ABSTRACT
Inan effort to understand mammalian olfactory processing, we have been describing the responses to systematically differentodorants in the glomerular layer of the main olfactory bulb of rats. To understand the processing of purehydrocarbon structures in this system, we used the [14C]2-deoxyglucosemethod to determine glomerular responses to a homologous series of alkanes(from six to sixteen carbons) that are straight-chained hydrocarbons withoutfunctional groups. We found tworostral regions of activity evoked by these odorants, one lateral and onemedial, that were observed to shift ventrally with increasing alkane carbonchain length. Furthermore, wesuccessfully predicted that the longest alkanes with carbon chain lengthgreater than our previous odorant selections would stimulate extremely ventralglomerular regions where no activation had been observed with the hundreds ofodorants that we had previously studied. Overlaps in response profiles were observed in the patterns evoked byalkanes and by other aliphatic odorants of corresponding carbon chain lengthdespite possessing different oxygen-containing functional groups, whichdemonstrated that hydrocarbon chains could serve as molecular features in thecombinatorial coding of odorant information. We found a close and predictable relationship among themolecular properties of odorants, their induced neural activity, and theirperceptual similarities.
Studiesby a number of laboratories using odorants that differ systematically inmolecular structures continue to support a combinatorial model of olfactoryprocessing initially proposed by Polak (1973), in which molecular features ofan odorant are recognized and encoded by the system (Kauer and Cinelli, 1993;Friedrich and Korsching, 1997, 1998; Joerges et al., 1997; Johnson et al.,1998, 1999, 2002, 2004, 2005a, b; Malnic et al., 1999; Rubin and Katz, 1999;Sachse et al., 1999; Johnson and Leon, 2000a, b; Uchida et al., 2000; Belluscioand Katz, 2001; Fuss and Korsching, 2001; Meister and Bonhoeffer, 2001;Igarashi and Mori, 2005). According to this model, odorants that share a molecular feature shouldbe recognized by the same type of olfactory receptors to produce overlappingneural activity. To test thisprediction, and to understand how the olfactory system responds to variousmolecular features, our laboratory has been studying responses to hundreds ofodorants in the glomerular layer of the rat main olfactory bulb (Johnson etal., 1998, 1999, 2002, 2004, 2005a, b; Johnson and Leon, 2000a, b; Linster etal., 2001a). In an effort to understand the processing of purehydrocarbon structures in this system, we determined in this study the responseto a series of alkanes, which are straight-chained hydrocarbons withoutfunctional groups.
The responses to the odorants studiedthus far collectively involve most of the glomerular layer, with the markedexception of an area in the midventral region. From studies of numerous homologous series of odorants up toeleven carbons in the chain, it was clear that larger odorants systematicallyevoked responses that were progressively ventral (Johnson et al., 1999, 2004;Johnson and Leon, 2000b). Wetherefore hypothesize that the previously unresponsive ventral area mightrespond to even larger odorants than we had yet studied (Johnson et al., 2002). Since straight-chainedalkanes remain volatile even at great molecular length, we used the [14C]2-deoxyglucose(2-DG) method developed by our laboratory to examine glomerular responses to ahomologous series varying from six to sixteen carbons, and predicted thatresponses to the larger odorants in this series would be observed in themid-ventral area of the bulb.
Eventhough glomerular responses have been observed to be organized topographicallyand have been found to be correlated with a number of molecular features ofodorous stimuli (Imamura et al., 1992; Katoh et al., 1993; Friedrich andKorsching, 1997, 1998; Joerges et al., 1997; Johnson et al., 1998, 1999, 2002,2004, 2005a, b; Johnson and Leon, 2000a, b; Fuss and Korsching, 2001; Meisterand Bonhoeffer, 2001; Takahashi et al., 2004; Igarashi and Mori, 2005), itremains unclear whether such chemotopic information is maintained throughdownstream perceptual processing, so that odorant-evoked responses in the olfactorybulb would predict perceptual phenomena such as odor recognition anddiscrimination. Indeed, fewstudies have examined directly the relationship between the similarity ofoverall glomerular responses and perceived odor similarity (Linster et al., 2001a;Cleland et al., 2002). Nevertheless, these studies have shown a high correlation between theevoked glomerular patterns and odor perception. Aspects of odorant chemistry also have been found tocorrelate with odor discriminability in a number of psychophysical studies(Laska and Freyer, 1997; Laska and Teubner, 1999; Laska et al., 1999, 2000;Linster and Hasselmo, 1999; Laska and Galizia, 2001; Laska and HŸbener, 2001;Linster et al., 2001a, b; Cleland et al., 2002). Together, these results suggest a close and predictablerelationship among odorous stimuli, glomerular responses and perceivedodors. In all of these cases,however, the odorants used involved molecules with oxygen-containing functionalgroups, which are well known to have profound impact on perceived odor. In fact, aliphatic odorants containingthe same oxygen-containing functional group were found to be discriminable evenwith just a one-carbon difference in their chain length (Laska and Freyer,1997; Laska and Teubner, 1999; Laska et al., 1999; Linster and Hasselmo, 1999;Laska and Galizia, 2001; Laska and HŸbener, 2001; Cleland et al., 2002).
Odorantspossessing oxygen-containing functional groups can interact with receptors byway of hydrogen bonds. Incontrast, alkane odorants can only interact with receptors by way of Van derWaals forces, which are much weaker than hydrogen bonds. The weakness of these interactionswould be expected to result in low-affinity binding. Given the likelihood of low-affinity receptor binding, itwas not clear whether the pattern of glomerular activity evoked by these alkaneodorants would reveal the same degree of specificity that has characterized thepatterns evoked by the numerous oxygen-containing odorants that we have studiedpreviously. It also was not clearwhether these alkanes would be perceived to have significantly distinctodors. Indeed, these odorantsoften are given the same Ògasoline-likeÓ odor description by humans (see, forexample, http://hazmap.nlm.nih.gov/). Therefore, we investigated the triad relationships among odorantchemistry, bulbar activation patterns, and perceived odors using a homologousseries of pure alkanes.
