Single-Neuron Comparison of the Olfactory Receptor Response to

6 days ago - The mammalian olfactory receptors (ORs) constitute a large subfamily of the Class A G-protein coupled receptors (GPCRs). The molecular ...
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Single-Neuron Comparison of the Olfactory Receptor Response to Deuterated and Nondeuterated Odorants Mihwa Na,†,‡ Min Ting Liu,†,§ Minh Q. Nguyen,∥ and Kevin Ryan*,†,‡,§ †

ACS Chem. Neurosci. Downloaded from pubs.acs.org by 5.189.201.248 on 10/20/18. For personal use only.

Department of Chemistry and Biochemistry, The City College of New York, 160 Convent Avenue, New York, New York 10031, United States Doctoral Programs in ‡Biochemistry and §Chemistry, The City University of New York Graduate Center, 365 Fifth Avenue, New York, New York 10016, United States ∥ Taste and Smell Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, United States S Supporting Information *

ABSTRACT: The mammalian olfactory receptors (ORs) constitute a large subfamily of the Class A G-protein coupled receptors (GPCRs). The molecular details of how these receptors convert odorant chemical information into neural signal are unknown, but are predicted by analogy to other GPCRs to involve stabilization of the activated form of the OR by the odorant. An alternative hypothesis maintains that the vibrational modes of an odorant’s bonds constitute the main determinant for OR activation, and that odorants containing deuterium in place of hydrogen should activate different sets of OR family members. Experiments using heterologously expressed ORs have failed to show different responses for deuterated odorants, but experiments in the sensory neuron environment have been lacking. We tested the response to deuterated and nondeuterated versions of p-cymene, 1-octanol, 1-undecanol, and octanal in dissociated mouse olfactory receptor neurons (ORNs) by calcium imaging. In all, we tested 23 812 cells, including a subset expressing recombinant mouse olfactory receptor 2 (Olfr2/OR-I7), and found that nearly all of the 1610 odorant-responding neurons were unable to distinguish the D- and H-odorants. These results support the conclusion that if mammals can perceive deuterated odorants differently, the difference arises from the receptor-independent steps of olfaction. Nevertheless, 0.81% of the responding ORNs responded differently to D- and H-odorants, and those in the octanal experiments responded selectively to H-octanal at concentrations from 3 to 100 μM. The few ORs responding differently to H and D may be hypersensitive to one of the several H/D physicochemical differences, such as the difference in H/D hydrophobicity. KEYWORDS: Olfactory receptor, odorant receptor, deuterium, GPCR, aldehyde, calcium imaging

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INTRODUCTION

analogous to the function of other Class A GPCRs, and recent in vitro data support this prediction.9 The lack of even one OR X-ray structure has made it difficult to rule out alternative theories like the “vibrational” theory, which is still discussed as an alternative to the mainstream chemical recognition, or “shape,” theory in contemporary textbooks.10 Since isotopic substitution of deuterium for hydrogen in an odorant makes a large change in a molecule’s bond vibrational frequencies, it has been proposed that replacing odorant hydrogen with deuterium should prevent the activation of receptors activated by the nondeuterated odorant.5,11 Some experiments in insects,12−14 whose receptors are unrelated to GPCRs,15 have shown perceptual differences between nondeuterated and deuterated odorants, though other studies have not.16 (Here, deuterated refers to odorants containing C−D bonds in place

Before it was understood that the molecular recognition step in odor detection by mammals was mediated by a large subfamily of G-protein coupled receptors (GPCRs),1 now called the odorant or olfactory receptors (ORs), many speculative theories on the molecular mechanism of olfaction were considered.2−5 Several of these postulated that the vibrational modes of an odorant were the main determinant of odor detection and character.4,5 After the discovery of the odorant GPCRs, but before the structural elucidation of nonolfactory GPCRs became common,6 most theories unrelated to GPCR function lost their appeal, but one vibrational theory survived and was modified to accommodate some details of GPCR function.5 Despite repeated successes in GPCR structural biology over the last 15 years,7 and the fact that almost half of the human GPCR genes encode ORs,8 no OR structures have been solved. This gap in our knowledge has made it difficult to know the precise molecular details of odor reception, though it is reasonable to expect that OR activation works in a manner © XXXX American Chemical Society

