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1 Monell Chemical Senses Center, Philadelphia; 2 Thomas Jefferson University, Philadelphia; 3 Veterans Affairs Medical Center, Philadelphia; and 4 University of Pennsylvania, Philadelphia, Pennsylvania 19104-3308
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Rawson, N. E., G. Gomez, B. Cowart, J. G. Brand, L. D. Lowry, E. A. Pribitkin, and D. Restrepo. Selectivity and response characteristics of human olfactory neurons. J. Neurophysiol. 77: 1606-1613, 1997. Transduction mechanisms were investigated in human olfactory neurons by determining characteristics of odorant-induced changes in intracellular calcium concentration ([Ca2+]i). Olfactory neurons were freshly isolated from nasal biopsies, allowed to attach to coverslips, and loaded with the calcium-sensitive indicator fura-2. Changes in [Ca2+]i were studied in response to exposure to individual odors, or odorant mixtures composed to distinguish between transduction pathways mediated by adenosine 3
5-monophosphate (cAMP; mix A) or inositol 1,4,5-trisphosphate (InsP3; mix B). Overall, 52% of biopsies produced one or more odorant-responsive olfactory neurons, whereas 24% of all olfactory neurons tested responded to odorant exposure with a change in [Ca2+]i. As in olfactory neurons from other species, the data suggest that odorant exposure elicited calcium influx via second-messenger pathways involving cAMP or InsP3. Unlike olfactory neurons from other species that have been tested, some human olfactory neurons responded to odorants with decreases in [Ca2+]i. Also in contrast with olfactory neurons from other species, human olfactory neurons were better able to discriminate between odorant mixtures in that no neuron responded to more than one type of odor or mixture. These results suggest the presence of a previously unreported type of olfactory transduction mechanism, and raise the possibility that coding of odor qualities in humans may be accomplished to some degree differently than in other vertebrates, with the olfactory neuron itself making a greater contribution to the discrimination process.
Human olfaction Subjects and psychophysical measurements
Biopsies were obtained from volunteers (n = 34) who first completed a medical screening questionnaire and psychophysical tests to assess olfactory function. Some biopsies were also obtained from surgery patients (n = 10) and are tabulated separately. Volunteers were recruited to include a cross section of the local population, age 18-64 yr, with 9 males and 20 females. Subjects provided informed consent by signing a document describing the nature and possible consequences of participation; those with potentially complicating diseases or medications were omitted from the study. Biopsies were obtained generally within 2 wk of initial evaluation. One compound from each of the two odorant mixtures used in the single-cell experiments was chosen for threshold testing: phenylethyl alcohol and lyral. These compounds appear to stimulate the olfactory system exclusively, because even at the highest concentrations tested they cannot be localized with the use of a lateralization test (C. Wysocki, personal communication) and are not detected by anosmics (Rawson et al. 1995 Biopsy and cell dissociation procedures
Biopsies of ~1 mm3 were obtained from the high middle turbinate and apposed septum from subjects after local anesthetic as described (Lowry and Pribitkin 1995 Measurement of [Ca2+]i and odorant exposure
Changes in [Ca2+]i were observed by loading cells with fura-2 and observing fluorescence emitted in response to dual excitation at 340 nm (Ca2+ sensitive) or 360 nm (Ca2+ insensitive). Details have been published elsewhere (Restrepo et al. 1993a Statistics
Only responses that began within 100 s of exposure, returned toward baseline after removal of the odorant, and could be repeated after washout were included in the analyses. Statistical comparisons were performed with the use of paired t-tests for baseline versus stimulated values and independent t-tests for comparisons of baseline values between groups with Statistica software (StatSoft).
Response rates to odorant stimuli
Psychophysical tests showed that all subjects were able to detect odorous compounds included in the mixtures used to stimulate isolated olfactory neurons (see METHODS).
