This is a preprint of an article
published in The Journal of Comparative Neurology, 2004, 480:234-249.
© 2005
Wiley-Liss, Inc
Local and Global Chemotopic Organization: General Features of the Glomerular Representations of Aliphatic Odorants Differing in Carbon Number
Brett A. Johnson*1, Haleh Farahbod1, Zhe Xu1, Sepideh Saber1, and Michael Leon1
1Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697-4550
Number of text pages: 48
Number of figures: 8 (4 color)
Number of tables: 3
Abbreviated title: Chemotopic glomerular representations
Associate Editor: Thomas E. Finger
Indexing terms: deoxyglucose, olfactory bulb, odors, imaging techniques
Correspondence to: Brett A. Johnson, PhD
Dept. of Neurobiology & Behavior
2205 McGaugh Hall
University of California
Irvine, CA 92697-4550
Telephone: (949)824-7303
Fax: (949)824-2447
Email: bajohnso@uci.edu
Supported by United States Public Health Service grant DC03545
ABSTRACT
To determine if there is a general strategy used by the olfactory system to represent odorants differing in carbon chain length, rats were exposed to homologous series of straight-chained, saturated aliphatic aldehydes, ethyl esters, acetates, ketones, primary alcohols, and secondary alcohols (32 odorants total). Neural activity across the entire glomerular layer of the olfactory bulb was mapped quantitatively by measuring uptake of [14C]2-deoxyglucose evoked by each odorant. Uptake was observed both in dorsal glomerular modules previously associated with the particular odorant functional groups and in more ventral and posterior modules. Aldehyde-evoked activity patterns were dominated by ventral modules that included the area receiving projections from octanal-responsive sensory neurons expressing the I7 odorant receptor. The dorsal area that has been the focus of optical imaging studies of aldehyde responses contained only minor activity. For all functional groups except for ketones, uptake within functional-group sensitive modules displayed local chemotopy, with longer odorants stimulating more ventral and rostral glomeruli. In more posterior regions, chemotopy was observed for all functional groups, again with uptake shifting ventrally and rostrally with increasing chain length. In addition to these local shifts in activity, correlations analysis of entire activity patterns revealed a global chemotopic organization for all odorant series, with each odorant evoking a pattern most similar to odorants possessing the same functional group but differing by only one carbon in length. Thus, global chemotopy and local modular chemotopy appear to be fundamental principles underlying the representation of odorants differing in carbon chain length.
Individual odorants are detected by a subset of odorant receptors in the nose, and the differential stimulaton of these receptors by different odorous ligands likely lays the foundation for odor discrimination and perception (Axel, 1995; Buck, 1996). Rodent sensory neurons expressing the same odorant receptor gene converge in their projections onto a limited number of glomeruli in the main olfactory bulb (Ressler et al., 1994;Vassar et al., 1994; Mombaerts et al., 1996). Monitoring the activity pattern of glomeruli while exposing animals to odorants therefore offers a read-out of the activation of odorant receptors (Johnson et al., 1998). Furthermore, different spatial relationships between activated glomeruli are predicted to result in different odor processing because center-surround, lateral inhibitory networks within the bulb can affect the relative contribution of individual receptors to the overall odor perception (Yokoi et al., 1995; Luo and Katz, 2003; Aungst et al., 2003). Thus, the determination of glomerular activity patterns across the entire olfactory bulb can enhance our understanding of odor sensory coding.
Several principles of odorant representations have emerged from recent imaging studies of glomerular activity evoked by odorant chemicals of systematically different structure (Leon and Johnson, 2003). First, each pure odorant stimulates multiple glomeruli, usually in several clusters, or modules, distributed across the bulb (Johnson et al., 1998, 2002). With the exception of a ventromedial module probably representing the septal organ projection (Johnson et al., 2002), modules are present as lateral and medial pairs corresponding to the paired lateral and medial projections of olfactory sensory neurons expressing the same odorant receptor gene (Johnson et al., 1998). Second, each glomerular module appears to have unique determinants for activity. For example, odorants possessing a carboxylic acid functional group stimulated rostral and dorsal glomeruli in the rat relatively independently of hydrocarbon structure, whereas changes in hydrocarbon structure caused the activation of different glomeruli in the posterior bulb (Johnson et al., 1999; Johnson and Leon, 2000a). Another example is that odorants with a ketone functional group did not activate the same rostral glomeruli as the acids, but instead stimulated more caudal and dorsal glomeruli (Johnson and Leon, 2000b; Johnson et al., 2002). Third, more subtle differences in odorant chemistry appear to be represented by the relative activity of neighboring glomeruli within the same module (Uchida et al., 2000). For example, homologous series of straight-chained carboxylic acids of increasing carbon number stimulated progressively more rostral and ventral glomeruli within the acid-responsive modules (Johnson et al., 1999), and this modular chemotopy reflects differences in molecular length within the series (Johnson and Leon, 2000a).
There is some indication that chemotopic progressions of activity across glomeruli within a module may be a general feature of the coding of straight-chained odorants of differing carbon number. Several laboratories have described apparent rostral shifts in activity with increasing aldehyde carbon number in optical imaging studies that access only the dorsal surface of the bulb (Rubin and Katz, 1999; Uchida et al., 2000; Meister and Bonhoeffer, 2001; Belluscio and Katz, 2001; Wachowiak and Cohen, 2001; Fried et al., 2002), although at least one recent study has failed to find such an organization (Bozza et al., 2004). Optical imaging techniques also have led to the impression that there are shifts with carbon number in dorsal responses to certain esters and alcohols (Uchida et al., 2000). In our studies using the 2-deoxyglucose (2-DG) method, we have found that chemotopic progressions may not be limited to the dorsal aspect of the bulb. Progressions also were found for carboxylic acids and esters in medial functional group-sensitive modules and in posterior odorant representations that are not accessible to optical imaging techniques (Johnson et al., 1998, 1999, 2002).