Received: August 14, 2018 Accepted: September 21, 2018

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DOI: 10.1021/acschemneuro.8b00416 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience of C−H bonds.) Likewise, in humans, some experiments,17 but not others,18 have detected perceptual differences between nondeuterated and deuterated odorants. Direct in vitro experiments on recombinant ORs expressed in heterologous cells have not detected any H versus D differences.19 The receptors are only one part of the mammalian olfactory system, albeit an important one, and other components, in the mucus, for example, might be able to distinguish H and D isotopologues and contribute to perceived differences. (Here we use the term isotopologue to indicate odorants with all nonexchangeable hydrogens replaced by deuterium.) It is therefore imperative to test the ORs in their natural cellular setting so that, if mammals can indeed perceive differences in H and D isotopologues, it can be known whether the differences arise through the receptors or through perireceptor, i.e., receptor-independent, processes. The vibrational theory of olfaction has been dismissed by some because, among other unorthodoxies, it has not always taken into account the need for a specific, energetically favorable physical association between the odorant ligand and the receptor, but instead considered only the vibrational modes of the odorant’s functional groups. More recently, variations of the vibrational theory have proposed that a ligand needs both to bind the receptor specifically and meet a vibrational requirement to activate it.11,20 This variation has been referred to as the “swipe-card” model, where a ligand may bind but fail to activate a receptor through isotope-dependent vibrational mismatch. Odorant deuteration should lead to a different combinatorial code21 of activated receptors and hence a different smell. This blending of traditional receptor theory with vibrational theory can be rephrased to make a testable prediction, namely, that deuterated odorants should be orthosteric antagonists for receptors activated by the nondeuterated odorant, and vice versa. As we are interested in the contribution that pharmacologic antagonism makes to the olfactory code,21 we became interested in testing this potential formula for odorant antagonist design, and decided to look for receptors that could distinguish deuterated from nondeuterated odorants in native olfactory receptor neurons, ORNs (also called olfactory sensory neurons, OSNs). Moreover, since tissue culture cells are different in many unknowable ways from ORNs, it is important to further test OR responses to deuterated odorants in primary sensory neurons to know whether the reported inability of ORs to distinguish deuterated odorants from nondeuterated odorants extends to the sensory neuron environment. In organic compounds, hydrogen and deuterium differ by several physicochemical properties.22 The question whether olfactory GPCRs can distinguish between hydrogen and deuterium for any reason is itself an interesting molecular recognition topic. Deuterium is considered to be an isostere of hydrogen.22 Of potential significance to olfaction, exchanging H for D also makes a compound less hydrophobic, i.e., less lipophilic, a difference sufficiently large to lead to changes in reverse phase HPLC retention times.23−26 Except for inorganic odorants, like ammonia and hydrogen sulfide, hydrophobicity is a key feature of odorant ligands, and one that has been correlated with odor intensity.10,27−29 Features that make small molecules more hydrophobic, few or no heteroatoms, for example, will also make them more volatile and thus more likely to be an odorant. Hydrophobicity is an important driver of ligand-protein binding in general30 and likely important to OR ligand binding. Thus, apart from considerations of the

vibrational theory, and in view of conflicting data from insect and human testing, we were interested in knowing whether the ORs could discriminate deuterated odorants from their nondeuterated forms on the basis of the former’s reduced hydrophobicity. Our results show that, in dissociated sensory neurons, outside of the context of the olfactory epithelium, and thus in the absence of perireceptor events, nearly all of the odorant-responding mouse ORNs were unable to distinguish the odorants we tested from their deuterated isotopologues. Interestingly, 0.81% of tested cells did show evidence of H/D discrimination over the 3−100 μM odorant concentration range and, among the octanal-responding cells, several were quite robust and unambiguous in their preference for the more hydrophobic H-octanal isotopologue.

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RESULTS AND DISCUSSION Heterologous Cell Versus Sensory Neuron Testing. In lieu of structural data, the prediction that deuterium substitution in an odorant ligand will alter the OR-mediated signal transduction response has been considered by some as a litmus test of the vibrational theory of olfaction.13,17,31 In animals, experiments that have detected an H/D difference might be detecting differences originating through the receptor or through perireceptor processes. This uncertainty makes it important to design experiments that probe the receptors directly. Fifteen odorants in their deuterated and nondeuterated forms were recently tested with one or more of their cognate ORs in the Hana3A tissue culture cell heterologous protein expression system using a luciferase reporter readout.19 In no case was a significant difference found between the nondeuterated and deuterated isotopologues. Heterologous expression is an established GPCR agonist testing method that avoids the difficulty of expressing a recombinant OR in ORNs, which requires a viral expression vector or the construction of a knock-in mouse.32−34 Nevertheless, the use of non-neuronal cells may be considered by some as limiting the scope of conclusions drawn from tissue culture experiments. Here we have studied primary mouse ORNs dissociated from the main olfactory epithelium to compare four nondeuterated odorants with their deuterated isotopologues. The odorants were: 1-octanol, 1-undecanol, octanal and the hydrocarbon p-cymene (Figure S1, IR spectra shown in Figure S2). This experimental system has complementary advantages and disadvantages with respect to other methods. As native primary cells of the olfactory epithelium, dissociated sensory neurons have all of the native accessory proteins, known and unknown, and at proper physiological concentration, needed to detect odors. Using the technique of calcium imaging optical recordings35−39 allows signal transduction to be monitored at the single-neuron level, in real time during transient exposure to the stimulus without the several hours lag time required by the Hana3A luciferase reporter.40 Another advantage is that when studied by calcium imaging, ORNs require high concentrations of the activating odorant, and typically have EC50 values in the micromolar to millimolar range. For this reason, and in contrast to live animal testing,31 trace impurities are unlikely to be responsible for any observed cellular activation. While the single OR chosen by each observed ORN (i.e., the one neuron, one receptor rule21) is not known in our experiments, most odorants, especially conformationally flexible odorants36 activate multiple receptors (e.g., octanal activates 33−55 rat ORs41) so that multiple ORs from the B

