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Department of Physiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
Submitted 24 August 2004; accepted in final form 18 September 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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). Because individual glomeruli represent a single odorant receptor (OR), the glomerular sheet of the OB forms OR maps (Buck 2000; Mori et al. 1999). The knowledge of spatial organization of OR maps may provide an important clue to understand the manner of processing and integrating signals from different ORs in the neuronal circuit of the OB. However, the principles of the spatial organization of OR maps are still poorly understood.
An individual OB has two symmetrical OR maps, lateral map and medial map (Nagao et al. 2000). Glomeruli in each map are parceled further into four zones in the dorsoventral direction (zones 1 to 4) (Nagao et al. 2000). Glomeruli in each zone are thought to receive olfactory axon inputs selectively from sensory neurons that are located within the corresponding zone of the olfactory epithelium, and therefore represent zone-specific ORs (Mori et al. 1999).
A variety of methods have been applied to map the odorant-evoked glomerular activity in the mammalian OB (Bozza et al. 2004; Imamura et al. 1992; Inaki et al. 2002; Johnson and Leon 2000; Johnson et al. 2002; Jourdan et al. 1980; Katoh et al. 1993; Meister and Bonhoeffer 2001; Rubin and Katz 1999; Stewart et al. 1979; Uchida et al. 2000; Xu et al. 2000, 2003). These studies clearly showed that individual odorants activate a specific combination of glomeruli. In addition, using the optical imaging method and large systematic panels of odorants, recent studies have begun to reveal the molecular receptive range (MRR) of individual glomeruli and to spatially map the MRR on the glomerular sheet in the dorsal surface of the rat OB (Meister and Bonhoeffer 2001; Rubin and Katz 1999; Takahashi et al. 2004a; Uchida et al. 2000). Because of the technical reasons, the optical mapping of the MRR properties has been limited to the dorsal surface of the OB.
On the dorsal surface, glomeruli responsive to odorants with similar molecular features (especially with similar polar functional groups) gather in close proximity and form molecular-feature clusters (Meister and Bonhoeffer 2001; Takahashi et al. 2004a,b; Uchida et al. 2000). It is not well understood whether similar molecular-feature clusters exist in other parts of the OB. On the dorsal surface, glomeruli that respond to aliphatic acids, aldehydes and amines form a cluster at the anteromedial part (cluster A). Glomeruli responsive to aliphatic alcohols, aliphatic ketones, and para-alkyl methoxy benzenes form another cluster in the anterolateral part (cluster B). A cluster of glomeruli responsive to phenol and its derivatives is located at the centrolateral part of the dorsal surface (cluster C). Glomeruli responsive to odorants with a keto group formed a cluster at the posterolateral part (cluster D). All the four clusters (A–D) are located in zone 1 of the OB (Takahashi et al. 2004a,b). Analysis of the MRR properties of these glomeruli indicates that the presence of one or more polar functional group(s) in the molecular structure is indispensable for activating the glomeruli in zone 1, although a specific combination of functional group(s) and overall molecular structure seems to be essential for activating individual glomeruli (Takahashi et al. 2004a).
Unlike a majority of odorants that contain one or more polar functional group(s), hydrocarbons lack any polar functional group. Yet hydrocarbons constitute a large family of odorants. For example, benzene and its derivatives (e.g., toluene and xylene) have gassy or kerosene-like odor, whereas cyclic terpene hydrocarbons (e.g., limonene and pinene) have citrusy or woody odor. Alkanes have ethereal odor (Arctander 1969). Does the olfactory system discriminate many hydrocarbon odorants based on the difference in hydrocarbon structure?
Optical imaging studies on the dorsal and dorsolateral surface of the OB have shown that hydrocarbon odorants activated only a few glomeruli in zone 1 but many glomeruli located just outside of the zone 1 (Takahashi et al. 2004a; Uchida et al. 2000). Furthermore, electrophysiological study (Katoh et al. 1993) showed that mitral/tufted cells responsive to hydrocarbon odorants were mainly located in the ventral regions (within zones 2–4) of the rabbit OB. These results suggest that hydrocarbon odorants are detected mainly by the glomeruli located outside of zone 1.