Local chemotopic progressions with carbon number would insure that the most similar odorants within any homologous series stimulate neighboring glomeruli. This in turn is thought to optimize the use of lateral inhibition to tune mitral cell projection neurons to a more limited range of odorant carbon number (Yokoi et al., 1995, Johnson et al., 1999; Uchida et al., 2000). Given the likely importance of this type of modular chemotopy, we have set out to explore the generality of the phenomenon using a large number of different homologous series involving multiple functional groups. We have applied our 2-DG mapping procedure to collect information regarding entire glomerular activity patterns averaged across groups of subjects into anatomically standardized data matrices, which lend themselves naturally to quantitative and statistical analyses of odorant-evoked responses (Johnson and Leon, 1996; Johnson et al., 1999).
Odorants were purchased from Fisher Scientific (Tustin, CA: Acros Brand), except for 2-hexanone, which was purchased from Sigma-Aldrich (St. Louis, MO: Fluka Brand). For each exposure, 100 mL of neat odorant was placed into a 125-mL gas-washing bottle fitted with a stopper assembly possessing an extra-coarse porosity diffuser. Research-grade, high-purity nitrogen gas was bubbled through the column of odorant at a flow rate of 250 mL/min. Part of the resulting vapor was mixed with ultra-zero grade air and directed toward the exposure chamber at a final flow rate of 2 L/min to achieve the desired dilution, while the remainder of the vapor was diverted to a vent. For each exposure, the system was equilibrated for at least fifteen minutes before an animal was introduced.
Table 1 shows the dilution and final vapor phase concentration of each odorant used in the study, together with each odorantÕs unique Chemical Abstract Service registry number and the number of rats used for each odorant. Vapor phase concentration was calculated from the odorantÕs vapor pressure. The value used for vapor pressure was the mode of all unique experimental and estimated values obtained from two Internet databases (Interactive PhysProp Database from Syracuse Research Corporation: http://www.syrres.com/esc/physdemo.htm and the Chemical and Physical Properties Database from the Pennsylvania Department of Environmental Protection: http://www.dep.state.pa.us/physicalproperties/CPP_Search.htm) as well as from two chemistry software packages (Molecular Modeling Pro v. 3.14 from ChemSW , Fairfield, CA and ChemDraw Ultra v.6.0 from CambridgeSoft, Cambridge, MA).
Aldehydes oxidize easily to form carboxylic acids of the same carbon number. We estimated the amount of acid contamination in our aldehyde material by measuring the pH of 100-mM aqueous solutions made with vigorous mixing immediately prior to analysis. We also measured the pH of different concentrations of propionic and caproic acids. As expected from the fact that these acids possess identical values of pKa (4.8), identical curves of concentration versus pH were obtained for propionic and caproic acids. Because all acid contaminants in the aldehydes also are expected to have pKa values of 4.8, we used the acid concentration versus pH standard curves to determine the concentration of acid in our aldehyde preparations. Measurements were made one day prior to any exposures and again one day after all odorant exposures were completed, using material that had served as odorant during the exposures.
The UC Irvine Institutional Animal Care and Use Committee approved all procedures involving animals. Litters of rats culled to 8 pups on the day after birth were transferred to clean cages together with the dam one hour prior to any odorant exposure to reduce carry-over of odors from soiled cages. Individual pups ranging in age from postnatal day 18 to 21 were weighed, injected subcutaneously at the back of the neck with [14C]2-DG (16 µL/g, 0.1 mCi/mL, 52 mCi/mmol, Sigma Chemical Company, St. Louis), and then placed into a clean, 1‑L glass jar. The odorant, or gas vehicle, entered the lid of the jar, which also was fitted with a vent, so that the concentration of odorant in the chamber steadily increased at the beginning of the exposure. The exposure was continued for 45 minutes, at which time the rat was removed and decapitated, and the bran was removed and frozen immediately in isopentane at –45¡C.
For each litter, the first pup was exposed to vehicle, and the rest were exposed to odorants. No two pups from the same litter were exposed to the same odorant, and the order of odorant presentation was varied across different litters in a pattern that avoided systematic relationships between odorants. The study was composed of several independently conducted experiments possessing their own vehicle blanks. In most cases, there was a single experiment for each homologous series, but acetates and ethyl esters were compared in a single experiment, and averages for ethyl acetate-exposed rats were used for both series. In the case of ketones, two experiments involving homologous series were conducted (Table 1). In the first ketone experiment, odorants were used at a fixed, high vapor phase concentration that we previously had found necessary to activate modules c and C in experiments using 2-hexanone (Johnson and Leon, 2000b). Larger ketones were not sufficiently volatile to achieve these high concentrations. To maximize the amount of uptake evoked by each of these odorants, we used them in a separate experiment at a fixed dilution rather than at a fixed vapor phase concentration (Table 1). To investigate further the relationship between ketone concentration and activation of modules c and C, a third experiment was conducted using two concentrations of 2-octanone (Table 1).
Sectioning, imaging, and mapping were carried out as described previously (Johnson et al., 1999), except that the procedure was greatly accelerated during the course of this work by the development of custom software. This software automatically pairs images of coronal, cresyl violet-stained sections with autoradiographic images of adjacent sections, and it provides tools for alignment and for tracing of the glomerular layer, midline, and subependymal zone. Sampling circles are automatically placed at the intersection of the traced glomerular layer and previously described gridlines (Johnson et al., 1999), and tools allow repositioning of these circles in the event of section tears or folds. The software then writes data files for each section individually. The software also provides tools for analyzing 14C-standards and uptake within the subependymal zone at standardized rostral-caudal positions within the bulb. Upon completion of both bulbs of a given brain, the software merges the individual data files into data matrices, standardizes the size of these matrices in relation to anatomical landmarks, and writes new files in various units, including as a ratio of glomerular layer uptake to subependymal zone uptake. Upon completion of all brains in a given study, the software further analyzes the data by subtracting the average data matrices of the vehicle-exposed rats from each matrix of odorant-exposed rats. These difference matrices are transformed into units of z scores relative to the mean and standard deviation of uptake across the glomerular layer in that brain. The software then averages these matrices across each odorant condition.
