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1Department of Physiology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033; 2School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan
Submitted 9 March 2004; accepted in final form 11 May 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Shepherd and Greer 1998). Individual glomeruli most probably represent a single odorant receptor (OR; Mombaerts et al. 1996; Ressler et al. 1994; Vassar et al. 1994). Thus the glomerular sheet of the mouse OB forms the maps of approximately 1,000 ORs. How are the numerous ORs represented spatially in the glomerular maps?
Spatial arrangement of OR-representing glomeruli is a crucial determinant for the map of odorant-evoked activity in the OB (Johnson et al. 1998; Leon and Johnson 2003; Meister and Bonhoeffer 2001; Rubin and Katz 1999; Uchida et al. 2000; Xu et al. 2003). The spatial pattern of the odorant-evoked glomerular activity in the mammalian OB has been analyzed with several different methods including 2-deoxyglucose uptake (Coopersmith et al. 1986; Johnson et al. 1998; Royet et al. 1987; Stewart et al. 1979), optical imaging of intrinsic signals (Meister and Bonhoeffer 2001; Rubin and Katz 1999; Uchida et al. 2000), Ca2+ imaging (Fried et al. 2002; Wachowiak and Cohen 2001), voltage-sensitive dye imaging (Spors and Grinvald 2002), and functional magnetic resonance imaging (fMRI; Xu et al. 2003; Yang et al. 1998).
These studies began to reveal the basic plan for the spatial organization of the OR maps. For example, glomeruli responsive to structurally similar odorants are clustered in specific regions of the OB (Imamura et al. 1992; Inaki et al. 2002; Leon and Johnson 2003; Meister and Bonhoeffer 2001; Uchida et al. 2000). However, it is not well understood how the represented ORs are spatially arranged in the glomerular maps of the OB.
To further analyze the spatial organization of the glomerular OR maps, we used the method of optical imaging of intrinsic signals that allowed us to measure the response to many (
70) different odorants in a single rat OB and thus to examine the range of odorants that activated individual glomeruli. For the initial mapping study, we used a large panel of odorants (72 different odorants) with a systematic variation of their molecular structure. By comparing the molecular structure of odorants that were effective in activating individual glomeruli, we deduced the characteristic molecular features that the effective odorants shared. We then mapped the characteristic molecular features on the glomerular sheet of the OB.
Because aromatic compounds such as phenols have semirigid molecular conformations, they are more suitable for the estimation of the characteristic molecular features than the open-chain aliphatic compounds, which can have various flexible conformations. In the latter part, we thus focused on the glomeruli in the cluster responsive to phenols and neighboring subclusters, and estimated the spatial representation of the characteristic molecular features. The results suggest the presence of a systematic topographical map of the characteristic molecular features in the dorsal zone of the OB.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Seventeen male Sprague–Dawley rats (200–300 g) were anesthetized with medetomidine [0.5 mg/kg, intraperitoneally (ip)], ketamine (67.5 mg/kg, ip), and pentothal sodium (25 mg/kg, ip). In addition, lactated Ringer solution (0.3 ml/h), containing glucose (25 mg/h), dexamethason (0.02 mg/h), and riboflavine phosphate (0.05 mg/h), was intravenously injected through the tail vein. Rats were mounted in a handmade stereotaxic apparatus that enables us to fix the animal's head at any angle. The skull overlying the dorsal and dorsolateral surfaces of the OB was removed. In some cases, dura overlying the OB was also removed. The exposed surface of the OB was covered with 1.5% agarose gel and a glass slip. Heart beat, respiratory rate, and the lack of pain reflexes were monitored continuously. All experiments were performed in accordance with the guidance of the Physiological Society of Japan and the animal experiment committee of the University of Tokyo.
Optical imaging of intrinsic signals
The optical imaging device and the experimental procedures for the measurement of intrinsic signals were described in detail previously (Uchida et al. 2000). Briefly, the intrinsic signals induced by odorant stimulation were imaged under 705-nm wavelength light illumination (Meister and Bonhoeffer 2001). Images of reflected light from the surface of the OB were collected using a CCD camera (CS8310, TELI, Tokyo, Japan) with a tandem-lens macroscope arrangement, digitized and stored with Pentium PC using a frame-grabber board (Pulsar, Matrox, Quebec, Canada). In the first series (5 rats) and the third series (7 rats) of experiments, head and body were tilted by about 40° with the recorded side up, and the CCD camera focused on the dorsolateral surface of the OB. In the second series of experiments (5 rats), the CCD camera focused on the dorsal surface of the OB. The images had a spatial resolution of 320 x 240 pixels (after 2 x 2 binning). In these experiments, we imaged a 4.2 x 3.1-mm region, giving a pixel size of 13.1 µm. Before imaging the responses, blood vessel patterns were imaged under 540-nm wavelength light illumination. In the recording session, the focusing depth of the CCD camera was adjusted to 50–150 µm below the surface of the OB. For each recording trial, data were collected for 8 s with a frame length of 500 ms (16 frames/trial). Odorant stimulation was applied from the beginning of the 4th to the end of the 16th frame. The interstimulus interval was 30 s. Odorants were prepared in a glass test tube, either in pure liquid form or with dilution in mineral oil. Odorant stimulation was performed by placing the opening of an odorant-containing test tube at a distance of 10 mm from the animal's nostril. The order of odorant application was arbitrarily changed in each experiment. Each odorant was tested more than 4 times per animal.
