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Department of Physiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
ABSTRACT I. INTRODUCTION II. ODORANT MOLECULAR FEATURES AND PERCEIVED ''ODOR'' III. TWO-DIMENSIONAL REPRESENTATION OF ODORANT RECEPTOR MAPS IN THE OLFACTORY BULB IV. INDIVIDUAL GLOMERULI REPRESENT A SPECIFIC COMBINATION OF MOLECULAR FEATURES A. MRR Property of Individual Glomeruli B. MRR Property of Individual Mitral/Tufted Cells V. CLUSTERING OF GLOMERULI DETECTING A SIMILAR COMBINATION OF MOLECULAR FEATURES A. Cluster A 1. Oxygen-containing functional groups 2. Nitrogen-containing functional groups 3. Molecular profiles B. Cluster B 1. Oxygen-containing functional groups and molecular profiles C. Cluster C 1. Oxygen-containing functional groups and molecular profiles D. Cluster D 1. Functional groups and molecular profiles E. Cluster E F. Cluster F 1. Oxygen-containing functional groups 2. Molecular profiles G. Cluster G 1. Oxygen-containing functional groups 2. Molecular profiles H. Cluster H 1. Molecular profiles 2. Oxygen-containing functional groups I. Cluster I 1. Molecular profiles 2. Oxygen-containing functional groups J. Comparison With Maps Obtained With Other Methods and in Other Species VI. ZONES AND CLUSTERS IN THE OLFACTORY BULB VII. TOPOGRAPHIC MAP OF CHARACTERISTIC MOLECULAR FEATURES VIII. MOLECULAR-FEATURE CLUSTERS MAY PARTICIPATE IN ODOR QUALITY PERCEPTION A. Cluster A and Odor Quality B. Cluster B and Odor Quality C. Cluster C and Odor Quality D. Cluster D and Odor Quality E. Cluster H and Odor Quality F. Cluster I and Odor Quality IX. MAPPING THE NATURAL ODOR IN THE MOLECULAR-FEATURE MAPS OF THE OLFACTORY BULB A. Spatial Representation of Spoiled Food Smells in the Odor Maps B. Spatial Representation of Urine Odors in the Mouse Olfactory Bulb X. CONCLUSION GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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The olfactory information is carried in a vast variety of odor molecules (odorants), small volatile compounds with molecular mass <300 Da. Each object, an apple for example, typically emits hundreds of different odorants. It has been estimated that more than 400,000 different compounds are odorous to the human nose (31). More surprisingly, no two compounds have ever been found to have exactly the same odor quality (9), suggesting that the olfactory system can detect and discriminate more than 400,000 different compounds. How does the olfactory system recognize and discriminate numerous different odorants? This is one of the basic and challenging questions in the field of olfactory research.
In addition to the question of odorant discrimination, olfactory research has been challenging the difficult puzzle of the molecular structure-odor relationship (85). Since the discovery of the methods for determining the molecular structure of odorants, chemists, especially fragrance/flavor chemists, have noted a variety of correlations between the molecular structure of odorants and their perceived "odor." In addition, psychophysical studies of olfaction were pursued to elucidate the rules for odorant structure-odor relationships (3, 9, 63, 81). It is surprising that there exist relationships between the molecular structure and "odor," in spite of the large gap between the two. The structure of odorants is at the molecular level in the range of nanometers, whereas the perceived odor of the odorants is the subjective description of human olfactory sense.
The knowledge of the structure-odor relationship is fragmental and far from complete. Furthermore, postulated relationship rules contain many exceptions. Nonetheless, studies on the structure-odor relationship have accumulated a large number of specific examples and provided theoretical frameworks of individual specific examples that can be tested experimentally in the olfactory system of both human and animals. These studies thus raised a notion that the information of molecular features detected and processed at the levels of olfactory sensory neurons and olfactory bulb (OB) may play a key role in the perception of odor quality of odorants in the brain. Thus olfactory research faces another important question: What is the neural basis for the molecular structure-odor relationship? To this end, the use of panels of odorants with systematic variation of molecular structure for sensory stimulation is crucial.
The OB is the first relay station of the central olfactory system in the mammalian brain (Fig. 1) and has a cortical structure with distinct layers and numerous glomerular modules (66, 100). Individual sensory neurons in the olfactory epithelium project a single axon to a single glomerulus in the OB. The glomerular sheet of the OB thus forms a map of olfactory axon terminals. The axonal projection of olfactory sensory neurons to the glomeruli is precisely organized in such a way that olfactory sensory neurons expressing a given odorant receptor (OR) converge their axons to a few topographically fixed glomeruli (Fig. 1) (13, 62, 68, 83, 122). Since individual glomeruli represent a single OR, the glomerular sheet of the OB forms a map of ORs.