Averaged z-score data matrices are visualized here as color-coded contour charts in our customary two-dimensional, ventral-centered format, which minimizes the impact of missing dorsal values caused by occasional loss of tissue on the cryostat knife (Johnson et al., 1999). These charts differ from the dorsal-centered maps preferred by others (Land, 1973; Stewart et al., 1979; Jourdan et al., 1980; Schwob and Gottlieb, 1986; Royet et al., 1987; Xu et al., 2003). To facilitate comparison between our data and data from other labs, we provide both dorsal-centered and ventral-centered charts of each of the present patterns, together with the rest of our odorant response archive, on our website (http://leonlab.bio.uci.edu/). Furthermore, because any two-dimensional map of a rounded three-dimensional surface necessarily distorts spatial information at the periphery of the map, our website also displays each pattern on a three-dimensional model of the glomerular surface.
Images of bulb sections used to compare 2-DG uptake to the location of the I7 odorant receptor were acquired using a Sony XC-77 CCD camera and NIH IMAGE 1.62 software. The autoradiography image was pseudocolor-enhanced (32 colors) using IMAGE, while the image of the cresyl violet-stained section was saved using a grayscale look-up table. Both images then were opened in Canvas 7 (Deneba Systems, Inc), where the grayscale image was sharpened, made partially transparent, and overlaid on the autoradiography image.
In previous studies, we had identified a number of odorant molecular features that were correlated with activity in particular glomerular modules (Johnson et al., 1998, 1999, 2000a,b, 2002). A number of these features were present in the various odorant series studied here, and it seemed important first to determine if the new patterns were consistent with our earlier models of glomerular specificity. The odorant-evoked activity patterns for all odorants in the present study are shown in Figure 1, which also shows a diagram of the previously identified glomerular modules. To simplify the activity patterns further, we calculated the average activity, in units of z score, under each of these previously defined modules and represented the values as circles of different sizes in Figure 2. We found that the new patterns indeed included modular responses consistent with our previous model of feature detection by glomerular modules (Johnson et al., 1998; Johnson and Leon, 2000b; Johnson et al., 2002).
The ethyl esters in the present study stimulated modules ÒaÓ and ÒAÓ (Fig. 1), previously identified as responding to ethyl and methyl esters (Johnson et al., 1998; Johnson and Leon, 2000b). There was some very slight stimulation of these same modules by aldehydes, consistent both with our data for high concentrations of pentanal (Johnson and Leon, 2000b) and with optical imaging studies (Rubin and Katz, 1999; Uchida et al., 2000; Meister and Bonhoeffer, 2001; Belluscio and Katz, 2001; Wachowiak and Cohen, 2001; Fried et al., 2002). However, much of the rostral activity evoked by larger aldehydes extended beyond our former borders of modules ÒaÓ and ÒAÓ into more ventral positions that we have labeled aÕ and AÕ in Figure 1.
Our previous work has shown that modules ÒaÓ and ÒAÓ are stimulated by low concentrations of carboxylic acids in a chemotopic manner, such that larger acids stimulate more ventral parts of the modules (Johnson et al., 1999; Johnson and Leon, 2000a,b). Because aldehydes oxidize easily into the corresponding acids, we were concerned that the slight stimulation in this area by these aldehydes might reflect contamination of the aldehydes by acids. This notion was reinforced by the finding that acids evoke module ÒaÓ and ÒAÓ responses at concentrations two orders of magnitude lower than the levels required by other odorants (Johnson and Leon, 2000b). We therefore made pH measurements to determine if acids were present in the preparations. Indeed, we found evidence for the presence of acid in our aldehyde material immediately after opening the reagent bottles, and the amount of acid increased over the course of our exposures, despite the use of nitrogen to volatilize the material (Table 2). We therefore suspect that much of the activity in modules ÒaÓ and ÒAÓ that is evoked by preparations of aldehydes actually may be due to the acid contaminants.
The greatest amount of activity evoked by the aldehydes in our series was located ventrally in areas overlapping with our modules ÒdÓ, ÒDÓ, ÒlÓ, and ÒLÓ (Fig. 1 and Fig. 2). Figure 3 shows a lateral focus within this area in a rat stimulated with octanal. It is striking that the location of this activity agrees well with the projection area of rat I7 odorant receptor-bearing sensory neurons (Vassar et al., 1994), which are known to respond with some specificity to octanal (Zhao et al, 1998; Araneda et al., 2000, 2004). The focus of 2-DG uptake shown in Figure 3 covered at least five glomeruli in a single coronal section. Additional glomeruli in more rostral and caudal sections also contributed to the octanal response. Other experiments have shown that the 2-DG technique can resolve activity in single glomeruli (Johnson et al., 1998, 1999). Activation of a larger cluster of glomeruli such as shown in Figure 3 is consistent with a model in which neighboring glomeruli have closely related odorant specificities, which is a corollary of the chemotopic progressions to be described below. The large cluster of glomeruli responding to octanal also is consistent with the presence of between 33 and 55 distinct octanal receptors as estimated from pharmacological profiles of rat sensory neurons (Araneda et al., 2004).
Aldehydes and primary alcohols stimulated paired modules ÒbÓ and ÒBÓ as previously described (Johnson et al., 2002). The larger acetates also stimulated these modules, consistent with the region responding to isoamyl acetate in a prior study (Johnson et al., 1998). There were several odorants in the present study that also stimulated modules ÒbÓ and ÒBÓ that had not been examined previously. These odorants included larger secondary alcohols with the hydroxyl group at the 2-position as well as ketones and larger ethyl esters (Fig. 1 and Fig. 2). None of the alcohols in the present study activated module ÒaÓ or ÒAÓ, unlike in a prior study where 1-pentanol evoked 2-DG uptake in these modules (Johnson and Leon, 2000b).
Dorsal modules ÒcÓ and ÒCÓ were stimulated by ketones ranging in carbon number from five to eight, which is consistent with previous indications that these modules recognize the ketone group (Johnson and Leon, 2000b; Johnson et al., 2002). However, we found that larger ketones did not activate these modules (Fig. 1 and Fig. 2). One possible explanation for this phenomenon is that these higher molecular weight ketones, which have low volatility, were not present at sufficiently high vapor phase concentrations to stimulate the modules. Previous results with 2-hexanone had shown that modules ÒcÓ and ÒCÓ were not activated below 75 ppm (Johnson and Leon, 2000b), and ketones with more than nine carbons in the present study were presented at concentrations below this value (Table 1). To determine if the effect of concentration on activation of modules ÒcÓ and ÒCÓ indeed applied to ketones other than 2-hexanone, we presented 2-octanone at 25 ppm and at 250 ppm. As shown in Figure 4, modules ÒcÓ and ÒCÓ were activated strongly at 250 ppm, but more weakly at 25 ppm. Therefore, it appears that modules ÒcÓ and ÒCÓ are particularly sensitive to the concentration of various ketone odorants.