Quantification of glomerular activity
Images were analyzed using IDL (Research Systems, Boulder, CO) and MetaMorph (Universal Imaging, West Chester, PA) software. Images of odorant-induced responses were obtained by dividing the magnitude of signals acquired during odorant stimulation (in most cases, frames 10–16) by that acquired before stimulation (frames 1–4). A Gaussian spatial filter was used to eliminate nonspecific global darkening and high-frequency noise of the differential image (cutoff frequencies, 
= 20.0/mm for high cut and = 0.2/mm for low cut). To achieve a better signal-to-noise ratio, these filtered images were averaged over 4–10 odorant presentations.
We used 72 odorants in the initial series of experiments (Fig. 1). By counting the number of glomeruli that were activated by at least one of the tested odorants, we identified 90–120 glomeruli within the imaged region. In some OBs, large blood vessels passed through the imaged region. We omitted the areas near the large vessel from subsequent analysis. To analyze the MRR properties of the glomeruli, hypothetical glomeruli (150 µm in diameter) were set on the center of these spots and the mean pixel value was measured. Glomerular activity was calculated by subtracting the prestimulating baseline value. The threshold of glomerular activity was set at 2 SDs above the mean pixel value of all images recorded from a rat. The glomerular activity usually ranged from 0.04 to 0.2% of the reflected light intensity. The intensity of glomerular activity was classified into 4 levels: 0.04–0.055% changes in light intensity is weak (shown by the smallest circle in Figs. 3, 4, and 6); 0.055–0.07% is modest (shown by a small circle); 0.07–0.085% is strong (a large circle); and more than 0.085% is very strong (the largest circle).
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In 3 experiments, Pontamine Sky Blue (4%, Nacalai Tesque, Kyoto, Japan) was iontophoretically injected into several points of imaged region after the optical imaging. The marker dye was ejected from a glass micropipette (tip diameter 1–2 µm) by applying a 10- to 50-µA negative current, pulsed at 500 ms ON, 500 ms OFF intervals for a period of 5 min. For olfactory cell adhesion molecule (OCAM) staining, rats were recovered from the anesthesia after the optical imaging and survived at least for a week. This procedure was necessary because of the disappearance of OCAM immunoreactivity after intensive olfactory stimulation (Yoshihara et al. 1993).
Under deep anesthesia, animals were perfused with 4% paraformaldehyde (Nacalai Tesque). The OB was dissected out, postfixed overnight at 4°C, and immersed in 30% sucrose solution at least 2 days at 4°C. Frozen sections of the OB (20 or 30 µm in thickness) were obtained with a sliding microtome. Sections were stained for OCAM using rabbit polyclonal antibodies (Yoshihara et al. 1997) and counterstained with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR).
An unrolled map of the glomerular layer of the OB was constructed as described previously (Nagao et al. 2000). Frontal sections were obtained at 100-µm intervals. A smooth line was traced along the center of the glomerular layer of each section. The line was flattened by opening it at the most ventral point. OCAM-positive and -negative glomeruli and dye-injected points were then mapped on the flattened line. The unrolled map was constructed by aligning the flattened traces of consecutive sections using the dorsal edge of the glomerular layer as a reference. The imaged region was determined with reference to the dye-injected points.
Odorants
Some 146 different odorants were used for stimulation of the olfactory epithelium. Odorants were purchased from Sigma-Aldrich (St. Louis, MO), Tokyo Kasei Organic Chemicals (Tokyo, Japan), and Nacalai Tesque (Kyoto, Japan). In the first series of the experiments, we used 72 different odorants that are listed in Fig. 1. In the third series of the experiments, we used aromatic compounds shown in Fig. 5 and many aliphatic and cyclic compounds. Aliphatic compounds used in the third series of the experiments were: 2 aliphatic acids (3COOH and 6COOH in Fig. 1), 1 aliphatic aldehyde (5CHO), 4 aliphatic alcohols (4OH, 6OH, 8OH, and 2-6OH), 1 diketone (3,4-Hxd), 5 aliphatic ketones (K3-1, K4-1, K5-2, K6-1, and K7-3), and 2 aliphatic ethers (4-1Eth and 3-3Eth). Cyclic compounds used in the third series of experiments were: 1 aliphatic–aromatic ketone (Acph), 5 cyclic ketones [(+)-Cvn, Mtn, (+)-Cpr, Ppt, and d-Plg], 1 cyclic ether (Cnl), and 6 hydrocarbons (Bzn, Tln, 
-Pn, Cpn, -Tpn, and cyclohexane).