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Figure 1. Ratiometric calcium imaging results from 5821 dissociated mouse ORNs exposed to deuterated and nondeuterated 1-octanol. (a,b) Representative cell responses from category 1 (positive response to H and D isotopologues). (c,d) Representative cell responses from category 2a (positive response to H and D isotopologues with variable H/D response at the lowest activating concentration, i.e. threshold effect). (e) Representative cell responses from a separate experiment where 765 ORNs were tested with repeated applications of nondeuterated 1-octanol. (f) Cell response showing category 2a type behavior to repeated applications of nondeuterated 1-octanol only, i.e., inconsistent responses at the threshold concentration, but consistent responses above the threshold concentration. This control experiment demonstrated that responses near the threshold are unreliable. (g) Category 2b type cell response (threshold concentration coincidently was the highest concentration used). (h−k) Calcium imaging traces of all four category 3 cells (different responses to H and D 1-octanol). (l) Percentages of cells in the four categories. Percentages from four biological repeats were averaged. Error bars show one standard deviation over four biological repeats (i.e., four mice). Heat maps summarizing the responses of all 162 1-octanol-responding ORNs can be found in Table S1.

capability to the end of the experiment. Stimuli were delivered by syringe to a stream of Ringer solution flowing over the cells. A total of 5821 functioning (i.e., forskolin-responding) mouse ORNs from four adult mice were monitored by the calcium imaging technique (see Methods). In total, 162 ORNs (2.78% of 5821 ORNs) responded to at least one concentration of 1octanol (scoring criteria: ≥10% of the forskolin response is scored as positive). The raw imaging traces of representative cells are shown in Figure 1. In Table S1, we show in heat map format the profile of all 162 1-octanol-responding cells normalized to the 10 μM forskolin response.

entire repertoire are probed in each experiment. Neither Hana3A cells nor dissociated ORNs include the nasal mucus, a feature that increases the likelihood that any H/D differences are receptor-mediated, rather than due to perireceptor processes like enzymatic modification of the odorant before receptor binding.42 The Response to Deuterated and Nondeuterated 1Octanol. We began by testing the deuterated and nondeuterated versions of 1-octanol at 3, 10, 30, and 100 μM, followed by forskolin, an adenylyl cyclase activator, to identify cells that maintained a functioning signal transduction C

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certain that the different response was not an all-or-nothing case, and so does not fit our expectation of a cell that is detecting a difference in H and D behavior. The last three cells (3/162, or 2%) in category 3 (Figure 1i−k) showed the opposite preference, responding to the deuterated 1-octanol but not the nondeuterated isotopologue. In this experiment, the syringe containing the nondeuterated 1- octanol had produced clear responses in other cells at other times during the experiment, ruling out a compositional error in the syringe solution. We note that these three cells were atypical in that the drifting baseline indicated either leakiness to calcium or difficulty pumping out calcium after activation. Nevertheless, these cells responded selectively to deuterated 1-octanol. Our overall conclusion is that 96.9% of responding neurons (categories 1, 2a and the cell shown in Figure 1h) responded to both D and H isotopologues of 1-octanol. These data do not support the discrimination of different bond vibration frequencies as a general mechanism for the detection of 1octanol, but we cannot rule out the possibility that a few 1octanol receptors in the mouse olfactome can discriminate H and D in the context of 1-octanol through some physicochemical difference. The Response to Deuterated and Nondeuterated 1Undecanol. It has been suggested that the greater the number of hydrogens in an odorant, the greater the likelihood that deuterium substitution will lead to a detectable H vs D response difference due to a vibrational effect,17 a trend we would also expect if the difference in hydrophobicity were the cause of different responses. We compared nondeuterated with deuterated 1-undecanol (a 35% increase in number of C−H bonds compared to 1-octanol). Here, we made duplicate applications of the 1-undecanol isotopologue at 3 and 10 μM, followed by a single pulse of each at 100 μM to judge whether the lower concentrations were close to the threshold (Figure 2). A total of 4849 forskolin-responding cells from 3 adult mice were assayed, and 51 cells (1.1%) responded. A representative calcium imaging trace is shown in Figure 2a and the categorized results are compiled in Figure 2b. The responses of all 51 responding ORNs are shown in heat map form in Table S1. In the case of 1-undecanol, no cells were found to discriminate between the H and D isotopologues (no category 3 cells). The Response to Deuterated and Nondeuterated Octanal. Aldehydes are known to activate higher numbers of ORNs compared to odorants with most other functional groups.37 We next compared the D and H isotopologues of octanal. A total of 9565 forskolin-responding cells were observed, and 699 cells (7.31%) responded to deuterated or nondeuterated octanal. Table S1 shows in heat map form the responses of all octanal-responding ORNs. On average, 69.7% (±14.3%, n = 3) and 19% (±6.7%, n = 3) of the odorantresponding cells fell into categories 1 and 2a, respectively, and thus were unable to distinguish the two forms of octanal (Figure 3a). Category 2b made up 10% (±6.6%, n = 3) of the responding ORNs; we cannot determine whether these are threshold cells (category 2a) or discriminating cells (category 3). Finally, 9 cells, i.e., 1% of the cells (±2%, n = 3, all responding cells were from a single mouse) showed unambiguous discrimination between deuterated and nondeuterated octanal and were assigned to category 3 (Figure 3a). The calcium imaging traces of all 9 ORNs able to discriminate the two octanal isotopologues are shown in Figure 3b−j. An example of category 1 cell from the same mouse,