As previously described (Johnson et al., 2002), activity patterns for various esters included modules Òf/FÓ and/or Òe/EÓ despite differences in length or location of the ester bond (Fig. 1 and Fig. 2). However, these modules also responded to a variety of alcohols and ketones in the present study, consistent with the unusually broad response range of these modules that was noted in our prior work (Johnson et al., 2002).
The previously defined glomerular modules also provided an unbiased statistical tool for evaluating the significance of differences between activity patterns within an experiment. In this analysis, we determined the maximal z score value within each module of each rat. Each module then was analyzed across odorant conditions using a one-factor ANOVA. To correct for the probability of spurious positives caused by testing 27 different modules, we then applied a false discovery rate correction (Curran-Everett, 2000) such that one module must satisfy P < 0.0019 (i.e., p < 0.05/27) for an experiment-wise P < 0.05 (additional modules then can be considered different by satisfying progressively less stringent criteria). As shown in Table 3, all odorant series yielded significantly different modules in this analysis.
To test for local chemotopic response progressions for different odorant series, we first determined centroids of 2-DG uptake within modules previously determined to respond to molecular features present in particular odorant series. These modules were not necessarily the modules exhibiting the greatest activation by the odorants. Centroids were calculated for each animal, and the significance of any difference in location across different odorants in a series was addressed using one-factor ANOVA. Figure 5 illustrates the relative positions of centroids within modules as ellipses. The centers of the ellipses represent the mean positions of the centroids and the two axes represent the standard error of the mean along the two dimensions used in calculating the centroids.
Despite the low levels of uptake evoked by aldehydes (or more probably, by acid contaminants in the aldehydes), analysis of centroids within modules (a + aÕ) and (A + AÕ) revealed robustly significant differences (F(5,16) = 4.80 and 9.23, respectively). In both cases, aldehydes of greater carbon number yielded centroids that were located more ventrally within the modules (Fig. 5). Centroids of aldehyde-evoked uptake within modules ÒbÓ and ÒBÓ also were significantly different across the aldehyde series (F(5,16) = 3.10 and 5.18, respectively). The larger aldehydes evoked more ventral activity in the lateral module ÒbÓ, and more ventral and anterior activity within the medial module ÒBÓ (the central location and higher variance of the centroid for pentanal in module ÒBÓ is probably due to an absence of actual odor-evoked 2-DG uptake in that module for any of the rats).
Responses to ester odorants also showed local chemotopic organization. The acetate odorants, which stimulated paired modules ÒbÓ and ÒBÓ, showed differences in the location of their centroids in module ÒbÓ (F(4,20) = 7.90). The larger acetates activated more ventral portions of the module (Fig. 5). The average positions of the centroids of the larger acetates also were more ventral within medial module ÒBÓ, but the differences just missed statistical significance at the P < 0.05 level (F(4,20) = 2.86 versus a critical F of 2.87). Ethyl esters, which stimulated paired modules ÒaÓ and ÒAÓ, differed significantly in the location of their centroids in both of these modules (F(6,28) = 6.69 and 7.99, respectively). The larger ethyl esters stimulated more anterior parts of the lateral module a (Fig. 5), which differs from the primarily ventral shift within this module that was seen for the aldehydes/acids. Within the medial module ÒAÓ, the larger ethyl esters activated more ventral areas (Fig. 5).
The primary alcohols yielded significantly different centroids within lateral module ÒbÓ (F(3,12) = 9.95), where the centroids shifted progressively anterior with increasing carbon number. In addition, the centroid for 1-octanol was located more ventrally than the other three odorants (Fig. 5). The centroids in the corresponding medial module ÒBÓ, however, were not significantly different (F(3,12) = 0.38) and overlapped completely.
Interestingly, ketones from five to eight carbons did not differ in their centroids within paired modules ÒcÓ and ÒCÓ (Fig. 5; F(3,15) = 1.03 and 1.06, respectively), despite the clear presence of evoked activity within these ketone-sensitive modules (Fig. 1). Thus, ketones represented an exception to the general finding of local chemotopy within functional group-related modules.
All odorants in all series evoked 2-DG uptake in more posterior regions of the bulb in addition to any stimulation of functional group-dependent modules (Fig. 1). The locations of these more posterior responses overlapped extensively across the different odorant series (Fig. 1). Within many of the odorant series, it appeared as though responses in these more posterior areas shifted anterior and ventral with increasing carbon number (Fig. 1), as had been observed in previous studies on carboxylic acid odorants (Johnson et al., 1999). To address this possibility quantitatively, we defined two broad areas, one lateral and one medial, that included most of these more posterior responses (red shading in the inset of Figure 1). We then tested for differences in centroids of uptake across carbon number within these two areas. Both medially and laterally, the areas almost entirely encompassed four previously defined modules (Òf/FÓ, Òe/EÓ, Òd/DÓ, and Òl/LÓ), and they included parts of other modules (Òh/HÓ, Òg/GÓ, and Òk/KÓ). The areas also extended somewhat more ventrally than any previously identified module due to ventral stimulation by large aldehydes, ethyl esters, and ketones.
As shown in Figure 6, within every odorant series there were significant differences across carbon number in the locations of centroids in at least one of the two more posterior areas. In most cases, both lateral and medial areas yielded highly robust differences. Increasing carbon number was associated with smooth ventral and anterior progressions in centroids for aldehydes, acetates, and ketones (Fig. 6). For ethyl esters, and 1- and 2-alcohols, the larger odorants in the series also gave clearly more anterior and ventral centroids, even though the progressions were not perfectly orderly with every step in carbon number (Fig. 6).