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It is ideal to compare the spatial patterning of MRRs at different odorant concentrations. However, because of the large panel of odorants and the limited time for the successful recording of glomerular responses (typically 6–8 h), we did not examine the effects of odorant concentration in the present experiments.
Estimation of the characteristic molecular features
In the third series of the experiments, we used for stimulus odorants a panel of aromatic compounds (having a benzene ring), including phenol and its derivatives (Fig. 5). Using Chem3D Ultra (version 7.0, CambridgeSoft, Cambridge, MA), we obtained the 3D structures of the aromatic compounds that were effective in activating individual glomeruli. We then estimated the characteristic molecular features of odorants by superimposing the 3D structures of the effective odorants. Because these compounds have semirigid structures, the most stable conformations were used for the molecular superposition. The superposition of the 3D structure of the compounds was performed by least-square fitting (SYBYL version 6.9 software, Tripos, St. Louis, MO). Because of the flat-sheet structure of the benzene ring, the superimposed 3D structures were represented in 2-dimensional (2D) models (see Fig. 6).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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) of individual glomeruli. Using a CCD camera that was positioned to image a wide area covering the dorsal and dorsolateral parts of the OB, we recorded glomerular responses to a large and systematic panel of odorants and determined the MRR of individual glomeruli. Figure 1 shows the panel of 72 odorants that were used in the first series of optical imaging experiments (5 rats). The panel consists of 12 structural classes of odorants: 6 aliphatic acids (shown red), 4 aldehydes (pink), 9 aliphatic alcohols (6 primary alcohols and 3 secondary alcohols, yellow), 3 cyclic alcohols (light brown), 7 phenols (green), 5 phenyl ethers (yellow green), 2 diketones (dark brown), 15 aliphatic ketones (blue), 3 aliphatic–aromatic ketones (dark blue), 10 cyclic ketones (pale blue), 3 ethers (purple), and 5 hydrocarbons (gray).
Figure 2, A–I show examples of optical images of intrinsic signals that were induced by the stimulation of olfactory epithelium with heptanoic acid (A, 7COOH), heptanol (B, 7OH), phenol (C, Phe), o-cresol (D, o-Cre), phenetole (E, Phele), butyl ethyl ketone (F, K4-2), menthone (G, Mtn), butyl methyl ether (H, 4-1Eth), and toluene (I, Tln). They were recorded from the OB of the same rat (Rat#1). Odorant-induced intrinsic signals consisted of either isolated spots or larger areas with relatively diffuse darkening. Within the large area, multiple peaks of darkening signal were usually observed. In the present study, we assumed that each isolated spot or each isolated peak in the larger area corresponded to the activity of a single glomerulus (Belluscio and Katz 2001). These records exemplify that each odorant activated a specific combination of glomeruli that were typically clustered in particular regions of the OB. Figure 2, C and D exemplify that structurally similar odorants activated a largely overlapping but slightly different combination of glomeruli. Figure 2J shows the position of 119 glomeruli (numbered from 1 to 119) that responded to at least one of the 72 odorants in the OB of another rat (Rat#2).
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The MRRs of individual glomeruli in the OB of Rat#2 are listed in Figs. 3 and 4. These figures show that individual glomeruli typically respond to a range of odorants having similar molecular structures. Examination of the relationship between the spatial position and the MRRs of these glomeruli in the first series of experiments (5 rats) clearly indicated that glomeruli with similar MRR tended to gather in close proximity (Figs. 2J, 3, and 4). To facilitate the analysis of the spatial representation of the glomerular MRR properties, we tentatively grouped the activated glomeruli into 7 clusters (clusters A–G, Fig. 2J) based on the similarity of the MRR and the spatial proximity of their position. We further grouped glomeruli within each cluster into several subclusters based on the detailed comparison of molecular structures of effective odorants.
CLUSTER A.
Glomeruli in the cluster A (glom#1–18 in Rat#2) were located at the most anteromedial part of the exposed surface (Fig. 2J). Consistent with previous studies (Meister and Bonhoeffer 2001; Uchida et al. 2000), these glomeruli responded to aliphatic acids (red in Fig. 3) and aldehydes (pink). They also responded to a subset of esters (data not shown; cf. Uchida et al. 2000). Glomeruli in the most posterior part of the cluster A also responded to diketones (Fig. 3). Thus the characteristic molecular features of odorants effective in activating the cluster A glomeruli are a carboxyl group (–COOH), a diketone group [–(CO)(CO)–], or an ester group [–(CO)O–], functional groups having 2 oxygens in a neighborhood. In addition, odorants having a single carbonyl group (–CO) at the end of the molecule were effective in activating the cluster A glomeruli.
