In order to comprehend the strategy of odor encoding by odorant receptors, we isolated 2740 mouse receptor neurons from four olfactory epithelial zones and classified them in terms of their sensitivities and tuning specificities to a chiral pair of odorants, S(+)-carvone (caraway-like odor) and R(-)-carvone (spearmint-like odor). Our approach revealed that the majority of receptors at the lowest effective stimulus concentration represented the principal odor qualities characteristic of each enantiomer by means of the principal odor qualities of the odorants for which the receptors were most sensitive. The chiral-non-discriminating receptors were newly recruited 3.7 times of R(-)-carvone-sensitive receptors and totally became 2.8 times (39/14) of R(-)carvone-sensitive receptors in the subpopulations when the stimulus concentration was increased 10-fold [corrected]. More than 80% of the responsive receptors (an estimated 70 +/- alpha types) exhibited overlapping sensitivities between the enantiomers. The signals from the non-discriminating receptors may be reduced to decode the characteristic odor identity for R(-)-carvone in the brain over an adequate range of stimulus strengths. The information processing of odors appears to involve the selective weighting of the signals from the most sensitive receptors. An analysis of the overall receptor codes to carvones indicated that the system employs hierarchical receptor codes: principal odor qualities are encoded by the most sensitive receptors and lower-ranked odor qualities by less sensitive receptors.

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.

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 Ca2+-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 Ca2+ 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 Ca2+ 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.

A new method is described for isolating olfactory receptor neurons suitable for simultaneous recording of odorant responses in several cells. This method, called “tissue printing” by Cassab and Varner, was used to isolate cells for measurement of odorant-induced increases in cytosolic-free calcium concentration ([Ca2+]i) using the Ca2+ indicator dye fura-2. A large number of receptors could be isolated from a piece of olfactory epithelium (about 300 μm square), preserving their normal morphology and relative local topology to that in the intact olfactory tissue. The probability that there are one or more receptor cells with odorant-induced responses in [Ca2+]i per preparation was 4 times higher with cells isolated by the tissue printing than with those obtained by the pipetting method. The responses of 2 receptor cells separated by 28 μm in the recording chamber differed for 2 odorants: isoamyl acetate and citralva. The method was useful for isolating receptor neurons without losing their morphological features and for investigating the spatial distribution of odorant responsiveness of each receptor over the olfactory epithelium.

OLFACTORY transduction involves second messenger-related enzymes and cAMP-gated, K+ and Ca2+ channels, which are known to be regulated by Ca2+. We report here that cytosolic free Ca2+ concentration ([Ca2+]i) in olfactory receptor neuron was increased by odorants or forskolin and Ca2+ influx contributed to the adaptation. The increases in [Ca2+]i were below two to three-fold of resting level and transient for 26 s (mean value, n = 18). The increases were due to two pathways: Ca2+ influx and release. The slow increases in [Ca2+]i by forskolin resembled those by citralva. It was suggested that the responses to citralva were accompanied by increases in intracellular cAMP and Ca2+ influx or release leading to transient increases in [Ca2+]i.