Aldehydes yielded more ventral and anterior centroids than did odorants of similar carbon number in any other series. In these other series there tended to be uptake remaining in dorsal and posterior parts of the areas (e.g., modules Òf/FÓ and Òe/EÓ), even for the larger odorants (Fig. 1), and this dorsal uptake likely prevented the centroid from extending as far ventrally.
A predicted consequence of these multiple local chemotopic progressions with carbon number is that the overall pattern of activity across the olfactory bulb would overlap more for odorants within a series that have a similar carbon number, and that patterns would differ more as the difference in chain length increases. To test this prediction, we correlated pairs of average data matrices within each study by treating matching cells of the paired matrices as X-Y pairs (Johnson et al., 2002). Every possible pair of average odorant-evoked activity patterns within a series was thus compared. The results are shown in Figure 7, where correlation coefficients have been converted into color-coded patchworks to make trends more immediately apparent.
Within each homologous series, each odorant evoked a pattern that was most similar to a pattern evoked by an odorant differing in length by only one carbon, which can be seen in Figure 7 as a clustering of higher correlations (darker squares) along the diagonal. Usually, the top two correlations for any given odorant involved odorants of one greater and one fewer carbon, and correlations generally fell off rather smoothly with increasing difference in carbon number. Therefore, a global chemotopic organization with respect to carbon number applies to all of these odorant series.
The activity patterns shown in Figure 1 are standardized into units of z scores relative to the mean and standard deviation of 2-DG uptake across the entire glomerular layer, which is an effective way to illustrate the pattern of activity independently of the amount of activity. However, to understand better the specificity of a glomerular module it also is relevant to consider the different amounts of stimulation caused by different odorant stimuli. For studies of 2-DG uptake, such information often is displayed as a ratio of glomerular layer uptake to subependymal zone uptake (GL/SEZ). The subependymal zone, which is comprised by immature neurons migrating into the structure, is considered a good control for different amounts of isotope injected into different rats and/or for different levels of circulating glucose (Coopersmith and Leon, 1984; Sallaz and Jourdan, 1992; Johnson and Leon, 1996).
To determine how carbon number influences amounts of activity in individual glomerular modules, we averaged GL/SEZ uptake over the modules that were determined to respond to features present in each of the homologous series. Then, we plotted the means and standard errors of these values as a function of carbon number (Fig. 8). Remarkably similar profiles were obtained for different homologous series and different glomerular modules. Uptake typically increased to a maximum at around seven or eight carbons across the homologous series of odorants. In most cases, uptake reached a plateau, but in the case of acetates (and possibly also with primary alcohols) uptake appeared to decrease again with further increases in carbon number. The results that are summarized in Figure 8 for individual glomerular modules largely recapitulated our impressions of the overall amounts of uptake across the entire glomerular layer when contour charts were produced using units of GL/SEZ (data not shown).
Local chemotopy is a general characteristic of the representation of homologous odorant series
Every homologous series of odorants that we used in the present study yielded chemotopic progressions of activity with carbon number in at least one region of the glomerular layer, and usually in several regions. The regions involved lateral-medial paired modules that when considered together covered almost half of the layer. These findings greatly extend our previous results with a homologous series of carboxylic acid odorants (Johnson et al., 1999) and indicate that orderly spatial arrangements of responses in the olfactory bulb might contribute generally and importantly to olfactory coding. Indeed, positioning glomerular responses to closely related odorants near one another in the bulb should optimize the use of lateral inhibition to tune or de-correlate projection neurons so that they respond to a more limited set of odorant molecules (Yokoi et al., 1995).
There are at least two possible mechanisms for these local chemotopic organizations of evoked responses in the glomerular layer. One possibility is that distinct odorant receptors with selective affinities for odorants of different carbon numbers are located in intermingled sensory neurons that project to glomeruli laid out in a chemotopic fashion in the bulb. The other possibility is that smaller and more hydrophilic odorants in any given homologous series might absorb almost completely to more dorsal (or central-channel) regions of the epithelial surface, whereas increasingly large and more hydrophobic odorants might be free to distribute increasingly ventrally (or laterally) in the epithelium (Hornung and Mozell, 1977). The chemotopic organization in the glomerular layer then could be the simple result of a topographic projection of the epithelium to the bulb (Astic and Saucier, 1986; Schoenfeld et al., 1994). These two possibilities would have a close relationship to the concept of ÒinherentÓ and ÒimposedÓ mucosal activity patterns, respectively (Moulton, 1976; Mozell et al., 1987).
Different aspects of our data support each of the ÒinherentÓ and ÒimposedÓ hypotheses. The spatial correspondence between octanal-evoked 2-DG uptake (Fig. 3) and the projection site associated with the I7, octanal-responsive odorant receptor (Vassar et al., 1994; Zhao et al.; 1998; Araneda et al., 2000, 2004) argues in favor of the importance of inherent receptor affinities in determining bulbar activity patterns. Further support comes from optical recording of excised rat mucosa following exposure to a homologous series of aldehydes in a manner intended to avoid differential sorption (Kent et al., 2003). The mucosal spatial activity patterns were clearly distinct for the different members of the series, suggesting an inherent specificity of different sensory neurons for different aldehydes (Kent et al., 2003), but they were not characterized by a simple, unidirectional shift in the location of the response that would correlate with the ventral shifts we have observed. In a prior study using carboxylic acids, we found that ventral shifts in 2-DG uptake in acid-responsive modules correlated much better with odorant molecular length than with hydrophobicity (Johnson and Leon, 2000a). Length might be expected to have a greater effect on specific receptor binding by way of steric factors, whereas chromatographic properties dictating mucosal absorption might be more related to odorant hydrophobicity. It remains to be determined whether the shifts we are reporting here for aldehydes, esters, alcohols, and ketones also will be correlated more with length than hydrophobicity. In any homologous series, length and hydrophobicity are perfectly correlated, so that dissociating the two properties would require the use of odorants possessing different hydrocarbon structures, as was done for acids (Johnson and Leon, 2000a).