Molecular features other than the functional groups are also important determinants for the activation of individual glomeruli in the cluster A. Based on the carbon chain length of the effective aliphatic acids, we tentatively classified the cluster A glomeruli into 3 subclusters: A-1, A-2, and A-3 (Figs. 2J and 3). Glomeruli in A-1 responded selectively to aliphatic acids with a long carbon chain (6COOH–8COOH in Fig. 3), those in A-2 to middle size acids, and those in A-3 selectively to short aliphatic acids (3COOH–6COOH). A majority of subcluster A-3 glomeruli also responded to diketones.
CLUSTER B. Cluster B (glom#20–37 in Rat#2) was located in the most anterior region of the lateral part of the dorsal OB (Fig. 2J). Glomeruli in cluster B selectively responded to aliphatic alcohols with a long carbon chain (6OH–8OH) and to a wide range of aliphatic ketones (Fig. 3). Additional experiments showed that a majority of cluster B glomeruli also responded to anisole derivatives that have a methoxy group (–O–CH3) and a carbon side chain arranged at the para-position of the benzene ring (data not shown; cf. Figs. 5, 6, and 7). Thus the characteristic molecular features of odorants effective in activating the cluster B glomeruli are elongated carbon chain structures with a hydroxyl group (–OH), an alkoxyl group (–O–R), or a carbonyl group (–CO) attached at one side of the molecule.
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CLUSTER C. Cluster C (glom#38–64 in Rat#2) was located at the central region of the lateral part of the dorsal OB (Fig. 2J). Glomeruli in cluster C characteristically responded to phenol family odorants (green in Fig. 3; an exception is glom#62). Many of them also responded to phenyl ethers (yellow green). Thus odorants effective in activating cluster C glomeruli have a benzene ring with a hydroxyl group, a methoxy group, or an ethoxy group (–O–CH2–CH3). Thus the characteristic molecular features include the combination of the benzene ring–like hydrocarbon structure and the functional groups.
Based on the detailed MRR properties and the positions of glomeruli, we tentatively divided the cluster C into 4 subclusters: C-1, C-2, C-3, and C-4 (Fig. 2J). In addition to phenol family odorants, C-1 glomeruli responded to aliphatic alcohols with a relatively short carbon chain (4OH–6OH). C-2 glomeruli invariably responded to salicyl aldehyde (Slc–CHO in Fig. 5; data not shown), a phenol derivative with a carbonyl group attached to the ortho-position. Glomeruli in subcluster C-3 responded relatively selectively to phenols and phenyl ethers. Glomeruli in subcluster C-4 also responded to short aliphatic ketones (blue) and aliphatic ethers (purple).
CLUSTER D. Cluster D glomeruli (glom#65–73 in Rat#2) were located at the caudal region of the lateral part of the dorsal OB (Fig. 2J). Although these glomeruli tended to respond to wide structural classes of odorants, odorants effective in activating them were mainly ketones: cyclic ketones, aliphatic–aromatic ketones, diketones, and a subset of aliphatic ketones with relatively short side chains (Fig. 4). Cluster D glomeruli were classified into 2 subclusters: medially located D-1 and laterally located D-2 (Fig. 2J). Many glomeruli in the D-1 subcluster responded to creosol (Creo in Fig. 1) and eugenol (Eug in Fig. 1; data not shown), phenol derivatives with a methoxy group attached at the ortho-position and a carbon side chain at the para-position. Because of the presence of a large blood vessel, only one D-1 glomerulus (glom#65 of Rat#2) was detected in the OB shown in Fig. 2J.
Subcluster D-2 glomeruli tended to respond to aldehydes, alcohols, and ethers in addition to a wide range of ketones. Thus odorants effective in activating the D-2 glomeruli have a carbonyl group or a hydroxyl group attached to a bulky carbon chain structure.
CLUSTER E. Cluster E (glom#75–88 in Rat#2) was located at the dorsalmost part of the lateral surface of the OB (Fig. 2J). We could not characterize the odorant-response specificity of the cluster E glomeruli because they did not respond systematically to any class of the 72 odorants examined (Fig. 4).
CLUSTER F. Cluster F was located at the rostroventral part of the exposed bulbar surface (Fig. 2J). Cluster F glomeruli (glom#90–103 in Rat#2) invariably responded to aliphatic ketones and hydrocarbons (Fig. 4). A subset of them also responded to secondary alcohols, phenyl ethers, diketones, and cyclic ketones. Thus odorants effective in activating the cluster F glomeruli include not only those with an oxygen-containing group but also hydrocarbon odorants that do not have any polar group. Among the hydrocarbon odorants, cluster F glomeruli preferentially responded to terpene hydrocarbons. We tentatively divided cluster F glomeruli into 2 subclusters: anteriorly located F-1 and posteriorly located F-2 (Fig. 2J). Compared with F-1 glomeruli, those in F-2 characteristically responded to a wider range of aliphatic and cyclic ketones including terpene ketones. The results suggest that the terpene hydrocarbon structure is one of the main determinants for the activation of F-2 glomeruli.