Each 1-octanol-responding cell was assigned to one of four categories. Category 1, the largest category of responding cells (91% ± 6%, n = 4) consisted of cells that responded to both the deuterated and nondeuterated isotopologues of 1-octanol in a dose-dependent manner, or were already close to the cell’s maximum response at 3 μM. Two representative imaging traces are shown in Figure 1a and b. The number and average percentage of cells assigned to each category are shown in Figure 1l. Cells in category 2a (Figure 1c, d) would have been included in category 1 except that at the lowest odorant concentration to produce a response, which we refer to as the threshold concentration, the cells responded differently to deuterated and nondeuterated 1-octanol. In total, nine cells, or 6% (±6%, n = 4) of the 1-octanol-responding ORNs displayed this behavior. In all category 2a cases, once above the threshold concentration, the cells responded to both deuterated and nondeuterated 1-octanol, like the category 1 cells. This behavior raised the possibility of a threshold effect where, at low ligand occupancy on the receptor, the probability of triggering a cellular response might be so low as to make cellular activation and calcium influx unreliable. Such sporadic responses of ORs to weak agonists have been previously observed43 and may be a receptor-level manifestation of approaching the just noticeable difference (JND).44 To test independently whether dissociated cells may respond randomly to odorant concentrations close to the threshold, in a separate experiment we exposed 765 dissociated mouse ORNs to nondeuterated 1-octanol at 3, 10, and 100 μM concentrations consecutively and in quadruplicate. Seventeen out of 765 (2.2%) ORNs responded to at least one application. The response of a typical cell that responded consistently to nondeuterated 1-octanol at the given concentration is shown in the upper panel of Figure 1e. Among the 17 responding cells, one cell (6%) gave inconsistent results to the 3 μM exposures, but responded consistently at the higher concentrations (Figure 1f). This control experiment showed that a different response to the H and D isotopologues near the threshold concentration can occur independently of any H/D differences. Thus, we interpret this behavior as an experimental threshold artifact unrelated to true H vs D discrimination. In total, 157 cells out of 162 responding cells (96.9%) that responded to 1-octanol, fell into categories 1 and 2a, and did not distinguish between deuterated and nondeuterated 1octanol. The third response category, category 2b, was made up of cells that responded to only one of the isotopologues but here the highest concentration tested happened also to be the threshold concentration. It is uncertain if such cells discriminated the deuterated from the nondeuterated isotopologues, or if they exhibit the threshold artifact. The calcium imaging trace of the one cell in this category (0.4%, ± 0.7%, n = 4) is shown in Figure 1g. The fourth and final category of 1-octanol-responding cells, category 3, consisted of four cells that showed the apparent ability to discriminate between the H and D isotopologues. All cells in category 3 are shown in Figure 1h−k. One of these cells, Figure 1h, responded to both H and D isotopologues but was slightly more sensitive to the H form. Because the two fluorescence values happened to fall on opposite sides of the 10% of forskolin response cutoff criterion, the D isotopologue was scored as negative and H isotopologue was scored as positive. The noise in this trace was low enough for it to be D

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olfaction. Agonist/antagonist structural differences can be subtle, but differences as subtle as isotopic substitution would be unprecedented and require serious consideration of alternative explanations. One such explanation would be the swipe card/vibrational theory model, which maintains that there must be a match between the vibrational modes of the odorant and the energy gap of an as yet undefined intramolecular OR electron donor/acceptor pair for activation to take place. If both D and H forms were binding, then the vibrational theory could offer an explanation why one form is completely nonactivating. In view of established GPCR structural biology, however, it is still not clear how odorantmediated electron transfer within a receptor could in any way be linked to G-protein activation. Another possible explanation would involve a primary kinetic isotope effect in the formation, after OR binding, of an odorant agonist tautomer, such as the enol form of the aldehyde. In that scenario, the OR would use general acid−base catalysis to accelerate the formation of the activating tautomer, while the C−D bond might slow the reaction to the point where it did not have time to form during the transient odorant exposure of the experiment. Lastly, we can never entirely rule out the possibility that a minor impurity unique to the nondeuterated octanal activated the cells profiled in Figure 3. That explanation would require impurities of unprecedented potency as measured by calcium imaging. (Gas chromatography traces for the odorants used are shown in the Supporting Information, along with the 1H NMR spectrum for octanal.) Whatever the case, it will be important in future work to adapt our experimental setup to test whether deuterated octanal antagonizes ORs with this response profile, and to identify the OR family member(s) expressed in such cells. The Response of OR-I7 to Deuterated and Nondeuterated Octanal in UbI7 Cells. Mature ORNs choose to express only one type of OR,21 and in our screens the receptor identity of responding cells is not known. To test further the aldehyde functional group and to compare H and D using a known receptor, we used the homozygous UbI7 (+/+) mouse.34,45,46 This knock-in mouse contains the coding region for the mouse OR-I7 (a.k.a. Olfr2, Olfr41, or MOR103−15) inserted at the olfactory marker protein (OMP) locus. OMP is expressed ubiquitously in mature ORNs,47 but the onset of expression during ORN development is relatively late.48 The OR-I7 receptor is activated by heptanal49 and octanal.50,51 It is expressed at low but uniform levels among the UbI7(+/+) ORNs but does not disrupt the normal expression of endogenous receptors.45,50,51 As the early papers describing this mouse34,46 lacked some detail in its characterization, we first assessed the OR-I7 mRNA in coronal sections of UbI7(+/+) and wild type (wt) mice by in situ hybridization (Figure 4a, b). As expected, the endogenous OR-I7 mRNA was expressed in a small fraction of wt mouse ORNs, while in the UbI7(+/+) mouse the OR-I7 mRNA was evident throughout the epithelium. In the higher magnification panels of Figure 4b, cells expressing OR-I7 endogenously can be discerned among the other cells as slightly darker, evidence supporting that UbI7(+/+) cells continue to choose and express their endogenous receptor (e.g., endogenous OR-I7) as well as the recombinant OR-I7.45 Infrequently, the endogenous receptor will be an octanal receptor other than OR-I7, but the number of these cells can be minimized by using a 1-octanol control, as described below. The activation of dissociated ORNs from UbI7(+/+) mice has been studied using patch-clamping techniques.50,52 Perhaps