MATERIALS AND METHODS
Odorants
Table1 contains detailed information regarding all odorants included in this study,which consisted of four independently conducted experiments. The initial experiment used ahomologous series of straight-chained alkanes varying from six to sixteencarbons. The experiments examiningodorant concentration and odorant purity tested various concentrations ofheptane and four samples of pentadecane from different manufacturers,respectively. 2-Dodecanone and8-pentadecanone were examined in a separate experiment to be compared to thealkanes. For each alkane, 100 mLof neat odorant was placed in a 125-mL gas-washing bottle equipped with anextra-coarse porosity diffuser. The ketones were first dissolved in mineral oil at a 1/20 dilution,before 200 mL was placed in a 500-mL gas-washing bottle. During each exposure, highly pureresearch-grade nitrogen was passed through the odorant column at a rate of 250mL/min (100 mL/min for ketones). The resulting vapor was mixed with ultra-zero grade air, after which themixture was delivered to the exposure chamber at a rate of 2 L/min (1 L/min forketones). The final vapor phaseconcentration listed in Table 1 for each odorant was calculated using the modeof vapor pressure data obtained from the following sources: PhysProp Databasefrom Syracuse Research Corporation (http://www.syrres.com/esc/physdemo.htm),the Chemical and Physical Properties Database from the Pennsylvania Departmentof Environmental Protection(http://www.dep.state.pa.us/physicalproperties/CPP_Search.htm), MolecularModeling Pro version 3.14 (ChemSW, Fairfield, CA), and ChemDraw Ultra version6.0 (CambridgeSoft, Cambridge, MA).
Odorant exposures
Allprocedures involving animals were approved by the Institutional Animal Care andUse Committee of the University of California, Irvine. Odorant exposures were conducted asdescribed in our previous studies (Johnson et al., 1998, 1999, 2002, 2004,2005a, b; Johnson and Leon, 2000 a, b). Eighteen- to twenty-one-day-old Wistar rats, culled to eight per litter,were used for all experiments. Inorder to reduce odors carried over from the soiled housing cage, each litterwas transferred to a clean cage with the dam at least one hour before anexposure. Prior to theintroduction of an animal, the exposure system first was equilibrated with theodorant of interest for fifteen minutes. Individual pups were injected subcutaneously at the back of the neckwith a dose of [14C]2-DG determined by body weights (1.6 µL/g, 0.1mCi/mL, 52 mCi/mmol, Sigma Chemical Company, St. Louis) before being placed ina clean, 1-L glass jar used as the exposure chamber. Each exposure lasted for forty-five minutes, with theodorant entering and exiting the glass jar through two vents in the jar lid, sothat odorant concentration increased steadily at the beginning of theexposure. Animals' brains thenwere immediately removed, frozen in isopentane at -45oC, and storedat -80 oC.
Thenumber of animals exposed to each odorant condition is shown in Table 1. The first pup obtained from each litterwas always exposed to the vehicle, which was air for the alkane experiments andmineral oil for the ketones. Notwo rats from the same litter were exposed to the identical odorantcondition. The order of odorantpresentation was pseudo-randomized across litters to avoid systematicrelationships among odorants, except for in the heptane concentrationexperiment, where exposures were done in order of increasing odorantconcentration within each day.
Measurementand analysis of 2-DG uptake
Histology,autoradiography, and activity mapping were performed as described in ourprevious studies (Johnson et al., 1998, 1999, 2002, 2004, 2005a, b; Johnson andLeon, 2000 a, b). Briefly, foreach coronal bulb section, an appropriate selection from a set of polar gridswas chosen according to the number of bulb sections between anatomicallandmarks and was applied to the sectionÕs autoradiographic image overlaid withthe image of the adjacent Nissl-stained section. The grids guided the collection of 2-DG uptake measurements fromthe glomerular layer throughout the entire olfactory bulb into standardizedmatrices. After the subtraction ofmeasurements from vehicle, each individual data matrix was converted toz-scores. Matrices from the sameodorant condition within a study were averaged to yield an average z-score patternfor that condition. However, forstatistical purposes, individual z-score patterns, rather than average z-scorepatterns from a single experiment were subjected to ANOVA followed by falsediscovery rate analyses (Curran-Everett, 2000; Johnson et al., 2002, 2004,2005a, b) to test for statistical differences among odorant-induced responsesin thirty predetermined areas termed glomerular modules, including twenty-sevenof them that were described in our previous studies (Johnson et al., 2002,2004, 2005a, b) and three new response modules that were defined based onglomerular responses to the longer alkanes in this study. The use of these predetermined modulesin the present study does not imply their functional significance within theolfactory system. Rather, itserves as a tool both to ensure an unbiased simplification of the activitypatterns for statistical analysis and to facilitate comparisons with previousstudies that were analyzed with respect to the same modules.
The centroid ofactivity within an outlined glomerular region was determined separately for thelateral and the medial aspects of each odorantÕs response pattern. To examine whether a significantdirectional shift of glomerular activation existed, dorsal-ventral centroidcoordinates for all alkanes were included in an ANOVA test.
Overall patternsimilarity among glomerular responses was analyzed by using Pearson correlationand principal components analysis. Each average response pattern for an odorant actually represented az-score array containing over 2,300 values. Using Pearson correlation (Johnson et al., 2002; 2004, 2005a, b), point-to-point comparisons could be made between any two patterns, andthe level of overall pattern similarity produced by two odorants was indicatedby the correlation coefficient (r). Correlation results from all possible paired comparisons within thishomologous series of alkanes were further subjected to principal componentsanalysis (StatView¨, SAS Institute, Cary, NC) to reveal theclustering of glomerular responses to these odorants. The components were extracted using the scree test and screeplot, with initial orthogonal solution matrices undergoing at least one obliquetransformation to optimize their structures. These procedures also were repeated with individual responsepatterns.
