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Odor Response Properties of Rat Olfactory Receptor Neurons

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Science  25 Jun 1999:
Vol. 284, Issue 5423, pp. 2171-2174
DOI: 10.1126/science.284.5423.2171

Abstract

Molecular biology studies of olfaction have identified a multigene family of molecular receptors that are likely to be involved in odor transduction mechanisms. However, because previous functional data on peripheral coding were mainly collected from inferior vertebrates, it has been difficult to document the degree of specificity of odor interaction mechanisms. As a matter of fact, studies of the functional expression of olfactory receptors have not demonstrated the low or high specificity of olfactory receptors. In this study, the selectivity of olfactory receptor neurons was investigated in the rat at the cellular level under physiological conditions by unitary extracellular recordings. Individual olfactory receptor neurons were broadly responsive to qualitatively distinct odor compounds. We conclude that peripheral coding is based on activated arrays of olfactory receptor cells with overlapping tuning profiles.

The molecular receptive range (1) of olfactory receptor neurons (ORNs) is defined by the specificity of their olfactory receptors (ORs) regarding the structure of odor molecules and by the number and diversity of ORs expressed by the same neuron. Evidence has been provided that ORNs can express only one receptor subtype (2), so that ORNs may have a narrowly tuned specificity (3, 4). Furthermore, the expression of ORs has been shown to be spatially segregated, and such an organization was proposed as defining the chemotopy of the olfactory mucosa (5). The rules governing the projection of ORNs to the olfactory bulb apparently support the spatial ORNs' segregation (6, 7). If one takes these assumptions and results literally, the qualitative discrimination of odor molecules follows the coding scheme of a “labeled line system” (8) that is an extreme version of the one ORN–one OR hypothesis (2). In contrast, if broad tuning is assigned to ORs (7), this hypothesis is consistent with functional data on the qualitative tuning of ORNs in vivo. However these data have been mainly collected in amphibians, where individual ORNs respond to structurally different odor molecules (9,10). One may envisage that there is a real gap between molecular data obtained in mammals and cellular data obtained in amphibians that may be ascribed to phylogenetic evolution, if one assumes that ORNs become more and more selective. Such an explanation is in disagreement with the broad responsivity of mammalian olfactory bulb mitral cells (11), and our knowledge of the response properties of individual ORNs in intact mammals is limited (12). Thus, the question of the range of the chemical receptive field of individual ORNs was addressed by means of classical extracellular recording techniques in anesthetized rats.

Individual ORNs were recorded in vivo in freely breathing (n = 19) or tracheotomized (n = 16) rats (13). Ninety ORNs were recorded, generally in the endoturbinate II, during periods ranging from 20 min to 2 hours. The electro-olfactogram (EOG) was simultaneously recorded as close as possible to the single-unit recording site. EOG is a transepithelial potential resulting from the summed activity of numerous ORNs, which provides direct and global information on both the intensity of the ORNs' response and the number of responding neurons. Sixteen pure odor compounds were used as stimuli. They were selected from those previously tested in the frog according to their effectiveness and their molecular structure (10). They were chosen as members of the qualitative groups established through several studies by Duchamp and collaborators and belong mainly to the terpene, camphor, aromatic, and straight-chained ketone groups (14). Ethyl vanillin that had never been tested in the frog in vivo was added to get information on the IP3 transduction pathway (15). Stimuli were odor pulses of 2-s duration delivered at 200 ml/min. They were applied directly near the surface of the turbinate with a dynamic multistage olfactometer (16), which ensured a precise control of the concentration range and allowed 12 discrete concentrations to be delivered. The compounds were delivered at concentrations ranging from saturated vapor pressure (SV) to SV/562. Depending on their SV values, the lowest and highest concentrations were between 3 × 10–8 mol/liter and 5 × 10–7 mol/liter and between 2 × 10–5 mol/liter and 3 × 10–4 mol/liter, respectively.