Figure 2. Ratiometric calcium imaging results from 4849 dissociated mouse ORNs exposed to deuterated and nondeuterated 1-undecanol. (a) Representative cell response from a category 1 cell (positive response to H and D isotopologues). (b) Percentages of responding cells in the four categories, as defined in Figure 1 and in the text. The percentages from three biological repeats were averaged. Error bars show one standard deviation over three biological repeats. Heatmaps summarizing the responses of all 51 1-undecanol-responding ORNs can be found in Table S1.

tested in the same experiment as these 9 cells, using the same odorant solution syringes, is shown in Figure 3k. This cell’s response to both isotopologues rules out the possibility of a compositional error in the syringe solutions. The behavior of these 9 ORNs is unexpected. Unlike the three cells responding differently to the 1-octanol isotopologues (Figure 1h−k), these cells responded robustly, unambiguously and with very little baseline drift, noise or other abnormality, and did so in response to the nondeuterated but not the deuterated octanal. One possible explanation is that the nondeuterated octanal is more hydrophobic and the ORs functioning in these cells are hypersensitive to the hydrophobic effect’s contribution to ligand binding. In this explanation, both deuterated and nondeuterated forms would be agonists but have significantly different binding affinities. We do not know if the hydrophobic effect could quantitatively explain the H/D difference observed in these cells. Another interpretation is that both the H and D forms of octanal bind the cell’s OR, but the deuterated octanal binds without activation, i.e., binds with zero efficacy, making it an antagonist. The 3−100 μM concentrations we used span the range where ORNs typically show some activation by cognate odorants, making the different behavior of the two ligands all the more unexpected because if one ligand were merely a weaker agonist we would expect to see at least a weak signal at the high end of the range. Calcium imaging does not measure binding; it measures the calcium influx that follows cellular activation that in turn results from agonist binding. An absence of activation can result from either binding with zero efficacy or no binding. Our data do not distinguish between these two possibilities. Understanding how structural differences determine whether a ligand is an agonist or antagonist is a longstanding question in E

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Figure 3. Ratiometric calcium imaging results from 9565 dissociated mouse ORNs exposed to deuterated and nondeuterated octanal. (a) Averaged percentages of cells from three biological repeats falling into the four categories, as defined in Figure 1 and in the text. Heatmaps summarizing the responses of all 699 octanal-responding ORNs can be found in Table S1. In these experiments, 1% of the cells (9 cells) fell unambiguously into category 3 (different responses to H- and D-octanal) over the entire 3−100 μM range. (b−j) Calcium imaging traces of all 9 category 3 cells. (k) Category 1 ORN from the same experiment using the same odorant solutions as the category 3 cells shown in (b)−(j).

endogenous OR-I7, but through endogenous non-OR-I7 octanal receptors, and these cells can be excluded from the OR-I7 H vs D octanal comparison. In a preliminary experiment, the percentage of 1-octanolactivated cells varied slightly from 2.68% for the wt to 5.2% for UbI7(+/+) (Figure 4d, 100 μM 1-octanol). Both of these percentages fall within the typical range for 1-octanol responses in dissociated ORNs from wt mice. For instance, in addition to the 2.78% responding cells in the 1-octanol experiments described in Figure 1, we previously found that at 30 μM, 1octanol activated 5.6% of dissociated wt mouse ORNs.38 The results of Figure 4d allow us to conclude that a normal percentage of 1-octanol-responding cells are functioning in the UbI7(+/+) mouse. To estimate the number of activated cells responding through OR-I7 in UbI7(+/+) cells, we will subtract

due to the low OR-I7 protein expression level, or the late onset of expression, in one report fewer than half the cells (44%) responded to octanal.50 Using calcium imaging on dissociated UbI7(+/+) ORNs (930 forskolin-responding ORNs, Figure 4c), we observed a 10-fold increase (from 3.7% to 35.7%) in the percentage of activated cells compared to wt (1,473 forskolinresponding ORNs) at 30 μM octanal, and a 6-fold increase (from 6.8% to 43.4%) at 300 μM. As a control stimulus, we chose 1-octanol, which is not a rodent OR-I7 receptor ligand.36,49,53 There are many rodent octanal receptors besides OR-I741 but the majority of these do not distinguish octanal from 1-octanol (e.g., 68% of endogenous octanal receptors were previously found also to be activated by 1-octanol38). Thus, a response to 1-octanol provides a crude indicator of UbI7(+/+) cells that are responding not through recombinant or F