CLUSTER G. Cluster G glomeruli (glom#104–118 in Rat#2) were located at the ventrocaudal part of the imaged region (Fig. 2J). Glomeruli in cluster G responded to hydrocarbons (Fig. 4). They preferentially responded to benzene family hydrocarbons rather than to terpene hydrocarbons. Many glomeruli in cluster G also responded to phenyl ethers, diketones, small aliphatic ketones, aliphatic-aromatic ketones, cyclic ketones, and ethers. Cluster G glomeruli were tentatively divided into 2 subclusters: ventrally located G-1 and dorsally located G-2 (Fig. 2J). G-1 glomeruli responded to aliphatic alcohols, whereas G-2 glomeruli did not respond to them.
Using the panel of stimulus odorants in Fig. 1, we examined in detail the MRR properties of clusters A–D in 10 rats (first and second series of experiments), and those of clusters E, F, and G in 5 rats (first series of experiment). Figure 2K indicates spatial arrangement of the clusters A–G in another rat OB (Rat#5). We consistently observed the characteristic odorant-class specificity of each cluster in all the OBs examined. Figure 2, J and K illustrate an example of interindividual comparison of the spatial arrangement of the clusters A–G. Although the shape and the tentative boundary of each cluster varied among different OBs, the glomerular clusters were always located at stereotypical positions and the spatial arrangement of the 7 clusters was conserved in all the OBs examined.
We also noted that some glomeruli at the boundary region between 2 neighboring clusters tended to show MRR properties that are intermediate between those of the 2 clusters. For example, glomeruli #38–43 in cluster C responded not only to phenols but also to aliphatic alcohols. In addition, the results in Figs. 3 and 4 suggest that the MRR properties of glomeruli within a given clusters resemble in part those of neighboring glomeruli in adjacent clusters.
A map of characteristic molecular features of odorants
The results shown in Figs. 2, 3, and 4 suggest that ORs represented by glomeruli in a same cluster respond to odorants that have similar molecular features. To examine this possibility, we need to know in detail the characteristic molecular features of odorants that were effective in activating individual glomeruli and to compare the characteristic molecular features among glomeruli in the same cluster and in the neighboring clusters. One possible method to estimate the characteristic molecular features is to characterize and superimpose the 3D structures of odorants effective in activating individual glomeruli. For this purpose, we made a third series of optical imaging experiments (7 rats) using a new panel of odorants that includes a wide variety of aromatic compounds (Fig. 5A), the structures of which are semirigid. Because of the limitation of the maximal number of odorants that can be examined in a single rat, however, we omitted many aliphatic compounds, which can have a variety of conformations. Using the new panel of stimulus odorants (see METHODS), we examined in detail the MRR properties of individual glomeruli in clusters B, C, and D.
Figure 5B shows examples of the MRR of individual glomeruli in the panel of aromatic compounds. We confirmed that individual glomeruli responded to a range of odorants with similar molecular structure. We also confirmed that glomeruli in a same subcluster showed similar MRR properties. For example, the MRRs of cluster B glomeruli (glom#9, #11, #19, and #20 in Rat#6) invariably and selectively covered the series of phenyl ethers that has a carbon chain attached at the para-position (surrounded by the bluish lines in Fig. 5B). The MRRs of glomeruli in subcluster C-2 (glom#40, #46, #47, and #55 in Rat#6) always covered a subset of phenols, a subset of methoxyphenols, and salicyl aldehyde (Slc–CHO) (surrounded by the reddish lines in Fig. 5B). The MRRs of subcluster D-1 glomeruli (glom#92, #99, and #104 in Rat#6) invariably covered eugenol family odorants (surrounded by the gray lines in Fig. 5B).
The characteristic molecular features of individual glomeruli were estimated by superimposing the 3D structure of the effective aromatic compounds. For example, glomerulus #46 in subcluster C-2 of Rat#6 responded strongly to guaiacol (Gua) and salicyl aldehyde (Slc–CHO); moderately to phenol (Phe), o-cresol (o-Cre), and o-chlorophenol (o-Chp); and weakly to m-cresol (m-Cre) and creosol (Creo) (Fig. 6A). Because these compounds have semirigid structures, the most stable conformations of the compounds were used for the molecular superposition. The superposition of the 3D structures of the compounds was performed by least-square fitting using the atoms numbered 1–7 in Fig. 6A. Superimposed 3D structures are shown in Fig. 6B. A total van der Waals (VDW) volume of the superimposed structures is also shown in Fig. 6C. It is likely that the superimposed 3D structures and their VDW volume give the information about the shape and physicochemical properties of the characteristic molecular features of the effective odorants. To make it clear, a 2D representation of the VDW volume is shown in Fig. 6D.