To examine how the hydrocarbon odorants are represented in the OR maps of the rat OB, we surgically exposed the lateral surface (part of zones 2–4 of the lateral map) of the OB and recorded the odorant-induced glomerular activity using the method of optical imaging of intrinsic signals. For the stimulation of the olfactory epithelium, we used systematic panels of hydrocarbon odorants and odorants that contain a polar functional group attached to the hydrocarbon structure.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Twenty-one adult rats (male Sprague-Dawley, 
300 g) were used for the imaging experiments. All experiments were performed in accordance with the guidelines of the Physiological Society of Japan and the animal experiment committee in the University of Tokyo. Animals were anesthetized with medetomidine (0.5 mg/kg ip), ketamine (67.5 mg/kg ip), and pentothal sodium (25 mg/kg ip). Animals were mounted in a custom-made stereotaxic frame that enabled us to rotate the frame so that animals lie on their side (Narishige, Tokyo, Japan). After the surgical removal of right eye, the skull overlying the caudal half of the lateral surface of the right OB was removed using a dental drill.
Odorants
Odorants (all 95–100% pure) were kindly presented by Takasago (Tokyo, Japan) and T. Hasegawa (Tokyo, Japan) or purchased from Sigma (St. Louis, MO), Tokyo Kasei (Tokyo, Japan), and Nacalai Tesque (Kyoto, Japan). The panel of 21 hydrocarbon odorants that were used for the initial series of experiments contained benzene, cyclohexane, ethylbenzene, o-xylene, m-xylene, p-xylene, butylbenzene, o-diethylbenzene, m-diethylbenzene, p-diethylbenzene, naphthalene, d-limonene, l-
-phellandrene, p-cymene, -cadinene, -pinene, camphene, myrcene, hexane, octane, and decane.
In the second series of experiments, the preceding panel of stimulus odorants was expanded to include the following 18 odorants: 9 odorants that have a polar functional group attached to the cyclic hydrocarbon skeletons (anisole, phenol, benzaldehyde, acetophenone, benzoic acid, d-menthol, l-carveol, d-pulegone, and d-carvone) and 9 open-chain odorants with a polar functional group (butanal, hexanal, octanal, 2-pentanone, 2-heptanone, 2-nonanone, methyl propyl ether, ethyl butyl ether, and dibutyl ether). Toluene was also used in the second series of experiments.
Intrinsic signal imaging
Detailed procedures for the optical imaging have been described previously (Takahashi et al. 2004a; Uchida et al. 2000). Briefly, intrinsic signals (absorption changes at 705-nm wavelength light) were optically recorded from the lateral surface of the OB through the glass coverslip window of an agarose gel chamber. Images were collected using a CCD camera (CS8310, Tokyo Electric Industry, Tokyo, Japan) and digitized with an IBM/PC-compatible equipped with a video frame grabber board (Pulsar, Matrox, Quebec, Canada). The imaged area was 4.2 x 3.1-mm region with a spatial resolution of 320 x 240 pixels. Images of surface blood vessels were photographed under 540-nm wavelength light using the CCD camera. The focusing depth was adjusted to 250 µm below the surface of the OB in the center of the imaged area. For each recording trial, data were collected for 8 s with a frame length of 0.5 s (16 frames per trial). Odorant stimulation was applied from the beginning of the fifth to the end of sixteenth frame. Interstimulus intervals were 30 s. Odorants (2 ml) were prepared at the bottom of glass test tubes (10-cm height) in pure liquid or solid form. Odorant stimulation was performed by placing an odorant-containing test tube within 20 mm of the animal's nostril. Representative odorants were sampled at the animal's nostril and their concentration was analyzed with gas chromatography (Hitachi Kyowa Engineering, Ibaraki, Japan). The concentrations of the odorants were: benzene, 5.2 ± 0.6 (SD) ppm; d-limonene, 0.37 ± 0.07 ppm; octane, 4.3 ± 0.2 ppm (n = 3). To check the consistency of the intrinsic signals during an experiment, responses to two standard odorants (benzene and d-limonene) were tested repeatedly. Experiments were continued until the responses to the control odorants decreased in intensity. In our experimental condition with surgical removal of the right eye, the successful recordings of intrinsic signals typically lasted for 4 h.