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Since then, a variety of methods have been used to spatially map the odorant-induced activities of glomeruli or mitral/tufted cells in the vertebrate OB. The mapping methods include 2-DG uptake (39, 41, 42, 52, 97, 111, 114); optical imaging with voltage-sensitive dyes (23, 45, 46, 112); electrophysiological recording from individual mitral/tufted cells (37, 44, 65); expression of immediate early genes such as c-fos (29, 78, 91), c-jun (7), zif268 (38), and activity-regulated cytoskeleton-associated (ARC) genes (56, 57); optical imaging of intrinsic signals (10, 36, 59, 88, 116, 120); expression of phosphorylated ERK (61); imaging with calcium-sensitive dyes (24, 124); imaging using pHluorin (11); and fMRI (128, 129).
The results clearly showed that a given odorant activates a specific combination of glomeruli that are located and distributed at stereotypical positions in the OB. Different odorants activate a distinct combination of glomeruli. The odorant-specific spatial positions of activated glomeruli are conserved across different animals of the same species. The principle of odor mapping is expressed widely by different vertebrate species (25, 76) and by invertebrates such as honeybees (26, 90), moths (32, 51), and flies (49, 125). Thus, either in the OB or in the antennal lobe, the insect analog of the vertebrate OB, the principle of odor mapping is conserved.
The mapping methods can be classified into two groups in terms of advantages and disadvantages in the mapping of odorant-induced activities in the OB. The first group of methods has the advantage of the ability to map the responses over the entire OB and includes the methods using 2-DG uptake, immediate early gene expression, phosphorylated ERK expression, and fMRI. Studies with these methods addressed the question about how individual odorants are represented in the space of entire glomerular sheet of the OB. For many different odorants, these studies revealed the odorant-specific spatial pattern of glomerular activities in the OB. These studies showed also that the odorant-induced activity maps are arranged in a symmetric fashion; one map (lateral map) in the rostro-dorso-lateral hemisphere of the OB and the other (medial map) in the caudo-medial hemisphere. However, except for the fMRI method, these methods map the response only to a single odorant in each animal.
The second group of methods can map responses to many different odorants in the same OB of an animal. These methods include optical imaging of intrinsic signals, optical imaging with Ca2+-sensitive dyes or voltage-sensitive dye, imaging with pHluorin, and fMRI. With the use of these methods, it is possible to determine the range of odorants that activate an individual glomerulus [molecular receptive range (MRR) of the glomerulus] (68) and to examine the spatial representation of the MRR property in the glomerular sheet of the OB. However, again except for the fMRI method, these methods allow us to map only the exposed surface of the OB. Because of this technical difficulty, we have been successful, so far, to image only the dorsal and postero-lateral surfaces of the OB.
The two groups of methods complement each other and have begun to reveal the basic spatial organization of the odor map in the OB. For the extensive knowledge of the spatial representation of individual odorants, see the recent reviews by Leon and Johnson (41, 52), Xu et al. (127), and Shepherd et al. (100). Detailed data of the spatial representation of individual odorants in the OB can be seen at the websites of the Leon laboratory (http://leonlab.bio.uci.edu/index.html), the Restrepo laboratory (http://www.uchsc.edu/rmtsc/restrepo/), and the OdorMapDB (http://senselab.med.yale.edu/senselab/OdorMapDB/default.asp).
Instead of reviewing the whole knowledge about the spatial representation of individual odorants, this review focuses on the question about the spatial arrangement of represented ORs in the OB. We review the emerging ideas regarding the spatial representation of MRR properties in the glomerular sheet of the mammalian OB. Recent studies with the optical imaging method have accumulated information on the MRR map, although the regions that are explored with this method cover only a half of the lateral hemisphere of the OB. The results so far obtained suggest that the mammalian OB represents systematically the characteristic molecular features of odorants, which reflect the systematic spatial representation of ORs. We suggest that the systematic representation of characteristic molecular features may participate in the initial grouping and processing of odorant molecular information at the level of the OB. The information carried by neuronal activities in the molecular-feature map of the OB may be utilized and processed at higher levels in the central olfactory system to form the neuronal representation of the olfactory image of objects and to perceive the odor quality of objects.