Human olfactory neurons respond to stimulation with odorant mixtures with either increases or decreases in [Ca2+]i
Changes in [Ca2+]i in response to odorant stimuli were measured in 179 human olfactory neurons (Table 1) identified by the presence of a single dendrite and olfactory knob with attached cilia. As shown previously (Restrepo et al. 1993b
All responding human olfactory neurons discriminated between mix A and mix B odorants
In previous experiments in our laboratory (Restrepo et al. 1993a Responses of human olfactory neurons to single odorants
Olfactory neurons (n = 16) from five subjects were tested with a set of individual odorants. As expected, few olfactory neurons responded to an individual odorant; however, olfactory neurons responding repeatedly to lilial (1 of 16) or isovaleric acid (2 of 16) were observed (Fig. 4). All responding olfactory neurons tested with more than one individual odorant responded to one odorant only (3 of 18).
Two distinct transduction pathways are involved in human olfactory transduction
To investigate the transduction pathways involved in the observed calcium responses, inhibitors were used in conjunction with odorants. L-cis-diltiazem (LCD; 20 µM) was used to block the cyclic nucleotide-gated channel (Kolesnikov et al. 1990 Some of our results are similar to those obtained in previous studies with olfactory neurons from other vertebrate as well as invertebrate species. For example, as found in other species (see reviews by Breer et al. 1994
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
the ability to detect many volatile chemicals in one's environmentappears to be a primitive system in an evolutionary sense, yet has proven to be biologically complex. It is commonly thought that olfaction is less developed in humans than most other species, yet we are able to detect and, with training, discriminate among thousands of odors at concentrations below the limit of detection of virtually any instrument (Amoore and Hautal 1983
). On the basis of molecular biological studies of putative olfactory receptor genes (Buck and Axel 1991), it has been estimated that humans may express as many as 1,000 different G protein-linked olfactory receptors that can be grouped into a number of classes and subclasses on the basis of nucleotide sequence similarities (Lancet and Ben-Arie 1993). The mechanisms by which the olfactory system utilizes these receptors to detect and decode a multitude of odors have been studied in a variety of invertebrate and vertebrate systems, but comparatively few studies have addressed this question at the cellular level in human olfactory neurons.
5-monophosphate (cAMP) and/or inositol 1,4,5-trisphosphate (InsP3) (see reviews by Restrepo et al. 1996; Shepherd 1994). The opening of cAMP- or InsP3-regulated cation channels results in influx of Ca2+ and a subsequent increase in intracellular calcium concentration ([Ca2+]i) (Restrepo et al. 1990, 1993a; Sato et al. 1991; Tareilus et al. 1995), leading to opening of Ca2+-activated Cl
or K+ channels (Kleene and Gesteland 1991; Kurahashi and Yau 1993; Morales et al. 1995). In those cells in which the concurrent change in membrane potential is a depolarization, voltage-sensitive channels open, triggering action potentials that are carried along the axon to the olfactory bulb.
; Firestein et al. 1993; Kang and Caprio 1995; Revial et al. 1982). This evidence that individual olfactory neurons are often nonselective has led investigators to propose that odor qualities are coded to a large extent by distributed patterns of neural activity interpreted at the level of the olfactory bulb (Kauer 1991).
; Restrepo et al. 1993b). However, a thorough study of the responsiveness of human olfactory neurons to odors has not been reported. In this manuscript we characterize the responses of human olfactory neurons isolated from olfactory tissue biopsies with the use of calcium imaging. Our studies indicate that, like olfactory neurons from other species, human olfactory neurons respond to odors utilizing at least two different second-messenger pathways. However, human olfactory neurons also respond to odorants with a decrease in [Ca2+]i, a response not observed in olfactory neurons from other species, and these cells appear to be more selective for odorants than are olfactory neurons from other species.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Threshold sensitivity was determined via a two-alternative, forced-choice staircase procedure described previously (Rawson et al. 1995). All subjects could detect both compounds on the side of the biopsy. Unilateral thresholds for phenylethyl alcohol on the biopsied side were 0.018-0.000024% vol/vol (median 0.00047%); for lyral, thresholds were 0.014%-0.0000042% vol/vol (median 0.00027%).