Data analysis
Images were analyzed using IDL software (Research Systems, Boulder, CO). A differential image was obtained by dividing the magnitude of signals acquired during odorant-stimulation (in most cases, frames 9–16) 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 3–10 odorant presentations. All final images were imported into Adobe Photoshop 6.0 for cropping and display. For superimposed display of multiple odorant-evoked responses, the threshold was set at 0.035% reflectance change, and the images were processed using 3 x 3 pixel median filter.
Because the average diameter of glomeruli in rat OB is 150 µm (Meisami and Sendera 1993), each darkening spot that showed >0.035% reflectance change and had dimensions larger than a circle with a diameter of 150 µm was defined as the response from one glomerulus. A number was assigned to each glomerulus in numerical order from posterodorsal to ventral parts in most cases. Response magnitude of an individual glomerulus was calculated by averaging the responses of the circular region with a diameter of 150 µm using MetaMorph software (Universal Imaging, West Chester, PA). Darkening on the blood vessels was excluded from the analyses. Because of the local pile-up of glomeruli, we cannot rule out the possibility that a spot originates from closely located or overlapped two glomeruli.
Superimposition of molecular structures
To deduce the common molecular features of hydrocarbon odorants effective in activating individual glomeruli, we superimposed the molecular structures of cyclic hydrocarbon odorants that activated the glomeruli >0.035% in magnitude using ChemOffice software (CambridgeSoft, Cambridge, MA). The benzene ring has a flat disk-like structure (Vollhardt and Schore 1998). Thus as a first approximation, the structure of the cyclic hydrocarbon odorants was described in a plane, placing the longest side chain downward (see supplementary figure1). Then all the structures of odorants effective in activating individual glomeruli were superimposed by overlaying the six-carbon ring. For -cadinene, the six-carbon ring with two side chains was used for overlaying. The six-carbon ring of the cyclic hydrocarbons was approximated as the disk-like structure of the benzene ring. In the case of -pinene and camphene, which have a bridged bicyclic structure, the six-carbon ring was approximated as cyclohexane ring in a plane, and the three-dimensional bridge structure was assumed (Whittaker 1972). If a glomerulus responded both -pinene and camphene, the structure of -pinene was represented. The deduced common molecular features of hydrocarbon odorants were shown at the position of each glomerulus.
Histology
In three experiments, a blue dye (Brilliant Blue 6B, Nacalai Tesque) was iontophoretically injected into some of the active spots using a glass micropipette after the optical imaging. Under deep anesthesia, animals were then perfused with 4% paraformaldehyde. Cryostat sections (20 µm) through the OB were stained for olfactory cell adhesion molecule (OCAM) using a rabbit polyclonal anti-OCAM antibody (Yoshihara et al. 1997), counterstained with 4', 6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR), and examined with a fluorescent microscope (BX60, Olympus, Tokyo, Japan). A flattened unrolled map of the glomerular layer of the OB was constructed as described (Nagao et al. 2000). OCAM-positive and -negative glomeruli and dye-injected points were then mapped on the flattened maps. The imaged region was determined with reference to the dye-injected points.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Surgical removal of the right eye enabled us to expose a substantial part of the lateral surface of the right OB (Fig. 1A). Because of the presence of the olfactory epithelium that covers the anterior part of the lateral surface of the OB, the optical imaging was performed from the posterior half of the lateral surface (the region surrounded by a red line in Fig. 1A). The imaged region extended ventrally to the bottom of the OB and caudally to the emergence of the lateral olfactory tract (LOT) at the brain surface (Fig. 1A).