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Figure 4. Mouse OR-I7 expression in UbI7 mice, and ratiometric calcium imaging responses to H- and D-octanal in dissociated wild-type (wt) and UbI7 ORNs. (a) In situ hybridization of wt mouse coronal sections using a probe for mouse OR-I7 mRNA shows scattered OR-I7 expression in the main olfactory epithelium. Dark spots indicate endogenous OR-I7 expression. (b) In situ hybridization of UbI7 mouse coronal sections using a probe for mouse OR-I7 mRNA shows uniform expression of OR-I7. The red arrowheads point to darker ORNs presumed to express both the endogenous and recombinant OR-I7. (c) Comparison of the dissociated wt (black bars) and UbI7 (gray bars) ORN responses to octanal by calcium imaging at 30 and 300 μM. 1473 wt and 930 UbI7 forskolin-positive ORNs were screened. (d) Comparison of the dissociated wt (black bars) and UbI7 (gray bars) ORN responses to 1-octanol by calcium imaging at 100 μM. Here, 5824 wt and 1852 UbI7 forskolin-positive ORNs were screened. (e, f) Calcium imaging responses of two representative UbI7 cells showing the expected calcium imaging profile of octanal activation through the OR-I7 receptor. Odorant concentrations, 300 μM. (g) UbI7 ORN responding to both octanal and 1-octanol. In cells displaying this profile, we cannot determine whether the octanal response is mediated solely by an endogenous receptor, or by both an endogenous receptor and recombinant OR-I7. These cells were therefore not used to form conclusions about OR-I7. (h) Three ORNs responded to 1-octanol but not to octanal, possibly due to low OR-I7 expression in a cell expressing an endogenous 1-octanol receptor that is not activated by octanal. These cells were therefore not used to form conclusions about OR-I7. Throughout this experiment, no ORN was observed to give different responses to H and D octanal. (i) Estimated number of assayed OR-I7-expressing UbI7 ORNs (endogenous plus recombinant OR-I7) and their calcium imaging response to deuterated and nondeuterated octanal. 2,026 cells responded to forskolin; 692 ORNs responded to both isotopologues of octanal. Of these 692 cells, 117 cells also responded to 1-octanol. The other 575 cells (692−117) have a high probability of responding to octanal through ORI7, and did not differentiate H- from D-octanal.

(notably, without exception, by both the H and D isotopologues). This group of 692 cells included 117 cells (5.8% of forskolin-responders) that were also activated by 1octanol. Since these 117 cells could be responding through an octanal receptor other than OR-I7, we exclude them from the H/D analysis. The remaining 575 ORNs (692−117), whose response profile matched that of OR-I7 (octanal yes; 1-octanol no) are likely responding through either the endogenous or the recombinant OR-I7 receptor. (Only three cells responded to 1octanol but not octanal.) The number of ORNs that responded to octanal and 1-octanol is summarized in Figure 4i and a heat map summary of the responses from all 692 cells can be found in Table S1. Since no cell, including those within

the number of 1-octanol-responding cells from the set of octanal-responding cells. We will then compare the H and D octanal responses within this 1-octanol-negative group of cells, which have the highest likelihood of responding through ORI7. In addition, we will use a single high concentration (300 μM) of the octanal H and D isotopologues. Since the EC50 of the rodent OR-I7 receptors is in the 1−10 μM range,36,38 using 300 μM octanal avoids the possibility of differences due to the threshold effect. We then tested 2026 forskolin-responding dissociated UbI7(+/+) ORNs with 300 μM deuterated and nondeuterated octanal and with the control of 300 μM (nondeuterated) 1octanol. A total of 692 cells (34%) were activated by octanal G

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Figure 5. Ratiometric calcium imaging results from 1551 dissociated mouse ORNs exposed to deuterated and nondeuterated p-cymene. (a) 1551 forskolin-positive ORNs were tested with 3−100 μM p-cymene. All six (0.4%) responding ORNs responded to both the deuterated and nondeuterated forms of p-cymene (category 1). (b, c) Calcium imaging traces of two of the six responding ORNs.

(category 2b, 4.7%), it could not be determined whether they truly responded to H and D isotopologues differently or if their threshold concentration, where responses are unreliable, coincided with the highest concentration tested. Category 2b cells likely express low-affinity ORs exhibiting the threshold effect (category 2a) but we cannot rule out that the discrimination would have persisted at higher concentrations. Our results show that in mouse ORNs, OR chemical recognition strategies do not generally include the ability to discriminate odors based on the small difference in hydrophobicity between H and D, or by the different bond vibrational modes of the C−H and C−D bonds, or by any other physicochemical difference between H and D. These results, like previous results with heterologously expressed ORs, suggest that if mammals can smell the difference between H and D odorants, the difference likely originates in receptorindependent processes. We note however that in our octanal and 1-octanol experiments, we found small numbers of cells able to differentiate H from D odorant isotopologues, and in the octanal case the cells appeared normal, healthy and responded strongly. We take this observation as physical evidence that one or a few mouse octanal ORs can distinguish H and D octanal. While this difference could result from sensing the different vibrational modes of the C−H and C−D bonds, it could also result from a difference in the hydrophobic effect contribution to ligand−receptor binding. In our view, the well-established but often overlooked difference in hydrophobicity between deuterated and nondeuterated receptor ligands calls into question the use of H and D isotopologues to test the vibrational theory of olfaction. Lastly, our results show that, in general, deuterated odorants cannot be expected to function as orthosteric binding site antagonists as predicted by the swipe-card vibration model.