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II. ODORANT MOLECULAR FEATURES AND PERCEIVED "ODOR" |
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The polar functional groups are known as "osmophores" (63) and strongly influence the perceived odor quality. As shown in Figure 2A, a homologous series of aliphatic acids share the same functional group (carboxyl group, -COOH) and have similar odor quality (pungent, sour, fatty, and rancid). A homologous series of aliphatic alcohols share the hydroxyl group (-OH) and have similar "fresh and sweet" note. However, odors of aliphatic acids are distinct from those of aliphatic alcohols. Thus the comparison of odors between the two homologous series suggests a major role of polar functional groups for the odor quality perception.
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The molecular profile is equivalent to molecular shape or three-dimensional structure and is known to have a strong influence on perceived odor. The benzene ring (Fig. 2C) is one of the characteristic molecular profiles shared by many benzene-family odorants. The similarity of perceived odor of benzene-family hydrocarbon odorants (e.g., benzene, o-, m-, and p-xylenes, toluene, etc.) suggests the importance of the benzene-ring profile in odor quality perception. Cyclic terpene hydrocarbons (e.g., limonene and pinene) share a molecular profile (Fig. 2C) and have citrusy or woody odor in common. Because hydrocarbons lack any polar functional group, the molecular profile may play a critical role for perception of the odor of hydrocarbon odorants.
In odor molecules, polar functional groups and molecular profiles overlap each other. A specific combination of polar functional groups and molecular profiles may determine the basic framework of molecular features of odorants that are critical for their perceived odor. For example, furan and its derivatives have a specific combination of polar functional groups and molecular profiles, as shown in Figure 3. The furans share the caramellic odor (Fig. 3).
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Recent studies show that individual ORs bind to a range of odorants having similar molecular features (54, 133). The range of odorants that bind to an OR is called the MRR (67, 68) of the OR. We define the characteristic molecular features of an OR as those molecular features shared by the odorants effective in activating the OR. Thus, at the molecular level, an individual OR presumably functions as a molecular-feature detecting unit. Because mice have a repertoire of 
1,000 OR genes, odorants are thought to be initially probed by 1,000 different molecular-feature detecting units.
The logic of molecular-feature detection at the OR level (molecular level) is converted to the cellular and neuronal network levels such that signals of distinct combinations of odorant molecule features are processed in parallel by a large number of molecular-feature detecting cells and modules (6, 66, 98).
At the cellular level, an individual sensory neuron in the olfactory epithelium may function as a molecular-feature detecting unit. Because each olfactory sensory neuron expresses a single OR, it may respond to a range of odorants having similar combinations of molecular features (4). Therefore, the characteristic molecular features of an olfactory sensory neuron can be determined by examining the MRR and molecular features shared by the odorants within the MRR.
At the neuronal circuit level, an individual glomerulus in the OB may function as a molecular-feature detecting unit. Because individual glomeruli represent a single OR, it is possible to determine the MRR and characteristic molecular features of each glomerulus by the same token.
In summary, initial olfactory processing at the levels of ORs, olfactory sensory neurons, and olfactory glomeruli is characteristic in such a way that odorant molecular features are processed in parallel by a large number of molecular-feature detecting units. The information regarding the molecular-features may be utilized by the higher olfactory systems to form the conscious perception of odor quality.
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III. TWO-DIMENSIONAL REPRESENTATION OF ODORANT RECEPTOR MAPS IN THE OLFACTORY BULB |
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1,800 in the mouse (87), 2,400–4,200 in the rat (58, 86), and 6,300 in the rabbit (86). An individual glomerular module consists of several thousands converging olfactory axons, 10–20 mitral cells, and 50–70 tufted cells (2, 86). Individual glomeruli represent a single OR (62, 83, 122). The glomerular sheet of the OB forms OR maps.
The mouse has a repertoire of 1,000 types of ORs. Each OB contains 1,800 glomeruli (87). Individual ORs are represented by a few (typically two) glomeruli. In rats and mice, each OB contains two mirror-image maps of ORs, the lateral and medial maps (62, 72, 83, 122). For a majority of ORs, each OR is represented by two sites: one site in the lateral map and a corresponding site in the medial map. For a small group of ORs, however, each OR is represented by a single glomerulus in one site (115). These glomeruli are located at the ventral midline of the OB.