; Rawson et al. 1995). For surgery patients, epinephrine was injected locally in addition to the general anesthetic and a single biopsy was obtained from the inferior half of the middle turbinates being resected for sinus surgery. It has traditionally been thought that the olfactory epithelium is located predominantly within the olfactory cleft (Lanza and Clerico 1995). The higher risk of obtaining tissue from this region because of its proximity to the cribriform plate led us to obtain biopsies from the middle turbinate and apposing septum, a region not generally characterized as containing olfactory epithelium. Nonetheless, nearly 50% (14 of 34) of the biopsies obtained from this region produced one or more odorant-responsive olfactory neurons. This finding is consistent with early reports suggesting that olfactory epithelium may also be present in the superior aspect of the middle turbinate (see Lanza and Clerico 1995 for discussion). Biopsies obtained from surgery patients were generally 2-3 times as large and produced odorant-responsive olfactory neurons 90% of the time. Cells were dissociated by brief incubation in cation-free mammalian Ringer solution containing 12-15 U/ml papain, as described previously (Restrepo et al. 1993b).
,b). Cells attached to coverslips were superfused with Ringer solution and exposed to odor mixtures or single odorants via the superfusion buffer or via a computer-controlled Solution Changer (Bio-Logic, RSC-100, Pullman, WA). Cells were tested with two odorant mixtures, mix A (hedione, geraniol, phenylethyl alcohol, citralva, citronellal, eugenol, and menthone) and mix B (lyral, lilial, triethylamine, ethylvanillin, isovaleric acid, and phenylethyl amine), or with individual odorants dissolved in Ringer solution at 1 or 100 µM as indicated. Some olfactory neurons were tested to determine the extent of calcium uptake following a challenge with the calcium ionophore ionomycin (5 µM). The [Ca2+]i of these olfactory neurons (53.6 ± 14.9 nM, mean ± SE) increased to 206.7 ± 15.7 nM after ionomycin [t(9) = 6.58, P < 0.05; n = 10]. Baseline and ionomycin-stimulated [Ca2+]i were similar in responding and nonresponding olfactory neurons. Baseline [Ca2+]i did not differ between olfactory neurons responding to mix A and mix B, those from males and females, or those from subjects and surgery patients. Depolarizing cells by increasing extracellular K+ leads to an influx of Ca2+ in >90% of rat olfactory neurons tested because of activation of voltage-sensitive calcium channels (Restrepo et al. 1993b; Tareilus et al. 1995). In contrast, depolarizing human olfactory neurons by replacing extracellular Na+ in the superfusion buffer with K+ produced an increase in [Ca2+]i in only two of eight olfactory neurons tested (
2 = 15.40, P < 0.001 vs. rat).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
), human olfactory neurons exhibiting an increase in [Ca2+]i in response to a mixture of odorants known to stimulate cAMP formation in rat ("mix A," 100 µM) were observed (Fig. 1, A and B; Table 1). Baseline and stimulated [Ca2+]i for these olfactory neurons was 37.2 ± 9.6 (SE) nM and 81.8 ± 21.0 (SE) nM, respectively [t(15) = 3.83, P < 0.01]. Responses were also observed to lower concentrations of mix A (1 and 10 µM) that in some cases were of similar magnitude, possibly because of saturation of the response.
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TABLE 1.
Response rates for biopsies and individual olfactory neurons
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FIG. 1.
Intracellular calcium concentration ([Ca2+]i) responses of human olfactory neurons to a mixture of odorants shown to elicit increases in adenosine 3 5-monophosphate (cAMP) in isolated olfactory cilia (Breer and Boekhoff 1991) (mix "A"). A: record from an olfactory neuron that exhibited an increase in [Ca2+]i in response to mix A (100 µM) that was blocked by L-cis-diltiazem (LCD, 20 µM). B: neomycin (1 mM) fails to block the rise in [Ca2+]i in another cell. C: record from an olfactory neuron that exhibited a decrease in [Ca2+]i in response to mix A (100 µM).
; Tareilus et al. 1995), a substantial number of human olfactory neurons (9 of 25) responded to mix A with a decrease in [Ca2+]i (Fig. 1C). The [Ca2+]i in these olfactory neurons decreased from a baseline of 67.9 ± 22.6 nM to 52.3 ± 17.4 nM after odorant exposure [t(8) = 5.06, P < 0.001).