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; Takahashi et al. 2004a) (Fig. 1C). The imaged region extended 3 mm in the dorsoventral axis and 2 mm in the anteroposterior axis. About 12% of the total glomerular sheet in the OB was imaged in the present study. The imaged region was located within the OCAM-positive zones (zones 2–4, yellow region in Fig. 1C) in the lateral map. Because the optical imaging with the CCD camera positioned perpendicularly to the lateral surface of the OB enabled us to simultaneously examine the glomerular activity in a substantial region of the central and ventral parts in exact focus (but not in the dorsal part due to the curved surface of the OB), we focused our analysis to the glomeruli in the central and ventral parts of the lateral surface.
Glomerular responses to hydrocarbon odorants
In the initial series of experiments, we examined the odorant-response specificity of individual glomeruli in the lateral surface using a panel of hydrocarbon odorants (21 odorants, see METHODS; molecular formulae and odor are listed in the supplementary figure). Based on the similarity in the molecular structure, the hydrocarbon odorants were tentatively grouped into four structural classes: benzene-family hydrocarbons (benzene, cyclohexane, ethylbenzene, o-, m-, and p-xylenes, butylbenzene, o-, m-, and p-diethylbenzenes), cyclic terpene hydrocarbons (d-limonene, l--phellandrene, p-cymene, -pinene, and camphene), hydrocarbons with two six-carbon rings (-cadinene and naphthalene) and open-chain hydrocarbons (myrcene, hexane, octane, and decane).
Figure 2 shows the optical images of glomerular responses in the central and ventral parts (surrounded by a red line in Fig. 2A) of the lateral surface induced by individual hydrocarbon odorants. Figure 2B, a–g, shows the glomerular responses to benzene-family hydrocarbons. Benzene (Fig. 2Ba) and cyclohexane (Bb) activated several glomeruli located in the posterocentral region of the lateral surface. Ethylbenzene, ortho-, meta-, and para-xylenes (B, c–f) activated several glomeruli in the posterocentral region, and some glomeruli in the anteroventral region. Figure 2C, a–g, shows the glomerular responses to cyclic terpene hydrocarbons. Cyclic-terpene-responsive glomeruli were clustered mostly in the anteroventral region of the imaged lateral surface (Fig. 2Cg). Thus many glomeruli in the ventrolateral surface responded to hydrocarbon odorants. Structurally similar hydrocarbons (benzene and cyclohexane; o-xylene and m-xylene; d-limonene, l--phellandrene, and p-cymene) activated a largely overlapping but slightly different combination of glomeruli.
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Glomeruli in cluster I responded to at least one of the cyclic terpene hydrocarbons. Many of the cyclic-terpene-responsive glomeruli in cluster I responded also to a subset of benzene-family odorants and open-chain hydrocarbons. -Cadinene, a hydrocarbon odorant with two six-carbon rings, activated some glomeruli in the cluster I. However, naphthalene, another odorant with two six-carbon rings, did not activate glomeruli in the cluster I. The cluster I glomeruli were distributed in the anteroventral region of the exposed lateral surface.
Figure 3, C and D, shows the spatial arrangement and MRR properties of glomeruli in the OB of another rat (rat 2). Compared with the OB of rat 1, the exposed surface of the OB extended more anteroventrally in this rat. Based on the criteria described in the preceding text, most of the hydrocarbon-responsive glomeruli could be classified into clusters H and I that were similar to those observed in rat 1. Although the shape of the tentative boundary of each cluster and the spatial extent of the exposed surface varied among different OBs, the basic MRR properties and the relative spatial position of the clusters H and I were conserved among all OBs (in 11 different rats) that were examined with the panel of hydrocarbon odorants (Fig. 4).
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). For this purpose, we used cyclic hydrocarbons (especially those that have a benzene ring) because of their semi-rigid disk-like molecular conformations, and approximated the molecular structure mostly in a plane (2-dimensional expression). We did not use open-chain hydrocarbons that can have a variety of molecular conformations.