In rats, ORNs were spontaneously active. About 40% of them fired spontaneously at more than 100 spikes per minute, which is a high rate when compared with rates reported in amphibians (10). Furthermore, they were highly responsive. Eighty-three percent of neurons were excited by one odor at least. When all odor tests were considered (n = 540), 53.5% induced excitatory responses, 5% induced suppressive responses, and only 41.5% did not evoke a response. According to the nature of the stimulus, the percentages of excitation induced by our odor set varied from 40 to 60%. Only ethyl vanillin was clearly different, with 15%. The same cells could respond to some odors by excitation and to others by inhibition. The excitatory and inhibitory response types were not associated with peculiar odors. Nevertheless, the excitatory response type clearly predominated in peripheral odor coding, each ORN being on average excited by four odors out of our whole odor set (17). The ORNs qualitative response spectra were poorly selective. Among the ORNs tested with the whole odor set, many of them were excited by several odors and some of them were even excited by all 16 odorants. The selectivity regarding odor subset 1 (17) is illustrated in Table 1, where ORNs are distributed as a function of the numbers of odors to which they responded with excitatory responses. More than 30% of ORNs responded to the six odorants of this subset. It is important to notice that these odorants are members of four distinct qualitative groups (9, 10): Camphor for the camphor group, limonene for the terpene group, anisole and acetophenone for the aromatic group, and isoamyl acetate and methylamyl ketone for the straight-chained ketone group. Lastly, whenever a cell responded to no odor of subset 1, it never responded to any odor of the whole odor set. Taken together, the present results and previous data collected in the frog lead us to propose the odors of the subset 1 as representative of the qualitative olfactory space.

Table 1

Distribution of responding ORNs according to their selectivity to odor subset 1.

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Twenty-one cells were tested with several concentrations of each odorant, and their response thresholds were estimated. We observed that when repeated stimulation was applied, both the EOG amplitude and the single cell response patterns were reproducible (18). About 50% of ORNs were observed to reach their response thresholds at concentrations higher than SV/10 (10–6 mol/liter to 10–5 mol/liter), whereas 32% showed supraliminary responses at the lowest available concentration, SV/562 (10–7 mol/liter to 10–8 mol/liter). Some ORNs responded to different odors, with thresholds dispersed over a wide concentration range (Fig. 1), whereas others responded with thresholds that were grouped in a narrow concentration range (Fig. 2). Increasing the concentration led us to describe the dynamics of ORN functioning (Fig. 3). For most neurons, bursts became more sustained and appeared earlier with an increase of odorant concentration. The responses evoked by the highest concentrations often consisted of an early high-frequency and long-duration burst or of a decremental initial burst followed by an incremental high-frequency burst and sustained rebound. The delay between the two response phases increased with concentration. Such a relation between the response pattern and the odorant concentration suggested that ORNs did not work at saturation, but within a dynamic phase of their excitability. Simultaneous recordings by the EOG supported this assertion, because EOG amplitudes increased with concentration, mirroring the recruitment dynamics of ORNs that participated in odor coding.

Figure 1

Spontaneous activity (upper trace) and representative responses of ORN50 (illustrated from raw data stored in computer files) to different stimuli (lower traces). This ORN was tested with all the odors of subsets 1 and 2 (17). All induced excitatory responses. Responses to ACE, CAM, and CYM are not shown because their thresholds were not estimated. ORN50's thresholds were widely distributed over the available concentration range. For ANI, MAK, and ISO, sustained responses were observed for the lowest concentration allowed by the olfactometer (SV/562). Their thresholds were thus lower than 10–7 mol/liter. For XON, the threshold was SV/100 (2.5 × 10–6 mol/liter). Thresholds were SV/10 for LIM, CIN, and VAN and SV/5 for CDN. *, stimulation artifact.