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FIG. 2.
Responses of human olfactory neurons to a mixture of odorants known to stimulate inositol 1,4,5-trisphosphate (InsP3) production in isolated olfactory cilia (Breer and Boekhoff 1991 ) (mix "B"). A: record from a cell that responded to 1 µM mix B with a homogeneous rise in [Ca2+]i. B: data from an olfactory neuron that responded to 100 µM mix B with an apically localized increase in [Ca2+]i. Filled circles: cell body. Filled squares: apical, dendritic portion. C: neomycin prevents the rise in [Ca2+]i induced by mix B (100 µM). D: neomycin also prevents the decrease in [Ca2+]i induced by mix B (100 µM) in another cell. E: blocking the cyclic nucleotide-gated channel with LCD (20 µM) does not affect the decrease in [Ca2+]i elicited by mix B (100 µM) in another cell. Mix B was applied with the use of the rapid solution exchanger in A and B, and by superfusion in the other figures.
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FIG. 3.
Pseudocolor image displaying the spatial distribution of the decrease in [Ca2+]i elicited by mix B in a human olfactory receptor neuron. [Ca2+]i is color coded linearly by the color palette displayed in the rainbow. In this image red corresponds to 200 nM [Ca2+]i, whereas blue corresponds to 100 nM [Ca2+]i. Left: olfactory neuron before stimulation. Right: olfactory neuron 10 s after stimulation with 100 µM mix B. This cell responded repeatedly to mix B with a decrease in [Ca2+]i, and this response was inhibited by 1 mM neomycin.
), we found that one third of responding rat olfactory neurons responded to both mixes A and B at a concentration of 100 µM. Similar results were obtained in independent experiments in rat olfactory neurons (Tareilus et al. 1995) with the use of similar odorants. These observations are consistent with electrophysiological investigations of odorant responses in other species showing that single olfactory neurons often respond to more than one odorant and may even respond to odorants of different chemical or perceptual classes (Boekhoff et al. 1994; Kang and Caprio 1995; Revial et al. 1982). In contrast, in 155 human olfactory neurons tested with odorant mixes A and B, 43 responded to either mixture, but not one responded to both.
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FIG. 4.
[Ca2+]i responses of human olfactory neurons to individual odorants. A: portion of a record from a cell that responded to lilial (100 µM), a component of mix B. This olfactory neuron responded to lilial several times, but not to isovaleric acid (IVA) or ethylvanillin, also components of mix B, or menthone, a component of mix A. B: another cell responds to IVA (100 µM). C: cAMP-gated channel inhibitor LCD (20 µM) does not affect the increase in [Ca2+]i elicited by 100 µM IVA (B and C are from the same cell). A total of 18 cells was tested with individual odorants at a concentration of 100 µM. The responsiveness to these odorants was as follows: ethyl vanillin (0 of 16 cells responding), lilial (1 of 16), isovaleric acid (2 of 16), menthone (0 of 11), 
-ionone (0 of 18), hedione (0 of 17).
), and neomycin (1 mM) was used to inhibit phospholipase C activity (Striggow and Bohensack 1994) and InsP3 production. As found previously, LCD reversibly inhibits the rise in [Ca2+]i induced by mix A (Fig. 1A) (Restrepo et al. 1993b). Similar to rat olfactory neurons (Restrepo et al. 1993a; Tareilus et al. 1995), activation of adenylate cyclase with forskolin elicited an increase in [Ca2+]i that could be blocked with LCD (data not shown). Addition of neomycin to inhibit InsP3 production did not alter the increase in [Ca2+]i in response to mix A (Fig. 1B; n = 3). LCD alone did not affect resting [Ca2+]i, nor did it block the decrease in [Ca2+]i observed in response to mix A in some cells (n = 3; not shown).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Restrepo et al. 1996; Shepherd 1994), our experiments indicate that at least two distinct second-messenger pathways mediate the odorant-induced changes in [Ca2+]i in human olfactory neurons. The presence of separate transduction pathways is evidenced by inhibition of responses to odorant mix B by the phospholipase C inhibitor neomycin, but not by the cAMP-gated channel blocker LCD, and inhibition of the responses to odorant mix A by LCD, but not by neomycin. This differential inhibition clearly shows that two pharmacologically distinct pathways are involved, and the known specificity of the inhibitors suggests that the second messengers cAMP and InsP3 are involved in human olfactory transduction. These results are consistent with previous pharmacological studies in rat (Tareilus et al. 1995).