Olfactory discrimination
Perceived odor similarity was assessed using a habituation assay similar tothat described in a prior study (Linster et al., 2001a). Adult male Wistar rats were handled andshaped in the behavior testing apparatus for seven consecutive days prior toexperimentation. The testingapparatus included a clean test cage similar to the home cages (30 cm L x 20 cmW x 19 cm H), and a clear, customized cage lid with evenly distributed 1-cmwide holes and a slit for a cage separator, which could be lowered verticallyto separate the length of the cage into two compartments.
Animals were food-deprived for two days, through the completion ofindividual behavioral testing, which was conducted under dim light during thedark phase of a reverse light-dark cycle. An animal was placed in a clean cage, and a plastic cap lined with cleanfilter paper was positioned over one of the holes in the cage lid. A ten-minute control period then beganimmediately, during which animals were allowed to investigate the cage. Upon the termination of the controlperiod, the cage separator was lowered to confine the rat to one side of thecage. An odorized cap was then puton top of the other side of cage, and the first experimental trial startedafter the separator was lifted. Each experimental trial lasted for two minutes, followed by a ten-minuteinter-trial period.
The same odorant was presented for the first three trials to familiarize andhabituate animals to this odorant. Test odorants then were alternated with the familiar odorant for theremaining trials. Threeexperimental series were conducted with different odorant sets (Table 2). Thepresentation order of test odorants was varied across animals in such a way toavoid systematic relationships among odorants. To control for performance fatigue, the final trial used anodorant different from the experimental odorant set based on molecularproperties and glomerular activity patterns. An additional experiment wasperformed to test the discriminability between two samples of pentadecane,Fluka and Acros. All trials wererecorded on videotape for subsequent analysis.
Behavioral data analysis
The total amount of time each animalspent investigating the odorant during each trial was determined. Investigation was defined as activesniffing within 1 cm of the odorized cap. Animals were excluded from further analysis if they failed to habituateto the familiar odorant by the third trial, or failed to investigate odorantsduring the first or the last trial. Habituation was indicated by the ratio of investigation time in thethird trial to that in the first trial, and was considered insufficient if theratio exceeded 0.15, which was the ratio averaged across animals from pilotstudies. We imposed these criteriabecause habituation to the first odorant was required for it to serve as thereference odorant to be compared with all subsequent test odorants, whereas alack of investigation in the first or the last trial may indicate differentkinds of behavioral problems. Ageneral low level of interest in or motivation for the behavioral task may beindicated if there were no odorant investigation during the first odorant presentation. Meanwhile, performance fatigue maycause a lack of investigative behavior in the final trial. These criteria were satisfied by 11-12out of 24 animals for each odorant series. Investigation time was sorted by the carbon numberdifference between test and familiar odorants. Data from the same carbon-number difference group in each ofthe three experimental series were collapsed together, and analyzed forstatistical differences with an overall ANOVA, followed by DunnettÕs posthoc tests.
Overall2-DG uptake pattern similarity between test and familiar odorants also wasexamined. Pairs of individualz-score patterns were compared using Pearson correlation as describedabove. Similar to the behavioraldata, the resulting Pearson coefficients were first sorted by the carbon numberdifference between test and familiar odorants, and then combined acrossexperimental series before being subjected to the same statisticalanalyses.
RESULTS
Chemotopic progression of predicted but novelglomerular activation
Eventhough responses to this homologous series had not been examined systematicallybefore the current study, based on our previous observations (Johnson et al.,1999, 2002, 2004; Johnson and Leon, 2000b), we predicted that these odorantswould produce chemotopically organized responses, with increasingly ventralglomeruli being activated by increasing carbon chain length. The molecular length of the longeralkanes was greater than the odorants that we previously had studied, and wetherefore expected that these alkanes would reveal a novel activation in theanterior to mid-ventral glomerular layer where other odorants had not beenobserved to evoke a response. Glomerular 2-DG uptake patterns evoked by the homologous series areshown in Figure 1, and were found to be significantly different using ANOVAtests (p < 0.05) for uptake inpreviously defined regions of the bulb followed by false discovery rateanalysis (Curran-Everett, 2000; Johnson et al., 2002, 2004, 2005a, b). Presumably because these alkanes do notcontain any functional groups with only their straight hydrocarbon backbone,none evoked observable activation in any glomerular regions previouslyidentified to respond to various functional groups (Johnson and Leon, 2000a;Johnson et al., 2002, 2004. 2005a,b), except for hexane (C6) and heptane(C7). The lateral-medial paireddorsal glomerular clusters activated by hexane and heptane (Fig. 1A) have beenactivated in other studies by aliphatic ketones and some aromatic odorants thatdo not share apparent odorant features (Johnson and Leon, 2000a; Johnson etal., 2002, 2004, 2005a, b). Octane(C8) evoked responses in a lateral-medial pair of more ventrally located foci,which were enclosed by the black outline (Fig. 1A). The location of these foci corresponded well with octane-inducedresponse predicted from studies of the olfactory epithelium (Mozell, 1966;Scott et al., 1996; Scott et al., 2000) and observed in studies of theolfactory bulb using electrophysiological recording of mitral/tufted cells(Imamura et al., 1992; Igarashi and Mori, 2005) as well as 2-DG (Johnson etal., 2005a). As we predicted, theactivity foci shifted more ventrally towards the center of the black outlinewith longer alkanes, extending to more ventral paired areas with dodecane(C12), and continuing until activity merged at the ventral midline withtetradecane (C14). The extremeventral activation can be appreciated more readily when a pattern (circled inpink) evoked by the 15-carbon alkane, pentadecane, was overlaid onto a3-dimensional glomerular layer model that has been rotated to show the responseon the ventral midline of the bulb (Fig. 1).