the 575 cells that have the highest probability of responding through OR-I7, responded differently to H- and D-octanal, we conclude that mouse OR-I7 is one of the (many) octanal receptors that does not distinguish deuterated from nondeuterated octanal in sensory neurons. The Response to Deuterated and Nondeuterated pCymene. As noted above, exchanging H for D in receptor ligands reduces the hydrophobicity to the extent that it shortens the reverse phase HPLC retention time, a parameter known to be sensitive to differences in hydrophobicity, and which has been used as a substitute for logP hydrophobicity measurements.54 The HPLC traces for the four odorants used in this work, with and without deuteration, are shown in Figures S3 and S4. In all cases, deuteration decreased the retention time, indicating a reduction in hydrophobicity. Many odorants have now been tested in animals or in vitro in their deuterated and nondeuterated forms. With the exception of cyclopentadecane and benzene19 all tested compounds have contained at least one heteroatom, usually oxygen or sulfur, and thus are likely to engage in polar interactions with their ORs. We wondered if the relative strength of polar interactions like hydrogen bonds might outweigh and mask the difference in hydrophobicity between H and D. We therefore tested the nondeuterated and deuterated forms of a hydrocarbon odorant, the monoterpene p-cymene. We screened 1551 forskolin-responding dissociated mouse ORNs using calcium imaging at 3, 10, and 100 μM nondeuterated and deuterated pcymene. In accordance with the expectation of weaker binding due to a lack of polar interactions with the receptors, only 6/ 1551 cells (0.4%, n = 1) were observed to respond to p-cymene (Figure 5 and Table S1). Despite the predicted reliance of pcymene receptor binding on the hydrophobic effect and van der Waals forces, none of the activated cells showed evidence of discriminating between the deuterated and nondeuterated forms of p-cymene.

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METHODS

Isolation and Dissociation of the Olfactory Epithelium. The use and care of the laboratory animals was carried out in compliance with a protocol approved by The City College of New York Institutional Animal Care and Use Committee. For testing 1undecanol, 1-undecanol-d23, 1-octanol, 1-octanol-d17, octanal, octanal-d16, p-cymene and p-cymene-d14, 6−13 week old male C57BL/6 mice (Charles River Laboratories) were used. For the octanal and octanal-d16 experiments, two 8−9 week old UbI7 mice were used. The procedure for the dissociation of the mouse olfactory epithelium was based on the method of Araneda et al.55 Mice were anesthetized with ketamine (Ketaset) at >100 mg/kg and xylazine (AnaSed) at >10 mg/

CONCLUSION We have assayed 23,812 mouse ORNs for their ability to discriminate deuterated from nondeuterated odorant isotopologues (Table S2). Of these, 1610 ORNs responded to at least one of the tested odorants and 94.5% (1522 ORNs) responded to both the deuterated and nondeuterated odorants (categories 1, 2a, and octanal-responding UbI7 ORNs), while only 0.81% (13 ORNs, category 3) responded selectively to a single isotopologue over multiple concentrations. For some cells H

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(e.g., 1 mM DMSO solution when a 1 μM Ringer solution is desired). The working concentrations are the final concentrations of the odorants applied to the cells. The odorants were diluted to the working concentrations in the Ringer solution 0.5−4 h prior to imaging. In Situ Hybridization. Olfactory epithelium from wild-type and UbI7 mice were harvested and fresh frozen in OCT (Tissue-Tek). In situ hybridization was performed on 10 μm frozen sections using full length I7 digoxigenin-labeled antisense RNA, as previously described.57 Brightfield photomicrographs were obtained using an Aperio Scanscope CS system (Leica).