To illustrate the spatial arrangement of the glomerular sheet that covers the oval OB, it is convenient to transform the three-dimensional glomerular sheet to a two-dimensional plane map with a defined scale and a coordinate reference system. Such a flattened map is useful for comparing and compiling data obtained by different mapping methods and in different individual animals. The map proved to be useful also for detecting the distortion of odor maps in Semaphorin 3A-deficient mice (118). Several methods have been proposed for the two-dimensional representation of odor maps in the mammalian OB, each having advantages and disadvantages (40, 50, 114, 129) (see also the websites of Restrepo's lab and OdorMapDB). For the readers who are not familiar with the three-dimensional anatomy of the mammalian OB, however, we recommend first to refer to the three-dimensional representation of odor maps, as shown, for example, by Leon's group (41).
We have generated the two-dimensional glomerular maps of rat and mouse OB as follows (Fig. 4) (38, 72, 118). Consecutive frontal sections of the OB are Nissl-stained or immunohistochemically labeled for NCAM to clearly see the glomeruli. In each section, we first define as reference points the dorsomedial edge (arrow heads) of the mitral cell layer. The glomeruli are then traced. The trace of the ring of glomeruli is flattened by opening it along its ventral edge. The traces are then aligned from anterior (top) to posterior (bottom) using the dorsomedial edge as a reference line to make an overall unrolled map of glomeruli.
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IV. INDIVIDUAL GLOMERULI REPRESENT A SPECIFIC COMBINATION OF MOLECULAR FEATURES |
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To examine whether individual glomeruli in the OB function as molecular-feature detecting units, it is necessary to determine the MRR of individual glomeruli and to compare the molecular structure of odorants that are effective in activating the glomeruli. For this purpose, it is essential to use a large panel of stimulus odorants with a systematic variation of molecular features. It is also critical to select those methods that enable us to examine the response of individual glomeruli in the same OB of an animal to numerous odorants in the panel.
Optical imaging of intrinsic signals is one of the useful methods to examine the MRR property of individual glomeruli (10, 36, 59, 88, 116, 120). It has been shown with this method that some glomeruli can discriminate even between enantiomers (89). Although the intrinsic signal-imaging method has several weak points that include small signal-to-noise ratio and the lack of histological correlation of the signal with glomeruli, the method provides a convenient way of recording the glomerular responses in the same OB to many different odorants (typically 70). Using the intrinsic signal-imaging method and a panel of systematically changing odorants, we thus examined the MRR property of a large number of glomeruli that were located in the dorsal, dorso-lateral, and postero-ventrolateral surfaces of the rat OB (36, 116, 117, 120). Comparison of MRR properties among the imaged glomeruli revealed the following.
Results 1 and 2 are consistent with the notion that individual glomeruli represent a single OR and that different glomeruli in each map of the OB may represent different ORs. These results suggest that each glomerulus can be characterized by its MRR property and by its characteristic molecular features. In other words, individual glomeruli represent the glomerulus-specific combination of molecular features. Result 3 will be discussed in detail in section V.
B. MRR Property of Individual Mitral/Tufted Cells
Mitral/tufted cells are principal neurons in the OB and project their axons to the olfactory cortex. Individual mitral/tufted cells extend a single primary dendrite to a single glomerulus and within the glomerulus receive excitatory synaptic inputs from many olfactory axons. Thus the MRR property of an individual mitral/tufted cell may strongly reflect the MRR property of its own glomerulus. Studies with single-unit recording from mitral/tufted cells basically support this hypothesis, although the MRR property of individual mitral/tufted cells is determined not solely by the MRR property of its own glomerulus but is modified by synaptic interactions within the local circuit of the OB (67, 68, 130).
Odorant-response specificity of individual mitral/tufted cells was examined using the systematic panel of odorants and extracellular single-unit recording in the rabbit OB (37, 44, 65) and in the rat OB (73, 117). Although there are many reports on the odorant-response specificity of mitral/tufted cells, it is critical to use a large panel of systematically changing odorants and to have the knowledge of odor maps in the OB (as described in sects. V–VII) to examine the MRR property of recorded mitral/tufted cells. Our results show that individual mitral/tufted cells in the dorsomedial region of the OB respond to a subset of aliphatic acids and aliphatic aldehydes with a similar hydrocarbon chain length. Mitral/tufted cells in the ventromedial region of rabbit OB respond to a range of aliphatic and aromatic compounds with similar molecular structures. Thus individual mitral/tufted cells typically respond to a range of odorants that share a specific combination of molecular features. These studies indicate that individual mitral/tufted cells are tuned to a specific combination of molecular features.