Thechemotopic progression of glomerular responses was examined further with acentroid analysis (Johnson and Leon, 2000a, b; Johnson et al., 2004, 2005a, b). The centroid of 2-DG uptake wascalculated separately for the lateral and the medial aspects of individualmatrices within the region outlined in black in Figure 1. The mean dorsal-ventral centroidcoordinate for each alkane is shown in Figure 2. In both the lateral (Fig. 2A) and the medial (Fig. 2B)regions, an overall ventral shift of centroid position was observed from eightto fourteen carbons within the homologous series of alkanes. Centroids were confirmed to bestatistically significant across the whole series in both the lateral (F(10,33) = 6.69, p< 0.001) and medial (F(10,33) = 8.01, p< 0.001) glomerular layer by an ANOVA analysis. However, we noticed that the most ventral centroid positionwas seen with tetradecane (C14), rather than the longest hexadecane (C16). In fact, the centroid position shiftedback dorsally again with pentadecane and hexadecane (Fig 2). This dorsal progression could be explainedby the presence of glomerular activation in the more dorsal region towards thetop and bottom of the black outline similar to that observed with octane (Fig.1A).
Minor impurities greatly impact uptake patterns
Accordingto the reliable chemotopic organization previously observed (Johnson and Leon,2000b; Johnson et al., 1999, 2002, 2004, 2005a, b), and found here for smalleralkanes, the more dorsal glomerular activation by the longest alkanes in theseries was surprising. Given thelow vapor concentrations of these large odorants, we considered the possibilitythat the dorsal responses were due to an odorous contaminant in pentadecane andhexadecane. To test this possibility, we examinedglomerular responses to pentadecane samples of different purity grades. In addition to the original 99% purematerial, two other samples of the same purity grade from different vendorswere tested along with a 99.8% pure odorant.
All three 99% pure samplesproduced activity both at the ventral midline and in the more dorsal and caudalregions outlined in Figure 3. However, the more dorsal and caudal activation was absent in theresponse to the 99.8% pure pentadecane, leaving only activation at the ventralmidline (Fig. 3). Modular ANOVAsfollowed by false discovery rate analyses revealed a significant differenceamong these activity patterns (p< 0.05). Specifically, whenresponses to the Acros (99% pure, our original source) and the Fluka (99.8%pure, the highest purity) samples were compared across glomerular modules usingpaired t-tests, significant differences (p < 0.05) were found only in areas included by theoutlines (Fig. 3). In addition, aPearson correlation comparison of these responses indicated a relatively lowlevel of overall pattern similarity (r = 0.42). These results supported the hypothesis that the unexpecteddorsal activation observed in the responses to the longest alkanes was producedby impurities present in odorants from different sources. Indeed, further analysis of theseodorant samples using gas chromatography-mass spectrometry (GC-MS) detectedminor contaminants in the 99% pure materials that were absent from the purerpentadecane sample. Moreover,different contaminants identified among the 99% pure samples suggest that theymay have been obtained by using distinct starting materials and/or productionprocesses, so that subsequent differences in impurities may have contributed tothe observed differences in their evoked neural responses. Given that there was a significantdifference and a low correlation between the glomerular patterns evoked by the99% pure pentadecane and the 99.8% pure pentadecane, we predicted that ratsmight discriminate spontaneously between these two odorant samples. A discrimination experiment wasconducted to confirm this prediction, and its results will be discussed below.
Overall pattern similarity among alkane-evokedglomerular responses
Studies of other homologousseries of odorants have shown that they evoke neural responses in the olfactorybulb that become increasingly dissimilar with greater differences in carbonnumber between odorants (Imamura et al., 1992; Katoh et al., 1993; Johnson etal., 1999, 2002, 2004; Rubin and Katz, 1999; Belluscio and Katz, 2001; Meisterand Bonhoeffer, 2001). UsingPearson correlation (Johnson et al., 2002, 2004, 2005a), point-to-pointcomparisons can be made between glomerular responses elicited by any two of theodorant conditions to examine their overall pattern similarity, which isindicated by a resulting single value, the correlation coefficient (r). The relationship among glomerularresponses to the homologous series of alkanes can be appreciated more easily bysubjecting a correlation matrix containing all possible paired comparisons ofthese responses to a principal components analysis. Three major clusters of odorants were revealed consequentlyin the olfactory space represented by these glomerular responses (Fig.4A). Hexane and heptaneconstituted one of the clusters, and were accounted for mostly by the secondcomponent (Fig. 4A). A secondgroup was associated with the first as well as the second components, andincluded octane, nonane, and decane, which were not closely clustered with anyother alkanes (Fig. 4A). Longeralkanes with at least 11 carbons in their hydrocarbon structures formed thelast cluster, and were separated from the other odorants largely by the firstcomponent (Fig. 4A). Thedemonstrated dissimilarity between neighboring alkanes and odorant clusterscorresponded to the glomerular activity patterns described above (Fig. 1A). Hexane and heptane evoked high levelsof uptake in the dorsal bulb both laterally and medially in regions whereoctane and all other odorants did not (Fig. 1A), a finding that probablyexplained both the dissimilarity between heptane and octane and the clusteringof hexane and heptane (Fig. 4A). Meanwhile,the clustering of the glomerular response for longer alkanes (Fig. 4A) may be aconsequence of the more ventral glomerular stimulation shared by the responsesto these odorants (Fig. 1A).
A separate principal components analysis was performed on another Pearsoncorrelation matrix containing paired comparisons of individual, rather thanaverage, response patterns (Fig. 4B). Overall, clustering of odorants was comparable to that observed inFigure 4A. However, individualdifferences between subjects exposed to the same odorant condition were greaterwith longer, more hydrophobic alkanes.