Figure 6, E–H show another example of the molecular superimposition. This glomerulus (#4 in cluster B of Rat#6) responded weakly to 4-ethyl anisole (4-Eanle) and 4-allyl anisole (4-Aanle). In addition, it responded to aliphatic compounds, octyl alcohol (8OH), butyl methyl ketone (K4-1), pentyl ethyl ketone (K5-2), hexyl methyl ketone (K6-1), and heptyl propyl ketone (K7-3). The characteristic molecular features were estimated based on the conformations of the 3D structure of the 2 aromatic compounds (4-Eanle and 4-Aanle). Then, to ascertain whether the 3D structures of the aliphatic compounds overlap well with the aromatic compounds, a set of energy-minimized conformers for each aliphatic compound was calculated and was superimposed on 4-ethyl anisole used as a template molecule. As shown in Fig. 6F, a conformer that overlaps well could be extracted for each aliphatic compound. A total VDW volume of the superimposed structures and its 2D representation are also shown in Fig. 6, G and H, respectively. A similar superposition based on the 3D structures of the effective odorants was carried out for the other glomeruli.
Figure 7 shows a map of the 2D structures of the characteristic molecular features in the OB of a rat (Rat#6). To facilitate the analysis, we labeled the presumed critical parts of the characteristic molecular features. The molecular features composed of a single methoxy or a single ethoxy group are indicated by blue shadows, those of a single hydroxyl group by yellow shadows. The molecular features that were composed of a combination of a single hydroxyl group and a single alkoxyl group arranged at the ortho-position are indicated by red surroundings, and those of a single hydroxyl group and a single carbonyl group arranged at the ortho-position by pink surroundings.
Interestingly, the map of characteristic molecular features (Fig. 7) showed a systematic and gradual change in the features according to the position of subclusters along the OB axes. For example, subcluster C-2 glomeruli typically responded to salicyl aldehyde (Slc–CHO in Fig. 5) that has a hydroxyl group and an aldehyde group (–CHO) arranged at the ortho-position (indicated by pink surroundings in Fig. 7). The characteristic molecular features of glomeruli in the C-2 subcluster thus include 2 oxygens in a neighborhood. Because the glomeruli in cluster A (glom#12, #51–53, #65, and #76) can respond to aliphatic acids, diketones, and esters that have 2 oxygens in a neighborhood, the results suggest that the characteristic molecular features of C-2 glomeruli partially resemble those of neighboring cluster A glomeruli.
When we consider overall molecular structure and the functional groups, salicyl aldehyde closely resembles benzoic acid and benzaldehyde (Bz–CHO in Fig. 5). In accord with this, additional experiments showed that benzoic acid and benzaldehyde activated several glomeruli in subclusters A-2 and A-3 (glom#12, #21, #30, and #51–53 in Fig. 7) that directly appose to subcluster C-2. This suggests that characteristic molecular features of the C-2 subcluster closely resemble those of the A-2 and A-3 subclusters.
Subcluster C-2 glomeruli also responded to methoxyphenols (indicated by red surroundings in Fig. 7) that have a hydroxyl group and a methoxy group arranged at the ortho-position around a benzene ring. The area occupied by the methoxyphenol-responsive glomeruli extended anterolaterally from subcluster C-2 to the medial part of subcluster C-3 and even to a part of subcluster C-1. This suggests that the subset of methoxyphenol-responsive glomeruli resembles each other in terms of the characteristic molecular features of the effective odorants.
Subcluster C-1 glomeruli characteristically responded to short aliphatic alcohols (4OH–6OH) in addition to phenols. A few C-1 glomeruli also responded to phenyl ethers having a methoxy group and a carbon side chain attached at the para-position (shown by blue shadows, glom#24 and #49 in Fig. 7). The glomeruli in the neighboring cluster B invariably responded to long aliphatic alcohols (6OH–8OH) and a majority of them also responded to phenyl ethers with a carbon chain attached at the para-position (blue shadows, glom#2, #4, #5, #8–11, #18–20, #25–27, and #35 in Fig. 7). The observation suggests that the characteristic molecular features of C-1 glomeruli partially resemble those of the neighboring cluster B.
Thus by the stepwise comparison of the characteristic molecular features of glomeruli in the chains of subclusters A-2, C-2, the medial part of C-3, C-1, and cluster B, we noted a systematic and gradual change in the characteristic molecular features. Another example is the characteristic molecular features of glomeruli in the neighboring subclusters C-4, D-1, and D-2. Glomeruli in subcluster C-4 characteristically responded to aliphatic and cyclic ketones in addition to phenols. Glomeruli in subcluster D-2 typically responded to the aliphatic and cyclic ketones, suggesting the similarity in the characteristic molecular features between the 2 neighboring subclusters.
The C-4 subcluster characteristically responded to phenols with a hydroxyl group and phenyl ethers with a methoxy group. The glomeruli in subcluster D-1 characteristically responded to eugenol family odorants, methoxyphenols that have a hydroxyl group and a methoxy group. This suggests that the characteristic molecular features of D-1 glomeruli partially resemble those of C-4 glomeruli.