For each glomerulus, we superimposed the two-dimensional structure of cyclic hydrocarbons that were effective in activating the glomerulus. For example, glomerulus 34 in Fig. 3B is located in the cluster I and responded strongly to d-limonene and l--phellandrene, modestly to p-cymene, and weakly to ethylbenzene, o-, m-, and p-xylenes, m- and p-diethylbenzenes (Fig. 5Aa). Figure 5Ab shows the superimposed molecular structure of these nine effective cyclic hydrocarbons. The superimposed structure indicated that characteristic molecular features of hydrocarbons for glomerulus 34 is a p-cymene-like structure (see supplementary figure).
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Structural determinants for the activation of the hydrocarbon-responsive glomeruli
Because glomeruli in clusters H and I can be activated by hydrocarbon odorants, we hypothesized that ORs represented by these glomeruli may primarily recognize the hydrocarbon skeletons of the odorants. Do the hydrocarbon-responsive glomeruli in these clusters respond also to the odorants that have a polar functional group attached to the hydrocarbon skeleton? To examine the effect of the presence of a polar functional group, we recorded the responses of the hydrocarbon-responsive glomeruli to odorants that have a polar functional group attached to the cyclic hydrocarbon skeletons (supplementary figure). Anisole, phenol, benzaldehyde, acetophenone, and benzoic acid were used to examine whether they activate the glomeruli that responded to benzene-family hydrocarbons. Cyclic terpene ketones (d-pulegone and d-carvone) and cyclic terpene alcohols (d-menthol and l-carveol) were used to examine the glomeruli that responded to cyclic terpene hydrocarbons.
Figure 6 (A–C) shows the responses of nine glomeruli (glomeruli 1–9 in Fig. 6B) in cluster H to the benzene-family hydrocarbons and to benzene derivatives with a polar functional group. These glomeruli were located in the center of the cluster H (Fig. 6, A and B) and responded to at least one of the benzene-family odorants (Fig. 6C, a–c). As shown in Fig. 6C, d–g, most of them responded also to one or more of the benzene derivatives with a polar functional group. For example, glomerulus 2 (rightward arrowhead) responded not only to benzene, toluene, and ethylbenzene but also to anisole and benzaldehyde. Glomerulus 6 (upward arrowhead) responded weakly to anisole in addition to the modest responses to toluene and benzene. Figure 6D indicates that MRR of individual glomeruli in the central part of the cluster H typically covers both benzene-family hydrocarbons and odorants with a polar functional group attached to the benzene ring. In all the 11 OBs examined, the MRR of individual glomeruli in the cluster H covered a range of odorants having similar hydrocarbon structure with or without a polar functional group. These results suggest that the common molecular feature of odorants for activation of the hydrocarbon-responsive glomeruli in cluster H is the benzene-related hydrocarbon skeleton.
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-phellandrene. Figure 6H shows MRR properties of 10 hydrocarbon-responsive glomeruli in cluster I. Most of the glomeruli responded to at least one of the terpene ketones and terpene alcohols in addition to the terpene hydrocarbons. Experiments from eight different OBs indicate consistently that hydrocarbon-responsive glomeruli in cluster I can respond also to odorants with a polar functional group attached to the terpene hydrocarbon skeleton. These results suggest that the common molecular features of odorants for activation of many glomeruli in cluster I involve the hydrocarbon skeleton shared by the effective terpene hydrocarbons, terpene ketones and terpene alcohols.
The preceding results indicate that characteristic molecular features of many glomeruli in cluster H involve benzene-like hydrocarbon structures, whereas those of cluster I glomeruli are cyclic-terpene hydrocarbon structures that have two side chains at para-position around the benzene ring. The characteristic molecular features of cluster I glomeruli thus appear to have more elongated and bulky hydrocarbon skeleton than those of cluster H glomeruli. To further compare the characteristic molecular features between cluster H glomeruli and cluster I glomeruli, we examined the responses of these glomeruli to three alkanes, open-chain hydrocarbons, with different carbon chain length (hexane, C6; octane, C8; and decane, C10; Figs. 3, B and D, and 7B). Because of their nonrigid structures, open-chain hydrocarbons can take many conformations. We hypothesized that hexane (C6) will activate glomeruli in clusters H and I by taking the benzene-like rolled-up shape, whereas octane (C8) or decane (C10) will preferentially activate glomeruli in cluster I by taking the terpene-like conformation. In agreement with this hypothesis, we observed that many glomeruli in cluster H tended to respond preferentially to hexane, the shortest among the three alkanes, whereas many glomeruli in cluster I responded either to all the three alkanes, or preferentially to octane and decane (Figs. 3, B and D, and 7, B and C).