Concentration-dependent glomerular activation
Differencesin the activation of the dorsal glomerular layer led to a more pronouncedseparation of the hexane-heptane cluster from the rest of the alkanes than wasexpected for odorants in a homologous series, especially between neighboringmembers such as heptane and octane. In this study, however, odorants were presented at equal dilutionsrather than at equal vapor concentrations in order to assure that there wouldbe responses to odorants at both ends of the series. Therefore, differences in odorant concentration mayhave contributed to differences in evoked activity patterns. In particular, we had previouslyobserved stimulation of the dorsal glomerular layer, similar to the areaactivated by heptane, with higher concentrations of 2-hexanone, which did notevoke activity in the same area at lower concentrations (Johnson and Leon,2000a; Johnson et al., 2004). Based on those results, we hypothesized that lower heptaneconcentrations might induce significantly less activity in the dorsal bulb,resulting in higher pattern similarity with octane.
Wetested five concentrations of heptane, with the highest level identical to theconcentration used in the original homologous series experiment (Table 1). Modular ANOVA tests followed by thefalse discovery rate analysis as described above revealed dorsal areas, pairedlaterally and medially, to be the only regions giving significantly differentresponses to different concentrations of heptane (Fig. 5). Indeed, strong activation of thesedorsal areas was only evident at 7500 ppm, the highest concentration that wetested (Fig. 5). This effectappeared to be more robust in the medial bulb, as indicated by the whitearrows, than in the lateral aspect (Fig. 5A). Interestingly, the response pattern evoked by 2200 ppm ofoctane (Fig. 1A, Table 1) showed the highest correlation (r = 0.82) with theactivity pattern produced by the most closely matched heptane concentration of2500 ppm.
Combinatorial coding of molecular features
Combinatorialmodels of olfactory processing predict that odorants with a shared molecularfeature should be encoded by the same neural elements to elicit overlappingneural responses (Friedrich and Korsching, 1997, 1998; Joerges et al., 1997;Johnson et al., 1998, 1999, 2002, 2004, 2005a, b; Malnic et al., 1999; Rubinand Katz, 1999; Sachse et al., 1999; Uchida et al., 2000; Belluscio and Katz,2001; Fuss and Korsching, 2001; Meister and Bonhoeffer, 2001; Leon and Johnson,2003). Because straight-chainedalkanes do not contain any functional groups other than their simplehydrocarbon structures, the molecular features that were recognized by theolfactory system to produce the observed responses must exist solely within thecarbon chain. Many aliphaticodorants that contain functional groups also possess the same straighthydrocarbon backbone, so that the alkanes would be predicted to evoke a similarglomerular activation as other aliphatic odorants that have differentfunctional groups, as long as they share a similar hydrocarbon chain.
Wetherefore compared glomerular responses to the eight-, twelve-, andfifteen-carbon alkanes with the responses to the corresponding aliphaticalcohol or ketone in separate experiments (Fig. 6). Similar ventral responses were observed for each pairedcomparison, and are indicated by white arrows of similar orientations. Octane(eight-carbon alkane) and 2-octanol (eight-carbon alcohol; Johnson et al.,2004) elicited overlapping responses in the octane activity foci describedabove (Fig. 1A). Overlapping areasof activation were also observed more ventrally between the twelve-carbondodecane (alkane) and 2-dodecanone (ketone), and at the ventral midline betweenthe fifteen-carbon pentadecane (alkane) and 8-pentadecanone (ketone; Fig.6). Since the shared carbon chainlength elicited a common glomerular response, despite differences in functionalgroups, this result indicated that molecular features recognized by the systemwere indeed contained within the aliphatic carbon chain.
It is noteworthy to mention thatlight mineral oil, in which the ketones were diluted, can contain about 2% offifteen- and sixteen-carbon alkanes (Fisher Scientific Technical Support).However, the volatility of these long alkanes is so low that their minorpresence in mineral oil is unlikely to result in high enough vaporconcentrations to evoke an olfactory response. Moreover, the subtraction of the mineral oil vehicleresponse from the responses produced by these ketones should have removed anypossible effects of long alkanes that may exist in the mineral oil. Therefore, the presence of fifteen- andsixteen-carbon alkanes in mineral oil cannot account for the shared glomerularrepresentation at the ventral midline produced by 8-pentadecanone andpentadecane.
Neurobehavioral correlates of straight-chained alkanes
To determine if there were ameaningful relationship between odor perception and the patterns of 2-DG uptakeevoked by alkanes, we compared glomerular activity patterns evoked by thealkane homologous series with the behavioral responses to these odorants usingan odor habituation assay that depends on the reliable willingness of rats toinvestigate novel odorants in their environment (Linster et al., 2001; Clelandet al., 2002). With repeatedexposure, however, the novelty of the odorant dissipates and rats are less andless likely to investigate that odorant (Fig. 7A). If a second odorant is then placed in their environment,rats are likely to investigate it if they regard the new odor as beingdifferent from the habituated odor. Using this assay, we tested whether the perceived odors became moredifferent with greater differences in alkane carbon number. Behavioral results including allanimals from each experimental series were presented after being sorted intodisqualified and qualified groups based on our criteria that were determined apriori (Fig. 7). Major differences in the behaviorbetween the qualified and disqualified groups of animals were exhibited duringthe habituation trials (Fig. 7A). However, both groups of animals expressed similar levels ofinvestigation of the control odorant that tested for motor fatigue (Fig. 7B),so that very few animals in this study were excluded for statistical analysesdue to a lack of responses to control odorants. These animals also exhibited comparable behavior during testodorant trials (Fig. 7C). Together, these observations support the notion that the analysiscriteria were unlikely to have selected for a group of animals that weresystematically different from the excluded group. Using only data from the qualified group (Fig. 8A),statistical significance was demonstrated with an ANOVA (F(4, 197) = 5.64, p = 0.00026), and DunnettÕs post hoc tests revealed that the meaninvestigation times for test odorants that differed by at least one carbon weresignificantly greater than investigation times for the familiar odorant withzero carbon difference. Thispattern of response was also evident when each habituated odorant wasconsidered separately (Fig. 7C). The mean investigation time for the two- and three-carbon differencegroups also was found to be significantly different from the one-carbondifference group. Overall,longer investigation time, presumably indicating more dissimilar perceivedodors, was observed with larger carbon differences between test and familiarodorants.