Thus the similarity in the characteristic molecular features was noted not only between 2 neighboring subclusters in a same cluster but also between 2 neighboring subclusters each belonging to a different cluster. We also noted that clusters A–D partially overlapped and were not completely segregated from each other. For example, we observed in many OBs that cluster A partially overlapped with cluster B so that a few glomeruli in the overlapped part responded to aliphatic acids and aliphatic alcohols (shown by AB in Figs. 2K and 7). These results suggest a gradual change in the characteristic molecular features according to the position of glomeruli within clusters A–D.
Bulbar zones and clusters
Glomeruli in the OB can be classified into zonal subsets (Mori and Yoshihara 1995; Mori et al. 1999; Schwob et al. 1986). To examine the spatial arrangement of clusters A–G relative to the bulbar zones, we dye-marked several points after the optical imaging of these clusters in 3 rats. The sections of the OB containing the marked points were labeled by anti-OCAM antibody to distinguish glomeruli in zone 1 (OCAM-negative) and those in zones 2–4 (OCAM-positive) (Fig. 8, A and B) (Yoshihara et al. 1997). From a series of the labeled sections, we made a flattened unrolled map of glomeruli (Nagao et al. 2000). Based on the positions of the dye-marked points, we then superimposed clusters A–G on the flattened glomerular map (Fig. 8C). The results showed that most glomeruli in clusters A–D were in the OCAM-negative zone 1, whereas those in clusters F and G were in the OCAM-positive zones 2–4. The boundary line between OCAM-negative and OCAM-positive zones was located near the lateral margin of clusters B, C, and D and presumably in cluster E. In a clear correspondence with the zonal classification of the clusters, clusters A–D glomeruli in zone 1 responded to odorants with polar functional group(s), whereas clusters F and G glomeruli (in zones 2–4) can respond to hydrocarbon odorants in addition to those having polar group(s).
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DISCUSSION |
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Present results indicate that similar characteristic molecular features map to neighboring glomeruli (Fig. 7). Thus one of the distinct properties of the OR map is the local clustering of glomeruli that represent similar characteristic molecular features. Such clustering of glomeruli is not evident in the antennal lobe of the fly brain (Wang et al. 2003), and thus may be the characteristic of the vertebrate OB. In the present study, we tentatively classified 7 clusters and 15 subclusters of glomeruli in the dorsal and dorsolateral surfaces of the OB. Because ORs having similar MRR properties may be highly homologous in amino acid sequence, the clustering of glomeruli with similar MRRs in the map is in good agreement with previous reports that olfactory sensory neurons expressing highly related ORs appear to project their axons to glomeruli that are in close proximity (Strotmann et al. 2000; Tsuboi et al. 1999).
Based on the amino acid sequences, numerous types of mouse ORs are classified into 228 families (Zhang and Firestein 2002). This raises a possibility that ORs represented in a same cluster or a same subcluster might belong to a specific OR family or a combination of OR families, which were presumably evolved by gene duplication and gene mutation. The clustering of glomeruli representing like ORs suggests that the gene duplication involves both the OR gene and the loci controlling the olfactory axon projection to the target glomeruli.
The imaged region covers a large part of the OCAM-negative zone (zone 1) and a small part of OCAM-positive zones (zones 2–4) of the lateral map (Fig. 8). Except for the characterless cluster E, the imaged region is covered by the clusters and subclusters that can be defined by the characteristic molecular features of effective odorants. Glomerular clusters with similar MRR properties are also present in the lateral and lateroventral surfaces of the OB (K. Igarashi and K. Mori, unpublished observations). These results suggest that the clustering of glomeruli having similar MRRs is present throughout the OR map, and is one of the basic plans for the spatial arrangement of glomeruli. The clustering of glomeruli handling similar but slightly different molecular structures may help to sharpen the discrimination of subtle difference in odorant structure (Imamura et al. 1992; Katoh et al. 1993; Mori et al. 1999; Yokoi et al. 1995).
The clusters and subclusters classified thus far were stereotypically arranged in the glomerular sheet and were conserved among different rats. Odorants that activate glomeruli in a specific cluster or subcluster in the rat OB tend to contain similar "odor " to human nose. For example, the anisole family odorants (Anle, p-Manle, 4-Eanle, and 4-Aanle in Fig. 5) that activate glomeruli in cluster B have an "aniseed odor " in common. Cluster C glomeruli were activated by phenols, which have a "phenolic odor. " Salicyl aldehyde, benzaldehyde, and benzoic acid activate glomeruli in subcluster C-2 and neighboring subcluster A-2. These odorants have an "almond-like odor. " Eugenol family odorants (Gua, Creo, 4-Egua, Eug, Ieug, and Dheug in Fig. 5) activated glomeruli in the medial part of cluster C and in subcluster D-1. These odorants contain a "clove oil odor " in common. We previously noted the correlation of the glomeruli in cluster A to the "pungent, sour, fatty, and rancid odor " (Uchida et al. 2000). These results thus corroborate the hypothesis that the clustering of glomeruli having similar MRRs might participate in the neuronal mechanisms responsive for odor quality perception. The map of the characteristic molecular features might provide the neuronal basis for the relationship between the odorant molecular structure and the subjectively perceived "odor quality " (Moncrieff 1967).