The ÒimposedÓ hypothesis would predict that, independently of functional group, chemotopic progressions with increasing carbon number should always be in the ventral direction in the olfactory bulb, because this would reflect access of more hydrophobic odorants to increasingly more ventral and/or lateral regions of the epithelium (Hornung and Mozell, 1977; Scott-Johnson et al., 2000). Indeed, we have found ventral progressions for three different pairs of modules and for five different odorant functional groups. This result seems unlikely to be spurious, given the number of other possible directions the shifts could take and still be effective if only the ÒinherentÓ hypothesis were to apply. Perhaps the most satisfying hypothesis is that length-specific receptors might be arrayed in the epithelium in such a way as to maximize their interaction with their optimal ligands given the different sorption profiles of those odorants (Scott-Johnson et al., 2000).
The presence of chemotopic representations in the posterior, medial bulb for all homologous series yet studied continues to explain why rats with large anterior and dorsal lesions of the olfactory bulb continue to be able to discriminate odorants of different carbon number (Bisulco and Slotnick, 2003).
Straight-chained ketones between five and eight carbons in the current study evoked activity in dorsal modules that we have labeled ÒcÓ and ÒCÓ, which is consistent with our previous findings associating the ketone functional group with activity in these areas (Johnson and Leon, 2000b; Johnson et al., 2002). Unlike the functional group-sensitive dorso-lateral modules ÒaÓ and ÒbÓ or their medial counterparts ÒAÓ and ÒBÓ, the locations of responses in modules ÒcÓ and ÒCÓ did not differ significantly for odorants of different carbon number. The absence of local chemotopy for ketone responses within modules ÒcÓ and ÒCÓ suggests that these modules may be organized differently than other modules.
Absence of local chemotopy as measured by uptake of 2-DG does not necessarily indicate that all ketones activated the same set of glomeruli within modules ÒcÓ and ÒCÓ. In individual sections, there are clearly glomeruli that do not take up 2-DG intermingled with active glomeruli in most glomerular modules. These intermingled inactive glomeruli also are observed using other imaging techniques (Meister and Bonhoeffer, 2001). The presence of such inactive glomeruli is not apparent in our final activity charts because the process of averaging across different bulbs and different rats tends to blur such details of the local pattern. This blurring is likely due to the fact that individual glomeruli vary slightly in their relative positions in the bulb (Strottman et al., 2000), in addition to probable experimental variation in tissue handling, section angles, and mapping. Thus, differences in activity in nearby glomeruli that might be apparent in an individual rat may not show up in our final analyses. Similarly, although chemotopic progressions within modules may appear to be smooth and continuous in our averaged data, progressions within individual rats may skip individual glomeruli (Meister and Bonhoeffer, 2001).
Representations of aliphatic aldehydes
There have been a number of recent optical imaging studies of the dorsal surface of rodent olfactory bulbs that have used homologous series of aliphatic aldehyde odorants as stimuli (Rubin and Katz, 1999; Uchida et al., 2000; Meister and Bonhoeffer, 2001; Belluscio and Katz, 2001; Wachowiak and Cohen, 2001; Fried et al., 2002). These studies were at least in part motivated by electrophysiological results showing responses by projection neurons located in the same area (Imamura et al., 1992; Yokoi et al., 1995). This work further has impacted a range of studies of neuronal specificity and psychophysics that use aldehyde odorants (Linster and Hasselmo, 1999; Laska et al., 1999; Linster et al., 2001; Kent et al., 2003; Xu et al., 2003). Our data confirms the presence of a small amount of aldehyde-evoked activity in the dorsal bulb, as well as chemotopic progressions of dorsal responses with carbon number. However, this dorsal activity pales in comparison to the strong activity evoked in more ventral regions (Fig. 1), where responses also are strongly chemotopic and overlap with the projection area of the well-studied rat I7 octanal receptor (Zhao et al., 1998; Araneda et al., 2000, 2004). These ventral responses have not been detected by optical imaging methods, which can access only the dorsal aspect of the bulb.
The dorsal parts of the bulb that respond slightly to aldehyde odorants are major components of the response to aliphatic acids even at lower vapor phase concentrations (Royet et al., 1987; Slotnick et al., 1989; Sallaz and Jourdan, 1992; Johnson et al., 1999; Johnson and Leon, 2000a,b). Aldehydes are unstable molecules in an oxygen atmosphere, and their spontaneous oxidation produces carboxylic acids of the same carbon number. Indeed, our preparations of aldehydes contained significant levels of acid even when the containers first were opened, and the amount of acid increased during our use of the material. It seems reasonable to be concerned that the dorsal activity that has received attention in optical imaging studies may actually be caused by acid contaminants in the aldehyde material. The chemotopic organization of these responses would be predicted because responses to carboxylic acid odorants in the dorsal bulb also are chemotopic (Johnson et al., 1999), and oxidation of a homologous series of aldehydes would produce a homologous series of acids.
Despite the above concerns, we cannot conclude from our data that dorsal glomeruli would not have a small response to aldehyde odorants if they could be presented in the absence of carboxylic acid contaminants. Furthermore, it remains possible that aldehyde oxidation could take place in the mucosa itself, in which case responses of the dorsal glomeruli would be inherent contributors to the perceived odor of aldehydes even if the aldehydes were to enter the nares without prior oxidation.
Olfactory glomerular responses to aldehydes also have been shown recently in an individual mouse using fMRI (Xu et al., 2003). The fMRI technique produced maps characterized by a diffuse signal distributed over most of the lateral aspect for hexanal, heptanal, and octanal. Any medial signal did not bear obvious relationships to the lateral pattern, despite the paired medial and lateral projection of homologous sensory neurons (Vassar et al., 1994; Ressler et al., 1994; Mombaerts et al., 1996). Local chemotopic progressions also were not evident in the fMRI data (Xu et al., 2003). Thus, the fMRI signal bore little similarity to our highly focal pattern of ventral activity that was paired in the lateral and medial aspects of the layer, that shifted chemotopically with carbon number, and that was highly reliable across all bulbs of all rats investigated. Indeed, fMRI patterns evoked by esters (Yang et al., 1998; Xu et al., 2000, 2003) also do not bear clear relationships either to 2-DG uptake patterns evoked by the same odorants (Johnson et al., 1998, and present study), or even to fMRI patterns attributed to the same odorants in different studies. Furthermore, the fMRI signal attributed to isoamyl acetate was found to shift in location over time in an unexplained manner (Xu et al., 2000). Both 2-DG uptake and fMRI signals are indirect measures of neural activity, and each method may have its own drawbacks. However, it seems likely that the greater order in the 2-DG data reflects true order in the organization of the olfactory system, and that the fMRI technique is being affected by something other than odorant-evoked neural activity in the glomerular layer. It may be the case that improved technical abilities of fMRI for use in small brain structures such as the bulb, as well as improved statistical analysis of fMRI activity patterns, ultimately will help reconcile the apparent differences between the two methods.