A recent study by Lin et al. (53) supports the idea that individual mitral cells in the mouse OB function as a molecular-feature detector. Urine contains numerous odorous compounds with a variety of molecular structures. Using the gas chromatography single-unit recording method, they demonstrated that individual urine-responsive mitral cells respond selectively to only one (or two) compound among many components of urine.
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V. CLUSTERING OF GLOMERULI DETECTING A SIMILAR COMBINATION OF MOLECULAR FEATURES |
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Figure 5A shows examples of MRR properties of glomeruli at the dorsal surface of the rat OB. For the detailed MRR properties of individual glomeruli, readers may refer to the data in Takahashi et al. (116) for the dorsal and dorsolateral surfaces and in Igarashi and Mori (36) for the postero-ventro-lateral surface of the OB. Examination of the relationship between the spatial position of glomeruli and their MRRs clearly indicates that glomeruli with similar MRR tend to locate in a close proximity and form clusters (Fig. 5, A and B). The clusters of glomeruli can be seen more clearly by the spatial mapping of characteristic molecular features using the method of superimposing three-dimensional structures of effective odorants, as shown in section VII. We tentatively classify four clusters of glomeruli (clusters A–D) in the dorsal surface, three clusters (clusters E–G) in the dorsolateral surface, and two clusters (clusters H and I ) in the postero-ventrolateral surface of the rat OB (Fig. 6). The MRR property of glomeruli in each cluster will be described in detail below. These descriptions form the basis for the spatial mapping of the MRR property in the glomerular sheet of the OB. The molecular structure of stimulus odorants is shown in Figure 2A and Figure 7.
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Glomeruli in cluster A are located at the anteromedial part of the dorsal surface (Fig. 6). Because it is relatively easy to image the dorsal surface, the MRR property of glomeruli in cluster A has been most thoroughly studied among the nine clusters.
1. Oxygen-containing functional groups
Glomeruli in cluster A respond to aliphatic acids and aliphatic aldehydes. When examined with a homologous series of aliphatic acids and aldehydes, individual glomeruli respond to subsets of these odorants with a similar carbon chain length. These glomeruli respond also to aliphatic acids and aldehydes with a branched hydrocarbon structure or with one or more double bonds in the carbon chain.
The cluster A glomeruli respond also to a subset of esters with a relatively long carbon chain of acid-part. Glomeruli in the most posterior part of cluster A respond also to diketones. A subset of cluster A glomeruli responds also to benzaldehyde and benzoic acid. Thus the molecular features of odorants that are effective in activating the cluster A glomeruli share either a carboxyl group (-COOH), a diketone group [-(CO)(CO)-], or an ester group (-COO-). These are functional groups that have two oxygen atoms located in proximity. In addition, odorants having a single carbonyl group (>C=O) at the end of the molecule are effective in activating the cluster A glomeruli.
2. Nitrogen-containing functional groups
A large subset of cluster A glomeruli responds also to alkylamines that contain an amino group (-NH2) (117). These results suggest that ORs represented by the subset of cluster A glomeruli respond to both fatty acids with carboxyl group (-COOH) and alkylamines with amino group (-NH2). Because it is difficult to conceive that a single part in the odorant receptive site of OR binds to both carboxyl and amino groups, we speculate that the represented ORs can recognize both carboxyl and amino groups at different parts of their odorant-receptive site. If that is the case, these ORs resemble fish ORs that detect amino acids [H2N-CH(R)-COOH] (15, 22, 24). This raises the possibility that the alkylamine-responsive glomeruli in cluster A represent fishlike class I ORs (see sect. VI) (17, 132).
Based on the carbon chain length of the effective aliphatic acids, we tentatively classify the cluster A glomeruli into three subclusters: A-1, A-2, and A-3 (Fig. 5B). Glomeruli in A-1 respond selectively to aliphatic acids and aldehydes with a long carbon chain, those in A-2 to acid and aldehydes with a carbon chain of middle size, and those in A-3 selectively to acids and aldehydes with a short carbon chain. However, this is not the case for the carbon chain length of the homologous series of alkylamines (117).
Benzoic acid and benzaldehyde (Fig. 12B) have a molecular profile of a benzene ring and an oxygen-containing functional group (carboxyl group or aldehyde group) in their molecular structure. These odorants activate several glomeruli in the subclusters A-2 and A-3.