Topographical map of the characteristic molecular features
By superimposing the 3D structures of effective aromatic compounds, we deduced the characteristic molecular features of glomeruli in cluster C and neighboring subclusters. Although the estimation of the characteristic molecular features is incomplete and the range of subclusters explored with this method were very limited, the map (Fig. 7) showed a systematic and gradual change in the characteristic molecular features according to the position of subclusters along the axes in zone 1 of the lateral map. This suggests the presence of the topographic map of the characteristic molecular features in zone 1 of the OB.
Although we tentatively grouped glomeruli into clusters and subclusters, the continual representation of the characteristic molecular features suggests that the glomerular map is not composed of mosaics of discrete clusters (and subclusters), each with completely different characteristic molecular features. Instead, 2 neighboring clusters show similarity in the characteristic molecular features, and in many cases partially overlap each other. Two neighboring subclusters, either in a same cluster or each in a different cluster, typically show similar characteristic molecular features. We propose that the glomerular map in zone 1 represents the characteristic molecular features in a systematic and continual way such that the characteristic molecular features gradually change along the axes in the map. We thus used the term "clusters" instead of the previously used term "domains" (Uchida et al. 2000).
ORs are 7-transmembrane G protein–coupled receptors (Buck and Axel 1991). Knowledge of the 3D structure of other G protein–coupled receptors such as rhodopsin (Okada et al. 2002; Palczewski et al. 2000) suggests that each OR might have a specific ligand-binding-site (odorant-receptive-site) structure formed by the bundle of transmembrane segments (Singer et al. 1995; Vaidehi et al. 2002). The distinct odorant-receptive-site structure of each OR might determine the odorant-response specificity of the OR. If this is the case and if the characteristic molecular features of individual glomeruli strongly reflect the odorant-response specificity of the represented ORs, present results imply that odorant-receptive-site structures of ORs might be gradually and systematically represented in the glomerular sensory map of the OB.
Previous and present studies of activity mapping showed that carbon chain length of aliphatic acids and aldehydes was systematically represented with a gradual shift of the position of activated glomeruli within cluster A (Inaki et al. 2002; Johnson et al. 1999; Meister and Bonhoeffer 2001; Rubin and Katz 1999; Uchida et al. 2000). From short to long, the chain length is represented by overlapping glomeruli whose position shifts gradually from subcluster A-3 through subclusters A-2 to A-1. Similarly, the chain length of aliphatic alcohols was represented by overlapping glomeruli whose position shifts from subcluster C-1 to cluster B. The systematic representation of the carbon chain length thus reflects the gradual change in the characteristic molecular features along consecutive series of subclusters in the OR map.
The systematic and continual representation of stimulus or receptor attributes is a common feature of mammalian primary sensory cortices. However, the map of the characteristic molecular features in the OB is unique among sensory maps in the brain. The presence of nearly 1,000 types of ORs might necessitate the central olfactory system to map the numerous characteristic molecular features of odorants to a large space of the glomerular sheet.
What is the functional significance of the systematic spatial map of the characteristic molecular features? Mitral cells in the OB read the glomerular map and then send their output to the olfactory cortex (Shepherd and Greer 1998). Individual mitral cells project a single primary dendrite to a single glomerulus and receive olfactory axon input within the glomerulus. In addition, individual mitral cells emit several secondary dendrites tangentially and receive inhibitory inputs by local interneurons from neighboring mitral cells that innervate neighboring glomeruli. Thus the output of a mitral cell is the consequence of the integration of the direct input from its own glomerulus and the indirect inputs from many neighboring glomeruli. Because individual mitral cells emit long (1 mm) secondary dendrites to a variety of directions (Orona et al. 1984), a mitral cell may integrate signals not only from glomeruli in the same subcluster and the same cluster, but also from those in neighboring clusters. In accordance with this, we observed that the activities of mitral cells are strongly influenced by the odorants that activated neighboring glomeruli (Nagayama et al. 2004). The integration of signals from its own glomerulus and surrounding glomeruli seems to be especially important for the processing of signals evoked by a complex mixture of odorants that occur in the natural environment. Spatial arrangement of the OR-representing glomeruli is thus a key factor to the manner of the olfactory signal processing in the neuronal circuit of the OB.
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Address for reprint requests and other correspondence: K. Mori, Department of Physiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail; moriken{at}m.u-tokyo.ac.jp).
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