The
olfactory system appears to be similar to the visual, auditory and
somatosensory systems in that sensory stimuli evoke a peak response that tapers
to a widespread background response that reaches across many millimeters of
cortex or bulb (Bakin et al., 1996; Chen-Bee et al., 2000; Grinvald et al.,
1994; Johnson and Leon, 2000b; Johnson et al., 1998; 1999; 2002; Masino et al,
1993). In all of these sensory
systems, though, it is the peak response that relates directly to the specific
stimulus, whether it is a point of light, a pure tone, a single whisker, or an
odorant feature. For example,
while there is an increase in background neuronal responses as far away as the
auditory cortex when a rat whisker is stimulated, it does not appear to evoke
an auditory perception (Brett-Green et al., 2001). At the same time, the peak response accurately indicated
which whisker was simulated.
Similarly, the measurement by 2-DG uptake of peak responses in the olfactory
bulb in response to a homologous series of odorants (Johnson et al., 1999)
accurately predicted the graded perceptual ability of rats to discriminate
among them (Cleland et al., 2002).
Moreover, peak glomerular responses also are in agreement with the
systematically graded responses to a homologous series of odorants in both
mitral cells (Imamura et al., 1992) and olfactory receptor neuron recordings
(Sato et al., 1994).
Bozza et al., (2004) recently reported that transgenic mice whose olfactory sensory neurons express synapto-pHluorin, a pH-sensitive protein that reports synaptic vesicle fusion, had no differential glomerular responses to a homologous series of aldehydes. However, although the responses they recorded were temporally associated with odorant presentation, the small region in the center of the dorsal bulb from which they were recording likely prevented them from seeing the peak responses to the odorants, given that even the most dorsal responses we observe for aldehydes (or their acid contaminants) are located more rostrally and rapidly shift ventrally away from the dorsal surface with increasing carbon number (this report; Johnson and Leon, 2000b; Johnson et al., 1999; 2002). To the contrary, Bozza et al. almost certainly were recording background responses, which are unlikely to be critical for odor coding and which are also unlikely to shift their pattern along a homologous series of odorants.
Bozza, et al. (2004) suggested that the chemotopy that we have reported previously might be an artifact of our averaging data across numerous bulbs. This is not the case, in that we typically see the altered locations of response in each individual bulb, as we have illustrated in a previous paper (Johnson et al., 1999). Also, the centroid shifts in the present study as well as in two prior studies (Johnson et al., 1999; Johnson and Leon 2000a) were found to be statistically significant, indicating that the changes in the mean positions across odorants greatly exceeded any variance between individual animals. The peak responses we observe often are associated with clusters of active glomeruli, which were not observed by Bozza et al. (2004). We consider their failure to see glomerular clusters to be a further indication that their imaged area did not contain the peak responses rather than that our averaging procedure somehow introduced a selection bias in favor of glomerular clusters. As in any data set, averaging across individuals prevents neuronal noise or background responses from being reflected in reported data, while the peak responses survive averaging to form the basis for statistical analysis. Indeed, rather than being an artifact, it would appear that chemotopy is a fundamental principle of olfactory coding, since molecules with different functional groups reflect this kind of organization in different glomerular modules.
Correlations between activity patterns and odor
psychophysics
Our quantitative comparisons of overall glomerular activity patterns showed a global chemotopic organization with respect to carbon number, such that within every homologous series, the pattern evoked by any given odorant was most similar to the pattern evoked by an odorant with either one additional or one fewer carbon atom (Fig. 7). If glomerular activity patterns were related to odor perception, one would expect the odors of these chemicals also to display this continuous pattern of relative similarity. To our knowledge, this expectation has been fulfilled for every homologous odorant series that has been evaluated psychophysically. In rats, progressions of perceived odor with carbon number have been shown for homologous series of aldehydes (Linster and Hasselmo, 1999; Linster et al., 2001; Kent et al., 2003), ethyl esters (Fletcher and Wilson, 2002), primary alcohols (Linster et al., 2001), and carboxylic acids (Linster et al., 2001; Cleland et al., 2002). In primates, smooth progressions in perceived odor have been shown for aldehydes (Laska et al., 1999; Keller and Vosshall, 2004), acetates (Laska and Freyer, 1997; Laska and Hubener, 2001; Laska and Seibt, 2002), primary alcohols (Laska et al., 1999), ketones (Laska et al., 1999; Laska and Hubener, 2001), and carboxylic acids (Laska and Teubner, 1998).
As articulated by Keller and Vosshall (2004), the continuous progression in the perceived odors of aliphatic aldehydes with increasing carbon number is not consistent with the vibrational theory of odor favored by Turin (1996), who indicated that even-numbered aldehydes should have one odor type while odd-numbered aldehydes should have another odor type. Our glomerular activity patterns also showed no evidence for greater overall similarity either within even-numbered or within odd-numbered aldehydes. Furthermore, inspection of individual aldehyde-evoked activity patterns did not reveal any focus of glomerular activity that might reflect the presence of a vibrational detector specifically recognizing either the even-numbered or the odd-numbered aldehydes. The continuous progressions of activity patterns with carbon number better support theories of odor involving recognition of odorant molecular features by sets of odorant receptors influenced in a more traditional way by steric and electronic factors.
Correlations between amounts of glomerular 2-DG
uptake, sensory neuron activation, and odor potency
When expressed as glomerular layer uptake/subependymal zone uptake, we found that activity increased with carbon number in every homologous series. In most cases, activity leveled off at around seven or eight carbons, but in some cases, activity appeared to peak and then to decrease with further increases in carbon chain length. These phenomena have parallels both in studies of sensory neuron activation and in psychophysical studies of odorant detectability.