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Because more glomeruli in cluster H responded to 6-carbons-alkane than to 8-carbons- and 10-carbons-alkanes (Fig. 7C, cluster H), we speculated that these glomeruli might preferentially respond to the polar aliphatic odorants with relatively short carbon chain. In contrast, glomeruli in cluster I might preferentially respond to the polar aliphatic odorants with a relatively long carbon chain because more glomeruli in cluster I responded to 10-carbons-alkane than to 8-carbons- and 6-carbons-alkanes (Fig. 7C, cluster I). To examine this possibility, we counted the number of glomeruli in these clusters that responded to each of the polar aliphatic odorants.
We then compared the averaged number of activated glomeruli (obtained from 5 different OBs) among the three members of aliphatic aldehydes (Fig. 7D), the three members of aliphatic ketones (Fig. 7E), and the three members of aliphatic ethers (Fig. 7F). The results showed that in all the three classes of aliphatic odorants, glomeruli in cluster I were activated preferentially by odorants with longer carbon chains (Fig. 7, D–F, filled columns). In contrast, glomeruli in cluster H were either activated preferentially by odorants with shorter carbon chain (Fig. 7E, open columns) or did not show significantly different responses to the three odorants with different carbon chain length (Fig. 7, D and F, open columns). These results are consistent with the hypothesis that the hydrocarbon skeletons of odorants play a key role in activating glomeruli in clusters H and I.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Hydrocarbon odorants are simple compounds containing only two elements, carbon and hydrogen, and do not have any polar functional group. Hydrocarbons with different molecular structure typically have different odor to human nose (Arctander 1969). How are the hydrocarbon odorants coded in the odor maps of the olfactory bulb?
In agreement with electrophysiological studies (Katoh et al. 1993) and 2-deoxyglucose (2-DG)-uptake studies that reported the "glomerular module j" as an area activated by some terpene hydrocarbons in ventrolateral region of the OB (Johnson et al. 2002), present results showed that many glomeruli in the ventrolateral zones (zones 2–4) responded to hydrocarbon odorants. The present study extended the previous 2-DG studies in elucidating the MRR properties of individual hydrocarbon-responsive glomeruli. The robust response of glomeruli in the ventrolateral zones to hydrocarbon odorants is in a striking contrast to the previous observations (Takahashi et al. 2004a; Uchida et al. 2000) that hydrocarbon odorants rarely activated glomeruli in the dorsal zone (zone 1). Electroolfactogram (EOG) studies showed that hydrocarbon odorants preferentially activated olfactory sensory neurons in the ventrolateral zones of the olfactory epithelium (Scott and Brierley 1999; Scott et al. 2000), suggesting that hydrocarbon-responsive ORs may be preferentially expressed in the ventrolateral zones. Because olfactory sensory neurons in the ventrolateral zones of the epithelium project their axons selectively to the glomeruli in the ventrolateral zones of the OB (Yoshihara et al. 1997), the present study is in good agreement with the EOG studies.
Based on the characteristic molecular features of individual glomeruli in the lateral surface of the rat OB, we tentatively classified the hydrocarbon-responsive glomeruli into two clusters, clusters H and I. Glomeruli in cluster H tended to respond to benzene derivatives that have no, one, or two side chain(s) at ortho- or meta-position around the benzene ring. Thus the glomeruli in cluster H might participate in the representation of gassy and kerosene-like odor quality (see supplementary figure). Glomeruli in cluster I tended to respond to cyclic terpene hydrocarbons in addition to the benzene derivatives. These glomeruli in cluster I thus may participate in the representation of citrusy or woody odor quality in addition to the gassy or kerosene-like odor quality.