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Cluster B is located in the anterior region of the lateral part of the dorsal surface of the OB (Fig. 6).
1. Oxygen-containing functional groups and molecular profiles
Glomeruli in cluster B respond to aliphatic alcohols with a relatively long carbon chain and to a wide range of aliphatic ketones. The cluster B glomeruli also respond to a subset of esters. Whereas cluster A glomeruli respond to a subset of esters with a relatively long carbon chain of acid part, cluster B glomeruli preferentially respond to a different subset of esters with a relatively long carbon chain of alcohol-part. A majority of cluster B glomeruli respond also to anisole and its derivatives that have a methoxy group (-O-CH3) and a carbon side chain arranged at the para-position of the benzene ring.
Thus the molecular features of odorants that are effective in activating the cluster B glomeruli are a combination of elongated carbon chain structures with a hydroxyl group (-OH), an alkoxyl group (-O-R), or a carbonyl group (>C=O) attached at one side of the molecule.
The glomeruli in the posterior part of the cluster B respond to a wider range of aliphatic ketones and aliphatic alcohols than those in the anteromedial part. We thus tentatively classify cluster B glomeruli into two subclusters: B-1 (the anteromedial glomeruli) and B-2 (the posterolateral glomeruli) (Fig. 5B).
Glomeruli in cluster C are located at the central region of the lateral part of the dorsal surface (Fig. 6).
1. Oxygen-containing functional groups and molecular profiles
Glomeruli in cluster C respond to phenol family odorants, molecules having a hydroxyl group attached to the benzene ring. Many of the cluster C glomeruli respond also to phenyl ethers. Thus odorants effective in activating cluster C glomeruli share the molecular features composed of a benzene ring with a hydroxyl group, a methoxy group, or an ethoxy group (-O-CH2-CH3). Thus the characteristic molecular features of those glomeruli include the combination of the benzene-like hydrocarbon profile and the hydroxyl or alkoxyl group.
Based on the detailed MRR properties and the positions of glomeruli, we tentatively divide the cluster C into four subclusters: C-1, C-2, C-3, and C-4 (Fig. 5B). In addition to phenol family odorants, C-1 glomeruli respond to aliphatic alcohols with a relatively short carbon chain (4OH-6OH). C-2 glomeruli invariably respond to salicylaldehyde (Fig. 12B), a phenol derivative with a carbonyl group attached to the ortho-position. Glomeruli in subcluster C-3 respond relatively selectively to phenols and phenyl ethers. Glomeruli in subcluster C-4 respond also to short aliphatic ketones and aliphatic ethers.
Cluster D is located at the caudal region of the lateral part of the dorsal surface (Fig. 6).
1. Functional groups and molecular profiles
Glomeruli in cluster D tend to respond to wide structural classes of odorants. Most notable are their responses to a variety of ketones: aliphatic ketones, aliphatic-aromatic ketones, diketones, and cyclic ketones. Compared with glomeruli in cluster B, the cluster D glomeruli tend to respond to a subset of aliphatic ketones with relatively short carbon side chains (Fig. 5B). We tentatively classify the cluster D glomeruli into two subclusters: D-1 and D-2. Medially located subcluster D-1 glomeruli respond to creosol and eugenol, phenol derivatives with a methoxy group attached at the ortho-position and a carbon side chain at the para-position. Laterally located subcluster D-2 glomeruli respond to aldehydes, alcohols, and ethers in addition to ketones.
Cluster E glomeruli are located at the dorsal-most part of the lateral surface of the OB (Fig. 6). We could not characterize the odorant-response specificity of cluster E glomeruli because these glomeruli did not systematically respond to any classes of odorants in our panel.
Cluster F is located at the rostroventral part of the dorsolateral surface of the OB (Fig. 6).
1. Oxygen-containing functional groups
Glomeruli in cluster F are activated by aliphatic ketones. A subset of glomeruli responds also to secondary alcohols, phenyl ethers, diketones, aliphatic-aromatic ketones, and cyclic ketones. We tentatively divide cluster F glomeruli into two subclusters: anteriorly located F-1 and posteriorly located F-2 (116). Compared with F-1 glomeruli, F-2 glomeruli tend to respond to a wide range of aliphatic ketones and cyclic ketones.
In addition to odorants with oxygen-containing functional groups, odorants effective in activating cluster F glomeruli include hydrocarbon odorants that do not have any polar functional group. Among the hydrocarbon odorants, the cluster F glomeruli preferentially respond to terpene hydrocarbons. Thus the terpene hydrocarbon structure (Fig. 2C) may be one of the main determinants for the activation of the cluster F glomeruli.