There have been several reports of increased sensory neuron responsiveness to larger members of homologous series. For example, using equal concentrations of aldehydes in a homologous series as stimuli, Kaluza and Breer (2000) found that higher concentrations of cyclic AMP were produced in rat olfactory cilia as chain length increased from five to eight carbons, followed by a plateau. Kafka (1970) found that locust sensory neurons were maximally stimulated by odorant molecules between five and seven carbons in length along a number of functional group series, and responsiveness typically declined for odorants of even greater carbon number. Similarly, Sato and coworkers (1994) found that the number of rat odorant sensory neurons tuned to a given odorant increased with carbon number from three to nine carbons in homologous series of alcohols and carboxylic acids. The fact that odorant carbon number affects levels of sensory neuron activity suggests that the influence of odorant carbon number on amounts of glomerular 2-DG uptake may reflect peripheral processes.
Thresholds for odorant detection also show a dependence on carbon number within a homologous series. In general, sensitivity of detection increases with increasing carbon number, but the exact shape of the curve apparently depends on the homologous series being studied. In humans, the sensitivity of odor detection for aldehydes increases with carbon number from one to eight, with a possible decline from eight to ten carbons (Nagata, 1993; Cometto-Mu–iz et al., 1998). Odor sensitivity for n‑alcohols progresses continuously with carbon number up to ten carbons in both rats and humans (Moulton and Eayrs, 1960; Cain, 1969; Cometto-Mu–iz and Cain, 1990; Nagata, 1993), whereas sensitivity for 2-ketones increases from three to seven carbons, but then plateaus between seven and nine carbons (Cometto-Mu–iz and Cain, 1993; Nagata, 1993). For ethyl esters, there is an apparent peak in human odor sensitivity at six carbons in a series from four to seven carbons (Nagata, 1993), and after a progression to a near plateau at around eight carbons, sensitivity to acetates increases again around twelve to fourteen carbons (Cometto-Mu–iz and Cain, 1991; Nagata, 1993). Similar results involving acetates were obtained for rats and monkeys (Moulton, 1960; Laska and Seibt, 2002). Thus, many of the homologous series showing evidence for peaks and plateaus in amounts of 2-DG uptake show similar behavior in relation to odor detectability.
In their efforts to construct a predictive model for calculating odor thresholds from odorant physical properties, Abraham et al. (2002) discovered that, in addition to terms describing physical transfer of odorant molecules between phases, they needed to add a term for the optimal length of compounds because small and large odorants were both less potent than otherwise predicted. The lengths showing the greatest odor potency corresponded to n-alcohols of six carbons and acetates of eight carbons (Abraham et al., 2002), which are close to the locations of peaks on our curves of carbon number versus amount of 2-DG uptake (Fig. 8). Abraham and coworkers (2002) noted that the size of these molecules is similar to the size of the proposed central binding pocket in odorant binding proteins of the nasal mucosa, which may explain the special potency of odorants with this number of carbons. It also is possible that odorant receptors themselves have evolved an overall greater sensitivity to molecules of this size, perhaps to optimize detection of individual odorants that are large enough to bear a reasonable amount of biologically significant chemical information. Odorants of this size may allow specific, high-affinity binding while still being reasonably volatile.
Conclusions
Mapping
glomerular responses to systematically differing odorant molecules has allowed
us to uncover emergent organizational principles such as chemotopic
representations that may not have been predictable from knowledge of odorant
receptor specificities and expression patterns alone. As previously discussed, these spatial progressions may
arise from orderly projections of sensory neurons expressing specific receptors
and/or from chromatographic properties of the olfactory epithelium. Straight-chained, saturated aliphatic
compounds represent only a small fraction of the odorants that the olfactory system
detects and discriminates, and odorant carbon number represents only one of
many variables that can differentiate aliphatic compounds from one
another. By continuing to relate
differences in odorant chemistry to differences in evoked spatial patterns of
glomerular activity, we hope to elucidate further organizational principles
involved in odor coding.
We thank Paige Pancoast, Jennifer Kwok, Edna E. Hingco, Linh Hoang, and Sakura Minami for technical assistance with sectioning and mapping. We thank Espartaco (Spart) Arguello and Dr. Robert Dielenberg for developing software to accelerate mapping. We further thank Spart Arguello for developing a database for our matrices, for writing software to analyze the matrices, and for creating and maintaining our website.
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Tables
Table 1. Dilutions and vapor phase concentrations of odorants.
|
Odorant |
CAS# |
Dilution
factor |
Vapor
concentration (ppm) |
Number
of rats |
|
Pentanal |
110-62-3 |
1362 |
25 |
4 |
|
Hexanal |
66-25-1 |
512 |
25 |
4 |
|
Heptanal |
111-71-7 |
185 |
25 |
3 |
|
Octanal |
124-13-0 |
62 |
25 |
4 |
|
Nonanal |
124-19-6 |
22 |
25 |
3 |
|
Decanal |
112-31-2 |
8 |
25 |
4 |
|
Ethyl acetate |
141-78-6 |
1537 |
75 |
5 |
|
Ethyl propionate |
105-37-3 |
630 |
75 |
5 |
|
Ethyl butyrate |
105-54-4 |
224 |
75 |
5 |
|
Ethyl valerate |
539-82-2 |
84 |
75 |
5 |
|
Ethyl caproate |
123-66-0 |
21 |
75 |
5 |
|
Ethyl heptanoate |
106-30-9 |
12 |
75 |
5 |
|
Ethyl octanoate |
106-32-1 |
8 |
29 |
5 |
|
Propyl acetate |
109-60-4 |
605 |
75 |
5 |
|
Butyl acetate |
123-86-4 |
206 |
75 |
5 |
|
Amyl acetate |
628-63-7 |
72 |
75 |
5 |
|
Hexyl acetate |
142-92-7 |
19 |
75 |
5 |
|
1-pentanol |
71-41-0 |
110 |
30 |
4 |
|
1-hexanol |
111-27-3 |
42 |
30 |
4 |
|
1-heptanol |
111-70-6 |
10 |
30 |
4 |
|
1-octanol |
111-87-5 |
8 |