In the dorsal zone of the OB, glomeruli with similar MRR properties gathered in close proximity and formed molecular-feature clusters (Takahashi et al. 2004a; Uchida et al. 2000). Present study showed that molecular-feature clusters of glomeruli exist also in the ventrolateral zones of the OB. Thus the clustering of glomeruli with similar MRR properties seems to be a conserved architecture across different zones of the OB. The cluster of glomeruli is in good agreement with the observation that olfactory sensory neurons expressing highly homologous ORs project their axons to neighboring glomeruli (Strotmann et al. 2000; Tsuboi et al. 1999).
Difference between zone 1 and zones 2–4 in the characteristic molecular features of individual glomeruli
Glomeruli in the OB are parceled into four zones (zones 1–4) (Mori et al. 1999; Nagao et al. 2000). Two molecular markers were so far used to selectively label glomeruli in zone 1 (NADPH diaphorase) (Alenius and Bohm 2003) or glomeruli in zones 2–4 (OCAM) (Yoshihara et al. 1997). In the present study, we used an anti-OCAM antibody to label glomeruli in zones 2–4 and found that hydrocarbon-responsive glomeruli formed clusters H and I within the OCAM-positive zones 2–4.
Figure 8 shows a schematic diagram illustrating the spatial map of the glomerular clusters A–I on the flattened glomerular sheet of the OB. This diagram is based on the result of the previous imaging study on clusters A–G (Takahashi et al. 2004a) and the present result on clusters H and I. In each cluster, one or two example(s) of the MRR of individual glomeruli is shown by the structural formulae of effective odorants (indicated by an arrow and surrounding line). Thick lines on the structural formulae indicate the common and characteristic molecular features that are shared by effective odorants for individual glomeruli.
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). Thus characteristic molecular features of individual glomeruli in the clusters A–D always included one or more polar functional group(s) (Fig. 8). In a striking contrast, MRR of individual glomeruli in clusters F and G (in zones 2–4) covered both hydrocarbon odorants and odorants with a polar functional group (Takahashi et al. 2004a). Present study greatly expanded the imaged region in zones 2–4 and found two large clusters of hydrocarbon-responsive glomeruli (clusters H and I). The deduced characteristic molecular features of individual glomeruli in the cluster H include benzene-ring-related hydrocarbon skeleton, whereas those in the cluster I include cyclic terpene hydrocarbon skeleton that has more elongated and bulky shape than the benzene-ring-related hydrocarbon skeleton (Fig. 8). In agreement with this result, glomeruli in cluster I responded preferentially to alkanes with relatively long carbon chain and to polar aliphatic odorants with relatively long overall chain length (Fig. 7).
The preceding results indicate a clear difference in the characteristic molecular features of individual glomeruli between the group of clusters A–D (in zone 1) and the group of clusters H and I (in zones 2–4). If the MRR of individual glomeruli reflects strongly the MRR of the represented OR, the results suggest that functional groups play a key role in activating ORs that are represented by glomeruli in clusters A–D, whereas overall hydrocarbon skeleton is of primary importance in activating ORs that are represented by glomeruli in clusters H and I.
Because clusters A–D are located in zone 1 and clusters H and I in zones 2–4, it is tempting to speculate that there might be a clear difference in the characteristic molecular features of individual glomeruli between zone 1 and zones 2–4. Although both the polar functional group and the overall hydrocarbon skeleton seem to be important determinants for activating individual glomeruli, common and characteristic molecular features of glomeruli in zone 1 might be the polar parts of odorants including one or more polar functional group(s). In contrast, common and characteristic molecular features of glomeruli in zones 2–4 might be the hydrocarbon part or relatively nonpolar parts of odorants. This hypothesis is supported by the previous observation that hydrocarbon-responsive glomeruli in clusters F and G are located in zones 2–4 (Takahashi et al. 2004a).