Cluster G is located at the ventrocaudal part of the dorsolateral surface of the OB (Fig. 6).
1. Oxygen-containing functional groups
Many glomeruli in cluster G respond to phenyl ethers, diketones, aliphatic ketones with relatively short side chains, aliphatic-aromatic ketones, cyclic ketones, and ethers. The cluster G glomeruli are tentatively parceled into two subclusters: G-1 and G-2. Ventrally located G-1 glomeruli respond to aliphatic alcohols, while dorsally located G-2 does not respond to them.
Glomeruli in cluster G invariably respond to hydrocarbons as well as odorants with oxygen-containing functional groups. Interestingly, cluster G glomeruli preferentially respond to benzene family hydrocarbon odorants rather than to terpene hydrocarbons. Thus the benzene ring structure (Fig. 2C) could be one of the main determinants for the activation of cluster G glomeruli.
Cluster H glomeruli extend from the posterocentral region to the posterodorsal region of the postero-ventrolateral surface of the OB (Fig. 6).
Individual glomeruli in cluster H respond to one or more of the benzene-family odorants. However, these glomeruli show no or only a weak response to cyclic terpene hydrocarbons. In addition, some of the cluster H glomeruli respond also to open-chain hydrocarbons.
2. Oxygen-containing functional groups
Most of the glomeruli in cluster H respond also to one or more of the benzene derivatives with a polar functional group, such as anisole (which has a methoxy group on the benzene ring), phenol (a hydroxyl group), benzaldehyde (an aldehyde group), or acetophenone (a carbonyl group) (Fig. 7).
Thus the characteristic molecular features of odorants effective in activating glomeruli in cluster H appear to be benzene-related hydrocarbon skeleton.
Cluster I is located at the anteroventral region of the postero-ventrolateral surface of the OB (Fig. 6).
Glomeruli in cluster I respond to at least one of the cyclic terpene hydrocarbons. Many of the cyclic terpene hydrocarbon-responsive glomeruli in cluster I respond also to a subset of benzene-family odorants and open-chain hydrocarbons.
2. Oxygen-containing functional groups
Several glomeruli in the ventral part of cluster I respond also to the cyclic terpene ketones (for example, d-pulegone or d-carvone) or cyclic terpene alcohols (for example, d-menthol).
Thus the characteristic molecular features of odorants in cluster I contain the hydrocarbon skeleton shared by terpene hydrocarbons, terpene ketones, and terpene alcohols.
Figure 8 summarizes the response specificity of clusters A–G in terms of chemical classes of odorants. The glomeruli responsive to a similar combination of chemical classes are located in close proximity and form molecular-feature clusters. The results shown in this section suggest that ORs represented in a same molecular-feature cluster or subcluster may selectively respond to odorants that have similar combination of molecular features. The molecular-feature clusters A–I are located at stereotypical positions in the glomerular sheet.
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With regard to the idea of molecular-feature clusters, it is interesting to compare our maps with the intrinsic signal imaging maps reported by other laboratories (10, 59, 88). Because the latter studies are designed to address questions other than the molecular-feature clusters, it is difficult to compare the reported maps of odorant-induced glomerular activity with our maps of MRR. Nevertheless, the reported maps at the dorsal surface of the rat and mouse OB are consistent with the idea of cluster organization, especially with regard to the position and odorant specificity of cluster A.
Using the 2-DG method, Johnson et al. (40) collected a huge number of measurements of glomerular activities in response to a large panel of odorants with systematic variation of the molecular structure. With the 2-DG method, it is possible to map the glomerular response only to a single odorant in each animal. Therefore, they compiled the maps from many different rats and found that almost the entire glomerular sheet is covered by glomerular "modules" (clusters of nearby glomeruli) and that each module responds to a particular molecular feature. Because of the difference in the measurement method and the definition of glomerular clusters, it is difficult to compare our maps of molecular-feature clusters with the maps of the glomerular "modules," especially in terms of the shape and boundary of each cluster. However, the overall comparison in terms of odorant specificity and spatial position suggests that the glomerular "modules" in the 2-DG studies are similar in general to the molecular-feature clusters in our study. For example, glomerular "modules" a and c (40) may correspond to clusters A and D in our study, respectively. The assembly of glomerular "modules" d, e, f, h, j, k, and l may roughly correspond to the assembly of clusters H and I. Thus the two different methods presumably shed light on the same basic spatial arrangement of glomeruli from different angles.