Figure 2

Spontaneous activity (upper trace) and responses (lower traces) of ORN19 to different stimuli delivered at the same concentration in terms of ratio of the SV of the different compounds (SV/562). Only odors that induced a response are shown. This ORN displayed sustained discharges at the lowest concentration that can be delivered by the olfactometer (from 2.5 × 10–8mol/liter to 5.2 × 10–7 mol/liter, according to the value of the SV/562 of each compound). Its response threshold was thus overpassed for all these stimuli. This figure indicates further that broad qualitative fields can be observed even at low concentrations; that is, that one may induce ORN responses to several different odorants without working at high concentrations.

Figure 3

Spontaneous (upper pair of traces) and odor-evoked (lower pairs of traces) EOGs and single-unit responses of ORN55 to increasing concentrations of LIM. This ORN had a low spontaneous firing frequency (about 1.2 spikes per second on the period shown). The lowest concentration shown (1.9 × 10–6 mol/liter) induced a small EOG but did not significantly modify the ORN firing activity. Increasing the concentration to 5.9 × 10–6 mol/liter (SV/20) induced a larger EOG and an ORN response characterized by a rhythmic discharge composed of four spikes. Thus, the response threshold of this ORN is between 1.9 × 10–6 mol/liter and 5.9 × 10−6 mol/liter. Increasing the concentration enhanced the ORN firing activity, which became a sustained tonic response pattern and then an initial high-frequency burst of activity followed by a silence and a rebound. EOG amplitude evolved in parallel: It was very small for 1.9 × 10–6 mol/liter and increased gradually, mirroring the global recruitment of the ORNs situated within the recording field of the electrode. Recordings also show that whereas the ORN burst discharge frequency increased with concentration, the latency of this discharge shortened with respect to the beginning of the odor pulse and thus appeared earlier and earlier with respect to the EOG kinetics. The concentration range used in this study overlaps the dynamic range of rat ORNs.

This study presents functional evidence that rat ORNs have broadly tuned chemical receptive fields. Their selectivity and sensitivity are in agreement with those of mitral cells previously reported in the same animal species (11). Rat ORNs tended to display a broader qualitative profile and a lower sensibility than those of the frog (19). Our finding that one ORN could display different sensitivities to different odors is consistent with a calcium imaging study that reports that single cells respond to additional odorants when concentrations are increased (20).

How can the the molecular biology of OR proteins be interpreted to take into account our functional results? Two molecular studies that have addressed the chemical tuning of odor receptors contain some divergent results. In insect Sf9 cells transfected with the OR5 receptor, Raming and co-workers (21) show that several odor molecules increase IP3 responses, and they conclude that OR5 receptors are rather poorly selective. In contrast, Zhao and colleagues (4) report, for the first time in rat ORNs, that increasing the expression of a single gene leads to greater responsiveness to octanal and other compounds with a very similar molecular structure. They conclude that the recombinant virus drove the expression of a gene coding for a selective OR.

Here the molecular receptive range of ORNs was identified in a biological preparation that was as close as possible to physiological conditions. If the hypothesis that each ORN expresses one OR is true, the fact that most ORNs responded to several distinct odor molecules demonstrates that ORs are broadly responsive. Another possibility could be that each ORN would expresses several ORs of a given subfamily (22), so that its qualitative response spectrum is the sum of the individual receptive fields of its ORs. We found neurons that displayed differential sensitivity to different odors, which suggests that, at the level of a single neuron, not only the categories of ORs and their specificity may differ but also their number and their affinity for odor molecules.

At the level of a single neuron, in terms of olfactory quality coding, our data are in agreement with those previously obtained in the frog. In contrast, they disprove the extreme concept that individual ORNs express only a single OR and respond to only one odor (8). Our electrophysiological data add to those of a recent study combining calcium imaging and single-cell reverse transcription polymerase chain reaction, which indicates that ORs are broadly tuned with various odors and, conversely, that various odors can be recognized by the same OR (23). Thus, we conclude that in vertebrates at the cellular level, the odor receptor binding process results in activation of ORN arrays with partially overlapping tuning profiles.

  • * To whom correspondence should be addressed. E-mail: pduchamp{at}olfac.univ-lyon1.fr

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