However, clusters F–I that were examined in the present or previous study cover only a small part of zones 2–4. Although the 2-DG studies reported the glomerular responses to hydrocarbons in the anterior part of zones 2–4 (Johnson et al. 2002), this region remained unexplored with the optical imaging method. Thus to examine the preceding hypothesis of the zonal difference in the characteristic molecular features, further experiments including the imaging of anterior part of zones 2–4 are necessary. It is also necessary to characterize in more detail the MRR property of individual glomeruli in zones 2–4 using a lager panel of stimulus odorants because we could not characterize many glomeruli in the posterolateral surface using the present panels of odorants.
Zonal organization of glomeruli and OR classes
OR genes of both mice and human are classified into two broad families, class I and class II (Glusman et al. 2001; Zhang and Firestein 2002). Recent studies showed that class I ORs (fish type, present in both fishes and tetrapods) tend to recognize relatively hydrophilic compounds, whereas tetrapod-specific class II ORs tend to recognize more hydrophobic compounds (Krautwurst et al. 1998; Malnic et al. 1999; Mezler et al. 2001). All class I ORs that are examined to date are expressed by olfactory sensory neurons (OSNs) in the dorsal part or the zone 1 of the olfactory epithelium (Bulgar et al. 1999; Malnic et al. 1999). In contrast, class II ORs have been found in all four zones. OSNs expressing a class I OR send their axons selectively to dorsal zone 1 of the OB (Conzelmann et al. 2000). Thus many glomeruli in zone 1 represent class I ORs. Glomeruli in zones 2–4 most probably represent class II ORs. The present and previous results on the difference in the characteristic molecular features between the group of clusters A–D (in zone 1) and the group of clusters F–I (in zones 2–4) might correlate in part to the zonal difference in the expression of class I and class II ORs.
Characteristic molecular features of glomeruli in clusters A–D (in zone 1) are the polar parts of odorants. A subset of these glomeruli might thus represent class I ORs that interact in a critical manner with the polar parts of odorants. Class II ORs represented by a different subset of glomeruli in zone 1 of the OB might also recognize primarily the polar parts of the odorants. Characteristic molecular features of glomeruli in clusters H and I (in zones 2–4) are the hydrocarbon parts or relatively nonpolar parts of odorants. Class II ORs represented by these glomeruli may thus interact preferentially to the nonpolar parts of the odorants. If this is the case, the zonal arrangement of glomeruli in the OR maps of the OB might reflect in part the evolutional history of OR genes. In the course of evolution from ancient aquatic animals that recognize water-soluble odorants into modern terrestrial animals that detect air-borne odorants, prototype OR genes presumably have evolved into class I and class II, and then the two classes took separate evolutionary paths (Freitag et al. 1998; Glusman et al. 2001). Class I ORs and a subset of class II ORs that have evoleved to respond preferentially to odorants with one or more polar group(s) might be represented by glomeruli in zone 1 of the OB. The other subset of class II ORs that have evolved to recognize preferentially the relatively nonpolar parts of odorants might be represented by glomeruli in zones 2–4.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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1 The Supplementary Material for this article (a figure) is available online at http://jn.physiology.org/cgi/content/full/00873.2004/DC1. 
Address for reprint requests and other correspondence: K. Mori, Dept. 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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on the scaling bar. Filled arrowheads and open arrowheads indicate glomeruli that responded >0.035 and <0.035%, respectively. D: MRR property of 9 glomeruli for 4 benzene-family hydrocarbons and 5 benzene derivatives with a polar functional group. E–H: the MRR of individual glomeruli in the cluster I tended to cover a range of odorants having the terpene hydrocarbon skeleton. E: a schematic drawing of the analyzed region in cluster I (thick line). Scale bar, 1 mm. F: positions of 10 hydrocarbon-responsive glomeruli in the imaged region. Two representative glomeruli (glomeruli 4 and 9) are indicated by an upward arrowhead and a downward arrowhead, respectively. Scale bar, 500 µm. G, a–e: optical images of glomeruli in response to d-limonene (Ga), l-