The idea of molecular-feature clusters is consistent with the evidence that olfactory sensory neurons expressing homologous odorant receptor genes project their axons to neighboring glomeruli (119). The molecular-feature clusters are found also in the fish OB (25, 47, 76). Furthermore, recent studies indicate that a primitive form of molecular-feature clusters is present also in the larval and adult antenna lobe of insects (16, 49, 51, 90). These results suggest that the clustering of glomeruli that detect similar molecular features is the basic spatial organization of the OB (and antennal lobe) and is conserved across a wide variety of species.
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VI. ZONES AND CLUSTERS IN THE OLFACTORY BULB |
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By combining the optical imaging method and the OCAM immunohistochemistry, we examined the spatial relationship between bulbar zones and the molecular-feature clusters in the rat OB (Fig. 10) (36, 116). Most glomeruli in clusters A-D are in the OCAM-negative zone 1, whereas those in clusters F-I are in the OCAM-positive zones 2–4. The boundary between zone 1 and zones 2–4 is located near the lateral margin of clusters B, C, and D.
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Comparison of the MRR property indicates a clear difference in the characteristic molecular features of individual glomeruli between the group of clusters A-D (in zone 1) and that of clusters F-I (in zones 2–4). Odorants effective in activating glomeruli in clusters A-D (in zone 1) have one or more polar functional group(s) in common. Thus the presence of a specific polar functional group tends to be of primary importance to activate glomeruli in zone 1. In contrast, odorants effective in activating glomeruli in clusters F-I (in zones 2–4) have one or more molecular profile(s) in common, suggesting that the overall molecular shape tends to be critical in activating glomeruli in zones 2–4. For example, characteristic molecular features of glomeruli in clusters H and I are the hydrocarbon profiles or relatively nonpolar parts of odorants (Fig. 10).
OR genes are classified into two broad families, class I and class II (27, 132). Class I ORs (fish type, present in both fish and tetrapods) tend to recognize relatively hydrophilic compounds with one or more polar functional group(s), whereas tetrapod-specific class II ORs tend to recognize more hydrophobic compounds (48, 54, 60). Class I ORs are expressed in zone 1 of the olfactory epithelium, while class II ORs are expressed in all the four zones (14, 17, 54). Thus glomeruli in zone 1 represent both class I ORs and class II ORs, while glomeruli in zones 2–4 are most likely to represent class II ORs. Thus the difference in the characteristic molecular features between the groups of clusters A-D (in zone 1) and F-I (in zones 2–4) might correlate in part to the zonal difference in the expression of class I and class II ORs. Further studies are necessary to examine the spatial relationships among the clusters, zones, and OR classes in the glomerular OR maps of the OB.
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VII. TOPOGRAPHIC MAP OF CHARACTERISTIC MOLECULAR FEATURES |
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Results 1 and 2 suggest that each glomerulus represents a specific combination of molecular features. They thus raise the possibility that each glomerulus can be characterized by determining the specific combination of molecular features that are shared by the odorants that are effective in activating the glomerulus. We define the term characteristic molecular features of a glomerulus as the specific combination of molecular features that are shared by odorants that are effective in activating the glomerulus (116).
One of the practically feasible methods for deducing the characteristic molecular features of an individual glomerulus is to superimpose the three-dimensional structure of odorants that are effective in activating the glomerulus and to determine the combination of molecular features that the effective odorants share. Figure 11 shows two examples of this method. It should be noted that the superposition of effective odorants is not intended to describe the odorant receptive site structure, but to effectively examine the spatial representation of MRR properties. Mapping the deduced characteristic molecular features on the glomerular sheet will make it easier to see the spatial arrangement of MRR maps.
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Figure 12 shows a map of the characteristic molecular features in the cluster C and neighboring clusters in the rat OB. To facilitate the analysis, the presumed critical parts of the combination of characteristic molecular features are labeled by color in this figure. For example, blue shadows indicate a combination of molecular features composed of a single methoxy (-O-CH3) group or a single ethoxy group (-O-CH2CH3) attached to a benzene ring profile. Yellow shadows show a combination of molecular features composed of a single hydroxyl group (-OH) attached to a benzene ring. Red surroundings indicate a combination of molecular features composed of a single hydroxyl group and a single alkoxyl group (-O-R) arranged at the ortho-position around a benzene ring. T