Our behavioral results have supported arelationship between the molecular similarity of odorants and their perceptualsimilarity. However, to be able tocompare neural activity and behavior results meaningfully, we also had todetermine whether a relationship existed between odorant properties andodorant-induced activity patterns. To test for this relationship, individual z-score patterns representingodorants involved in the behavioral experiments were compared for overallpattern similarity using Pearson coefficients (Johnson et al., 2002,2004). The resulting Pearsoncoefficients were sorted according to the carbon number difference between testand familiar odorants as with the behavioral data, and then were averaged foreach group (Fig. 8B). Again, ANOVAfollowed by the DunnettÕs tests showed that odorants differing by at least onecarbon from the familiar odorants produced significantly less correlatedglomerular response patterns (F(4,253)= 10.56, p =0.0021). In general, decreasingpattern similarity was observed with increasing carbon number difference,except for a slight increase observed for the four-carbon group. Interestingly, the four-carbon groupshowed a similar reversal in the behavior data (Fig. 8A). This group, with the largest carbonnumber difference in the current study, included only comparisons betweenpentadecane and undecane. Both ofthese odorants elicited glomerular activation (Fig. 1A) in the dorsal regionsoutlined in Figure 3. Theirconsiderably overlapping activation in that particular region may be responsiblefor higher pattern similarity, as well as perceptual similarity (Fig 8A),between the two odorants.
In Figure 8C, the meaninvestigation time (Fig. 8A) was plotted against the pattern similarity data(Fig. 8B). Increasing patternsimilarity was associated with decreasing investigation time, which indicatedincreasing perceptual similarity. Indeed, a regression analysis yielded a significant negative correlationbetween the two (r =-0.96, p =0.0067).
In addition, we tested the ability of rats to discriminate between the 99.8%and the 99.0% pentadecane in a separate experiment. Rats (n = 15)habituated to the purer sample were found to spend significantly longer time (t = 2.25, p= 0.016) investigating the99.0% test odorant (mean investigation time = 1.67s, s.e.m. = 0.65) thanthe 99.8% sample (mean investigation time = 0.18s, s.e.m. = 0.09), indicating that they could spontaneously discriminatebetween different purity grades of the same compound. Again, differences in glomerular response patterns evoked bythese odorants predicted differences in perception (Fig. 5B). Itis also interesting that such a minor contaminant dramatically alters anodor.
DISCUSSION
Predictions of a combinatorial code
If odorants are coded by the combination of theirmolecular features, then one should predict that shared features would evoke ashared neural response. Indeed,the glomerular responses evoked by pure straight-chained hydrocarbons wereshared by odorants with that feature accompanied by different combinations ofother molecular features. Thesedata therefore strongly support the notion of a combinatorial code forolfaction.
Glomerular response patterns may emerge from acombination of the inherent and imposed interactions between the olfactory epitheliumand odorants
Wehave observed a chemotopic organization of responses to a homologous series ofstraight-chained alkanes that extended into a ventral glomerular region (Fig.1) that had not been stimulated by any of the hundreds of odorants examined inour previous studies (Johnson et al., 1998, 1999, 2002, 2004, 2005a, b; Johnsonand Leon, 2000a, b). Consistentwith prior results, increasingly ventral glomeruli tended to respond to longerodorous molecules (Johnson et al., 1999, 2004; Johnson and Leon, 2000b). Alkanes of greater length than wereused in this homologous series probably would not be sufficiently volatile forreceptor activation, which may explain why pentadecane stimulated the most ventralregion of the bulb. It also mayexplain why hexadecane did not stimulate the extreme ventral aspect of the bulb(Fig. 1A); that molecule may not be volatile and the glomerular activity thatwas seen may have been evoked by volatile contaminants. Larger alkanes, such as octadecane(C18), are actually solids at room temperature (PhysProp Database from SyracuseResearch Corporation: www.syrres.com/esc/physdemo.htm). It is interesting to note that mouseurine, which evoked activity in partially overlapping ventral glomerularregions (Schaefer et al., 2002), have been shown recently to containnon-volatile components probably of great molecular length (Kimoto and Touhara,2005).
Among odorants in a homologous series, a number of molecular propertiesusually co-vary, so that the exact property associated with the change inglomerular response often becomes difficult to pinpoint. Thus far, only one study (Johnson andLeon, 2000b) has been conducted to demonstrate that molecular length, ratherthan other molecular properties, correlated with the ventral shift ofglomerular activation observed with a homologous series of carboxylic acids(Johnson et al., 1999). Studiesthat further examine the molecular features responsible for chemotopicresponses produced by other homologous series are warranted.
This chemotopy along the dorsal-ventral axis, which exists locally withinresponse modules and globally throughout the olfactory bulb, may be explainedby the distribution of olfactory receptor types in the olfactory epithelium(Hornung and Mozell, 1977; Schoenfeld and Knott, 2004), as well as themaintenance of that topography by way of axonal projections to the glomerularlayer (Astic and Saucier, 1986; Ressler et al., 1994; Schoenfeld et al., 1994;Vassar et al., 1994; Schoenfeld and Knott, 2002; Schoenfeld and Knott,2004). Indeed, preferentialresponses of the dorsal glomerular layer to small odorants with an oxygenmoiety (Johnson et al., 1998, 1999, 2002, 2004, 2005a, b; Johnson and Leon,2000a, b) coincides with the dorsal epithelial expression of Class I olfactoryreceptors (Conzelmann et al., 2000; Zhang and Firestein, 2002; Zhang et al.,2004), which appear to have high affinity for small and/or hydrophilic odorants(Malnic et al., 1999; Sanz et al., 2005). Such odorant-evoked responses due to the intrinsic response specificityof olfactory receptors have been referred to as inherent responses (Moulton,1976; Mozell et al., 1987).
