to one or two odorants in each series with similar carbon chain lengths and showed a decrease of sensitivity with increasing stereochemical discrepancy, similar to nFA/nAA discriminating ORNs. 4. In 10 of 20 non-nFA/nAA discriminating ORNs, the sensitivity to nFA was > 10 times greater than to nAA, and 80% of them were localized in a central region of olfactory epithelium on the septum wall where ORNs preferentially project to the dorsomedial or centromedial regions of the olfactory bulb. In addition, the sensitivity to three series of n-aliphatic odorants with an added amino group was examined. Sensitivity became higher as the electronegativity of the functional groups increased, suggesting that a hydrogen bond might partly mediate affinity in one type of non-nFA/nAA discriminating ORNs. 5. The diversity in odorant tuning specificity and sensitivity of the individual ORNs indicated that their receptor sites were finely tuned to the stereochemical structures of numerous odorants by changes in the three-dimensional size and intermolecular positions of the hydrophobic domains for hydrophobic bond, as well as the proton-acceptor or donor for the hydrogen bond and the electrical charge for the ionic bond. 6. The subpopulation of ORNs tuned to an individual odorant increased as the length of carbon chain of the odorant increased from three to nine. This tendency was more marked for nFA than for nAA in the case of non-nFA/nAA discriminating ORNs. 7. Data obtained by the in vitro approach using the tissue-printing method suggested that three or more subtypes of ORNs, which were similar in some cases and significantly different in other cases, were located within close proximity to one another.

Enantiomeric pairs of mirror-image molecular structures are difficult to resolve by instrumental analyses. The human olfactory system, however, discriminates (−)-wine lactone from its (+)-form rapidly within seconds. To gain insight into receptor coding of enantiomers, we compared behavioural detection and discrimination thresholds of wild-type mice with those of ΔD mice in which all dorsal olfactory receptors are genetically ablated. Surprisingly, wild-type mice displayed an exquisite “supersensitivity” to enantiomeric pairs of wine lactones and carvones. They were capable of supersensitive discrimination of enantiomers, consistent with their high detection sensitivity. In contrast, ΔD mice showed selective major loss of sensitivity to the (+)-enantiomers. The resulting 108-fold differential sensitivity of ΔD mice to (−)- vs. (+)-wine lactone matched that observed in humans. This suggests that humans lack highly sensitive orthologous dorsal receptors for the (+)-enantiomer, similarly to ΔD mice. Moreover, ΔD mice showed >1010-fold reductions in enantiomer discrimination sensitivity compared to wild-type mice. ΔD mice detected one or both of the (−)- and (+)-enantiomers over a wide concentration range, but were unable to discriminate them. This “enantiomer odour discrimination paradox” indicates that the most sensitive dorsal receptors play a critical role in hierarchical odour coding for enantiomer identification.