; Tareilus et al. 1995). Odor-induced decreases in [Ca2+]i have not been described in studies in amphibians (Nakamura et al. 1994; Sato et al. 1991), and are rare in catfish olfactory neurons (1 of 140 catfish olfactory neurons) (Restrepo and Boyle 1991). Current models of olfactory transduction do not provide a mechanistic explanation for the odorant-induced decrease in [Ca2+]i reported here. In all current models, odorants elicit an increase in [Ca2+]i. Although not sufficient to propose a model accounting for the odorant-induced decreases in [Ca2+]i, our data can be used to rule out plausible models. For example, high basal levels of either cAMP or InsP3 in some olfactory neurons might cause the second-messenger (cAMP or InsP3)-gated channels to be open at steady state such that odorants trigger channel closing leading to a decrease in Ca2+ influx. However, in mix A-responsive olfactory neurons, the observed effect is not due to inhibition of the cyclic nucleotide-gated channel, because LCD alone did not affect [Ca2+]i in cells that responded to mix A with a decrease in [Ca2+]i (data not shown). Furthermore, it is unlikely that an increase in cytosolic Ca2+ buffering or in uptake of Ca2+ by intracellular organelles mediates the odorant-induced decreases in [Ca2+]i, because these decreases were not transient, persisting throughout stimulation (Figs. 1C and 2, D and E). Therefore the most likely explanations are either an increase in Ca2+ efflux or a decrease in influx through a pathway other than the second-messenger-gated channels.
). The decreases we observed did not tend to be localized to the dendrite, and baseline [Ca2+]i did not differ significantly between cells exhibiting increases and those exhibiting decreases. A Ca2+ ATPase has been implicated in the hyperpolarization induced by the chemoattractant cAMP in paramecia (Wright et al. 1993), but the role of such a pump in vertebrate chemoreception has not been investigated. It is possible that the decrease in [Ca2+]i is occurring after an increase that is brief and transient, faster than the first image taken after stimulation (typically 7-10 s). However, even if this were true, the mechanism for such an odorant-stimulated decrease in [Ca2+]i and its possible role in olfactory transduction in humans remain to be investigated.
; Tareilus et al. 1995). Indeed, studies with invertebrate and vertebrate species (Boekhoff et al. 1994; Firestein et al. 1993; Kang and Caprio 1995; Restrepo et al. 1993a; Revial et al. 1982; Tareilus et al. 1995) suggest that many olfactory neurons are not narrowly selective, but respond to qualitatively different odorants.
; Tareilus et al. 1995).
). Although rats can detect at least some odorants used in physiological studies (Slotnick et al. 1991), most animal studies do not combine behavioral and cell biological methods to establish the sensitivity of the same animals to the odorants used. It is unlikely that the rats raised in a laboratory and fed only rat chow had prior experience with odors like those used in our and other cell biological studies. Thus it might be that the response characteristics of rat olfactory neurons to odorants to which the animals had prior exposure would be more like those we have seen in human olfactory neurons when odorant mixtures are used that are part of a typical human environment.
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ACKNOWLEDGEMENTS |
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The authors thank E. Varga, L. Roben, and H. Rees for excellent technical assistance and G. Beauchamp, S. Kemp, J. Pierce, S. Snyder, J. Teeter, and C. Wysocki for valuable suggestions and discussions.
These studies were supported by grants from the National Institutes of Health, the Human Frontier Science Program, and by a grant from the Veterans Affairs Department.
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FOOTNOTES |
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Address for reprint requests: N. E. Rawson, Monell Chemical Senses Center, 3500 Market St., Philadelphia, PA 19104-3308.
Received 13 June 1996; accepted in final form 19 November 1996.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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