However, it appears that mucosal sorption of odorous molecules also maycontribute to the observed chemotopy within the olfactory bulb. Mozell and colleagues (Mozell, 1964,1970; Mozell and Jagodowicz, 1973; Hornung and Mozell, 1977; Mozell et al.,1987; Hahn et al., 1994; Kent et al., 1996; Keyhani et al., 1997) have demonstratedthat smaller and more hydrophilic odorants are absorbed rapidly by the aqueousmucosa upon entry into the nasal epithelium, and therefore are likely to havelimited access only to the dorsal central channels towards the entrance of thenasal cavity (Schoenfeld and Knott, 2004). On the other hand, larger and more hydrophobic odorants,including octane, were found to be able to disperse more freely throughout theolfactory epithelium than the hydrophilic ones. This way, increasingly hydrophobic odorants that aresufficiently volatile, such as longer alkanes in this study, may be more likelyto access the entire epithelium, even the most peripheral parts that containreceptor neurons projecting to the most ventral glomerular layer. Our results therefore support possiblechromatographic properties of the epithelium, and the resulting epithelialresponses have been referred to as imposed responses (Moulton, 1976; Mozell etal., 1987).
Responses to various odorants may be affected differently by their inherentand imposed interactions with the epithelium. More hydrophilic, and therefore more rapidly absorbedodorants were found to produce responses dominated by the imposed activity,whereas more hydrophobic odorants evoke activity that is more reflective oftheir inherent interactions (Mozell et al., 1987). However, both the inherent and imposed components may worktogether to facilitate the processing of odorous molecules at this level(Moulton, 1976; Mozell et al., 1987). An example of this synergism would be the affinity of Class I receptorsfor hydrophilic molecules and their localization in the dorsal olfactoryepithelium where the majority of such molecules are likely to be absorbed uponentry into the aqueous mucosa (Hornung and Mozell, 1977; Schoenfeld and Knott,2004). This intrinsic organizationof the receptors may allow a subset of the receptors to respond optimally tosmaller and/or hydrophilic odorants due to both their location and their high-affinityresponses to odorant features (Malnic et al., 1999; Mezler et al., 2001; Zhangand Firestein, 2002). Odorant-evoked epithelial responses examined by others also suggest moreperipheral responses to more hydrophobic odorants (Ezeh et al., 1995; Bozza andKauer, 1998; Araneda et al., 2000; Scott et al., 2000; Scott-Johnson et al.,2000; Bozza et al., 2002). Together, these results support the notion that olfactory receptors withhigh affinity for specific molecular features may be expressed in an epitheliallocation that maximizes each receptorÕs interaction with its preferentialodorous ligands (Scott et al., 2000; Schoenfeld and Knott, 2004).
Only at the highest concentration tested here did heptane stimulate thedorsal glomerular layer that corresponds to projections from the central channels(Fig. 5). Similarconcentration-dependent activation of the same dorsal regions also had beenreported with 2-hexanone (Johnson and Leon, 2000a) and 2-octanone (Johnson etal., 2004). These dorsalglomerular regions, previously defined as modules c (lateral) and C (medial),were thought initially to respond preferentially to ketones (Johnson and Leon,2000a; Johnson et al., 2002). Subsequently, reliable activation in these areas has been observed witha wide variety of chemicals including aromatic odorants, some cyclohexylmolecules, secondary alcohols, an ester, as well as the smaller alkanes in thisstudy (Johnson et al., 2002, 2004, 2005a, b). Even though these odorants possess different functional groupsand appear to be structurally distinct, there is clear evidence of responsespecificity within these regions (Johnson et al., 2002, 2004, 2005a, b;Takahashi et al., 2004). Furthermore, higher levels of activation in these dorsal modules weregenerally evoked by odorants of six to eight carbons in chain length (Johnsonand Leon, 2000a; Johnson et al., 2002, 2004, 2005a, b). These particular dorsal modulesreceiving input from the central channel of the olfactory epithelium may beparticularly responsive to high concentrations of odorants that possessspecific molecular features. Again, the intrinsic differential responsiveness of the olfactoryepithelium to odorant features appears to interact with the imposition of thephysical characteristics of odorant molecules to produce the observed glomerularresponse patterns.
Odorant hydrophobicity as a source of variance ininter-individual responses
Our principal components analysis suggested that inter-animal variabilitywas greater for the larger alkanes. The possible inherent and imposed interactions between the olfactoryepithelium and odorants discussed above also may account for this overallgreater individual variance. Ifthese odorants could gain access to more areas in the epithelium, then theirchances to interact with more types of receptors distributed throughout theepithelium would be increased, even though some of these interactions may beweaker and more inconsistent due to lower affinity binding that may produceindividual variability.
The importance of odorant volatility and purity
In this study, we observed an apparent exception to the typical chemotopicprogression with responses to the longest alkanes. Because chemotopically organized glomerular responses havebeen so reliable at both the local and global level, such a surprisingexception led us to examine alternative explanations. Here,the unexpected glomerular activation was explained by the impurity of theodorant preparations. A similarcontaminant effect has been demonstrated previously with the stimulation of theextreme rostral glomerular layer by carboxylic acids present in the aldehydepreparations as a byproduct of oxidation (Johnson et al., 2004). It may be surprising at first that sucha small difference in purity between the two grades of pentadecane used in ourexperiment would be significant for this system. Nevertheless, if the contaminant were appreciably morevolatile than the label compound, then a large impact would be expected. At the dilution that pentadecane waspresented to animals, there would be essentially no change in its vaporconcentration (approximately 0.4 ppm) from 99% to 99.8% pure. However, a contaminant that wereapproximately 5000 fold more volatile than pentadecane, for example, would havechanged in its vapor concentration from 20 ppm when using the less purepentadecane to only 4 ppm with the purer preparation.
