Junzo Hirono

Two types of increases in free Ca2+ evoked by odor in isolated frog olfactory receptor neurons

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.

Sensitivity-dependent Hierarchical Receptor Codes for Odors

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.

Relationship between Peripheral Receptor Code and Perceived Odor Quality

The discrimination of thousands of odorants is mediated by several hundred olfactory receptors (ORs). It is generally accepted that the main strategy in encoding odor quality is a combinatorial receptor code scheme, in which odorants are discriminated by different sets of ORs. In the present study, we classified 12 test odorants by their receptor codes and perceived odor qualities to examine whether odorants showing similar receptor codes are also similar in their odor qualities. Similarities of receptor codes between odorants were estimated by the overlapping responses of murine isolated olfactory sensory neurons. In contrast, we conducted a human sensory test to classify the test odorants according to their odor qualities. Despite the difference in species, the groupings of the test odorants were well conserved between receptor code and odor quality. These findings indicate that odorants that are discriminated by murine receptor codes are perceived as different odors by humans and further suggest that similarity of receptor codes correlates with that of odor quality, at least in our test odorants at the concentrations tested.

Architecture of odor information processing in the olfactory system

Since the discovery of the superfamily of approximately 1000 odorant receptor genes in rodents, the structural simplicity as well as the complexity of the olfactory system have been revealed. The simple aspects include the one neuron-one receptor rule and the exclusive convergence of projections from receptor neurons expressing the same receptors to one or two glomeruli in the olfactory bulb. Odor decoding in the olfactory cortex or higher cortical areas is likely to be a complicated process that depends on the sequence of signal activation and the relative signal intensities of receptors overlapping for similar but different odors. The aim of the present study was to investigate odor information processing both in receptors and in the olfactory cortex. At the receptor level, the similarity and difference in receptor codes between a pair of chiral odorants were examined using the tissue-printing method for sampling all the epithelial zones. In order to dissect odor-driven signal processing in the olfactory cortex by reducing cross-talk with the non-olfactory activities, such as cyclic respiration or other sensory inputs, an in vitro preparation of isolated whole brain with an attached nose was developed, and the methodologies and resulting hypothesis of receptor-sensitivity-dependent hierarchical odor information coding were reviewed.

Heterologous functional expression system for odorant receptors

Heterologous functional expression system for odorant receptors (ORs) is essential for investigating the structure-activity relationship (SAR) of various ligands. Different systems that coexpressed ORs with different G-protein alpha subunits (Galpha) demonstrated inconsistent effects on weak agonists and antagonists, but retained original relative sensitivities to potent agonists. In order to maintain the binding specificity of Galpha to ORs, we constructed a chimeric Galpha(15_olf), which contained the Galpha(15) sequence with the conserved C-terminal region of Galpha(olf). The Ca(2+) responses of the HEK293 cells that coexpressed OR-S6 with Galpha(15_olf) were more robust and reproducible compared to those of cells that coexpressed OR-S6 with Galpha(15). Furthermore, Galpha(15) sometimes induced unstable Ca(2+) responses that limited the accuracy of quantitative comparison of peak responses. Our results showed that a heterologous expression system that coexpressed ORs with Galpha(15_olf) and receptor transporting proteins was suitable for SAR analysis of various ligands.

Supersensitive detection and discrimination of enantiomers by dorsal olfactory receptors: evidence for hierarchical odour coding

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.

1912 Tuning specificities to various odorants in mouse olfactory receptor neurons

A. Iwama, A. Yamada, T. Kimura, E. Kono, T. Sekiguchi Oscillatory activities were studied by recordings of field potential at multiple sites in an olfactory center, the procerebral lobe, of the terrestrial slug Limaz marginatus brain. The cell mass of the procerebral lobe shows an about 1 Hz oscillation in its local fieldpotential. Multiple site-recordings showed that the terminal mass of the lobe also exhibited an oscillation synchronized with the cell mass oscillation, and that the terminal mass oscillation was decoupled from the cell mass oscillation by an application of serotonin or by a taste stimulation with quinidine sulfate to the lip. These results suggest that the terminal mass oscillators differ from the cell mass oscillators and such a novel oscillator system is localized in the terminal mass. Both types of oscillations were also modulated by an odor stimulation to the olfactory epithelium. Therefore, it is likely that a dual oscillator system in the procerebral lobe is involved in processing of olfactory information in slugs.

Study of Olfactory Transduction by Analysis of Dynamics of Odor-Induced [Ca2+]i Increases in Receptor Neurons

The regulation of odor-induced changes in cytosolic free Ca2+ ([Ca2+]i) and odor-responsiveness was studied by analyzing the dynamics of the fluorescence intensity of intracellular fura-2 in the knobs and/or somata of isolated olfactory receptor neurons. Isolated olfactory receptor cells were enzymatically obtained from the olfactory epithelia of wild bullfrog or newt anesthetized with MS-222, or from an adult female mouse anesthetized with Ketalar 50, using the tissue-printing method. Fura-2 was loaded to isolated cells by incubation with fura-2/AM solution. The measurement of [Ca2+]i was carried out at seven positions, as described in our previous reports [1,2]. Odorants dissolved in normal Ringer’s solution (NR) were applied to the cells by perfusion of the bath solution.

Optical Recordings of Calcium Responses in Neighboring Olfactory Receptors Suggest that the Distribution of Subtypes of Receptors is Heterogeneous

The cellular distribution of odor responsiveness in the olfactory epithelium is unclear. To obtain evidence for this distribution, we examined the odorant responsiveness of neighboring olfactory receptor neurons in mouse and frog. Using our new isolation method, “tissue printing” [1], we prepared samples in which many neurons were isolated while their original spatial relationship in intact tissue was retained. The enzymes used in the isolation procedure were papain (1 mg/ml, 5–10 min, for frog) and trypsin (0.025%, 12 min, for mouse). We made measurements of cytoplasmic free calcium concentration ([Ca2+]i) in isolated neurons, using the Ca2+ indicator dye, fura-2. The fluorescence intensity of fura-2 was recorded in the somata of several cells within the optical field of view (210 × 235 µm2) at \(\tfrac{1}{3}\)-s intervals. Each fluorescence intensity was the integrated value of 5 × 5 pixels (2 × 2.4 µm2 on a cell) in eight video frames (\(\tfrac{1}{{30}}\)-s intervals). The odor stimulus solutions used were: citralva (CT; 100µM), isoamyl acetate (AM; 10 mM), pyrazine (PY; 1 mM), and several fatty acids in normal Ringer’s solution. Cell viability and physiological intactness were confirmed by among others, determining the responses to high concentration potassium solution and forskolin, determining the stable resting level of [Ca2]i, and investigating cell morphology.