kg via intraperitoneal injection. After the unconscious mice were verified to have lost the reflex response to toe pinch stimulation, they were euthanized by decapitation. The olfactory epithelium was dissected from the heads on an ice tray and placed in chilled divalentcation-free-Ringer solution (145 mM NaCl, 5.6 mM KCl, 10 mM HEPES, 10 mM glucose, 4 mM EGTA, pH 7.4). The tissue was dissociated in 4.6 mL of divalent-cation-free-Ringer solution containing 2.62 U/ml Dispase II (Roche), 0.54 mg/mL Collagenase (Life Technologies), 3.26 mg/mL bovine serum albumin (Roche), and 0.1 mg/mL deoxyribonuclease II (Sigma-Aldrich) for 1 h at 37 °C with continuous shaking on an incubator shaker. The tissue was then placed in 37 °C culture media and agitated by brief vortexing (setting 7/10). The culture media consisted of DMEM/F12 supplemented with glutamine (Corning), 10% FBS (VWR), 1× insulin transferrin selenium ethanolamine (Life Technologies), 1% 100 U/mL penicillin, 100 μg/mL streptomycin (Cellgro), and 100 μM ascorbic acid. The dissociated cells were placed on concanavalin A (Sigma, 10 mg/mL) coated coverslips placed in 35 mm Petri dishes for 15 min at room temperature. After the cells had settled and adhered to the coverslip, 2 mL of culture media was added to each Petri dish and the dishes were placed in a 5% CO2 incubator at 37 °C for 1−4 h. Cells were then washed with Ringer solution (138 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose, pH 7.4), loaded with Ringer solution containing 6.25 μM Fura-2 AM and 0.02% pluronic acid F-127 (both Life Technologies), and left for 45 min in the dark at room temperature. The coverslips were then placed on the bottom of the imaging chamber (RC24-E, Warner Instruments), mounted on an inverted fluorescent microscope (Axiovert 200, Zeiss) using the platform and the stage adapter (Warner Instruments). The chamber was continuously washed with the Ringer solution at ∼1 mL/min using a peristaltic pump (Rainin) and a vacuum pump to sip away any excess that might overflow the imaging chamber. Stimuli were manually injected into the chamber from 5 mL syringes through a perfusion manifold (Warner Instruments). Each injection consisted of ∼0.40 mL of the stimulus and lasted 8 s. Calcium Imaging. For ratiometric calcium imaging, the inverted fluorescent microscope was equipped with a 10×/0.50 Fluar objective lens, a filter wheel, filter set for 340 and 380 nm, CCD camera (Model number 01-EXI-BLU−K-F-M-14-C, QI Imaging), and MAC 6000 controller system (Ludl Electronic Products Ltd.). This imaging system was custom-assembled by Advanced Imaging Concepts (Princeton, NJ). Time-lapse images were acquired every 4 s at 340 and 380 nm excitation and 510 nm emission, and the fluorescent intensities (F) were compiled for each cell using Metamorph software (version 7.8.6.0, Molecular Devices). The data shown are the F340/ F380 ratios vs time, graphed using Excel software after the experiment. For each experiment, odorant applications were followed by 10 μM forskolin to identify cells that maintained their signal transduction capability to the end of the experiment. Each odorant-induced response of an ORN was normalized to the forskolin-induced response of the ORN. The normalized responses from all odorantresponding ORNs were gray scale coded and summarized in heat map form in Table S1. Odorants. The deuterated odorants were purchased from CDN Isotopes. 1-Octanol (Spectrum Chemical), octanal (Sigma-Aldrich), and octanal-d16 (CDN Isotopes) were freshly purified before use by flash chromatography56 using a Teledyne Isco CombiFlash Rf-200 flash chromatography system, and solvents were removed completely before storing briefly under argon at 4 °C. For example, 100 mg of both octanal and d-octanal were chromatographed using the CombiFlash flash chromatography system with a gradient of 0% to 5% ethyl acetate in hexanes. Gas chromatography−mass spectrometry (GCMS QP-2010, Shimadzu) analysis showed chemical purities of >98% for the octanal and 1-octanol isotopologues. Gas chromatography analysis (GC-2010 with FID detector, Shimadzu) on 1undecanol (TCI), 1-undecanol-d23, p-cymene (Alfa Aesar), and pcymene-d14 showed purities of >99%. (Figure S5). All odorants were diluted in DMSO (Alfa Aesar) on the day of the experiment into concentrations 1000-fold higher than the working concentrations

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.8b00416. Odorant structures; IR spectra of odorants; LC-MS data for nondeuterated and deuterated 1-octanol, 1-undecanol, and octanal; HPLC traces of nondeuterated and deuterated p-cymene; gas chromatography traces of tested compounds and 1H NMR of octanal; calcium imaging traces from uncategorizable cells from 1undecanol and octanal experiments; calcium imaging traces from ambiguous cells from UbI7 experiments; calcium imaging results for all responding cells (heat map format); summary of all tested ORNs, responding ORNs, and category assignment (PDF) Results for all ca. 1600 cells tabulated in heatmap form (XLS)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kevin Ryan: 0000-0001-5304-2026 Author Contributions

M.N. customized the fluorescence microscope, designed the experiments, carried out calcium imaging experiments, and cowrote the manuscript. M.T.L. purified and characterized all odorants. M.Q.N. carried out in situ hybridization experiments. K.R. directed the project, designed the experiments and wrote the manuscript. Funding

This work was funded by the U.S. Army Research Laboratory and the U.S. Army Research Office under Grant Number W911NF-13-1-0148 (to K.R.). Additional support was provided by grants 5G12RR003060 from the National Center for Research Resources, and 8G12MD7603 from the National Institute On Minority Health and Health Disparities. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank L. Belluscio for providing the UbI7 mice and Xinghua Ma for cymene HPLC. The HPLC/MS analysis was kindly provided by Rinat Abzalimov of the CUNY ASRC Biological Mass Spectrometry Facility. We thank John Lombardi for helpful discussions. I

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ABBREVIATIONS OR, olfactory/odorant receptor; ORN, olfactory/odorant sensory neuron; D, deuterium

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