The finding that hexadecane evoked very little response in the extremeventral aspect of the bulb is consistent with the notion that hexadecane has alow enough volatility to preclude a glomerular response. Given that hexadecane is even lessvolatile than pentadecane, its evoked activity pattern is likely to bedominated by the same dorsal responses to contaminants more volatile thanitself.
Correlations among odorous stimuli, neural responses,and odor perceptions
After studyinghundreds of odorants, we are gaining enough of an understanding of therelationship between odorant properties and their induced neural activities tomake successful predictions of glomerular responses based on odorant molecularstructure. Correlations betweenactivity patterns and odorant chemistry also have been reported in otherstudies using a variety of methods (Friedrich and Korsching, 1997, 1998; Joergeset al., 1997; Johnson et al., 1998, 1999, 2002, 2004, 2005a, b; Malnic et al.,1999; Rubin and Katz, 1999; Sachse et al., 1999; Johnson and Leon, 2000a;Uchida et al., 2000; Belluscio and Katz, 2001; Fuss and Korsching, 2001;Meister and Bonhoeffer, 2001). Similarly, a close relationship between odorant properties and odordiscriminability has been demonstrated through a number of psychophysicalstudies using various homologous series (Laska and Freyer, 1997; Laska andTeubner, 1999; Laska et al., 1999; Linster and Hasselmo, 1999; Laska and Galizia,2001; Laska and HŸbener, 2001; Linster et al., 2001b; Cleland et al.,2002). Fewer studies have beenconducted in intact animals to compare both neural and perceptual responses toodorants that varied systematically in their molecular structures, although arecent study using an odor habituation paradigm to test odor similarityexplored the relationship between glomerular and behavioral responses to ahomologous series of carboxylic acids (Cleland et al., 2002). Since this behavioral assay does notrequire extensive training of animals, it can reveal spontaneousdiscriminability among odorants that may correlate more meaningfully withglomerular responses observed in animals na•ve to the odorants presented. A significant correlation between thetwo types of responses observed in that study suggested that glomerular patternsimilarity could predict perceived odor similarity (Cleland et al., 2002). This relationship was further supportedby a later study comparing epithelial and discrimination responses to analdehyde homologous series (Kent et al., 2003). Similarly, the strong correlation between neural andbehavioral responses in our current study is consistent with the idea thatchemotopic information represented within the olfactory bulb may be used forperceptual processing. Indeed, ourresults also are consistent with the response specificity to alkanes recordedfrom neurons in the anterior piriform cortex, the primary projection area ofthe olfactory bulb (Wilson, 2000). In conclusion, these data indicate that odorant molecular features,their evoked glomerular activity, and their odor perception are functionallyrelated.
In summary, wehave demonstrated that even pure hydrocarbons, which can use only weak Van derWaals forces to bind to olfactory receptors, evoke highly specific responsesthat can be seen at the glomerular layer. Systematic differences in these spatially specific responses werepredicted based on systematic differences in the molecular features of thehydrocarbon chain. Indeed, theglomerular responses were systematic enough to allow us to pick out anomaliesthat were based on odorant contaminants, rather than the target odorant. We then went on to show that the degreeof difference in glomerular responses was proportional to the magnitude ofperceptual difference in rats. Ourdata further suggest that intrinsic differences in responsiveness to odorantsby different olfactory receptor neurons interact with differences imposed bythe physical characteristics of specific molecules to produce the olfactorycode. Finally, odorants withoxygen-containing functional groups that shared the same hydrocarbon featurealso shared portions of the glomerular response that were present for thealkane alone, a finding that is consistent with the idea of a combinatorialolfactory code.
Acknowledgements
We thank thefollowing individuals for their technical assistance: Andrew Chen for 2-DGactivity mapping, Talla Motakef and Sakura Minami for their work on thebehavioral tests, and Espartaco (Spart) Arguello for the development andmaintenance of a database for our activity patterns, analysis software, and ourlaboratory's website. We alsowould like to recognize Dr. John Greaves, Director of the Mass SpectroscopyFacility at the University of California, Irvine, for his expert advice on ourGC-MS analyses.
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Table 1. Information and Exposure Conditions of Odorants.
| | Odorant | CAS # | Molecular Formula | Vendor | Dilution in Air | Vapor Concentration (ppm) | Number of rats | |
| | Hexane | 110-54-3 | C6H14 | F | 1/8 | 24885 | 4 | |
| | Heptane | 142-82-5 | C7H16 | A | 1/8 | 7535 | 91 | |
| | Heptane | 142-82-5 | C7H16 | A | 1/24 | 2500 | 5 | |
| | Heptane | 142-82-5 | C7H16 | A | 1/75 | 800 | 5 | |
| | Heptane | 142-82-5 | C7H16 | A | 1/240 | 250 | 5 | |
| | Heptane | 142-82-5 | C7H16 | A | 1/750 | 80 | 5 | |
| | Octane | 111-65-9 | C8H18 | A | 1/8 | 2231 | 4 | |
| | Nonane | 111-84-2 | C9H20 | A | 1/8 | 705 | 4 | |
| | Decane | 124-18-5 | C10H22 | A | 1/8 | 229 | 4 | |
| | Undecane | 1120-21-4 | C11H24 | A | 1/8 | 66 | 4 | |
| | Dodecane | 112-40-3 | C12H26 | A | 1/8 | 29 | 4 | |
| | Tridecane | 629-50-5 | C13H28 | A | 1/8 | 6 | 4 | |
| | Tetradecane | 629-59-4 | C14H30 | A | 1/8 | 2 | 4 | |
| | Pentadecane | 629-62-9 | C15H32 | A | 1/8 | 0.4 | 92 | |
| | Pentadecane | 629-62-9 | C15H32 |