Coding of olfactory information: Topography of odorant receptor expression in the catfish olfactory epithelium

Cell. Vol. 72, 667-660,

March 12, 1993, Copyright

0 1993 by Cell Press

Coding of O lfactory Information: Topography of Odorant Receptor Expression in the Catfish O lfactory Epithelium John Ngai,*t Andrew Chess,* Michael M. Dowling,” Nicholas Necles,s Eduardo Ft. Macagno,* and Richard Axel’ *Department of Biochemistry and Molecular Biophysics Howard Hughes Medical Institute College of Physicians and Surgeons Columbia University New York, New York 10032 *Department of Biological Sciences Columbia University New York, New York 10027

Summary Discrimination among the vast array of odors requires that the brain discern which of the numerous odorant receptors have been activated. If individual olfactory neurons express only a subset of the odorant receptor repertoire, then the nature of a given odorant can be discerned by identifying which cells have been activated. We performed in situ hybridization experiments demonstrating that individual olfactory neurons express different complements of odorant receptors and are therefore functionally distinct. Thus, a topographic map, defining either the positions of specific neurons in the epithelium or the positions of their projections, may be employed to determine the quality of an olfactory stimulus. Neurons expressing specific receptors appear to be randomly distributed within the olfactory epithelium. These data are consistent with a model in which randomly dispersed olfactory neurons with common receptor specificities project to common glomeruli in the olfactory bulb. Introduction How does the vertebrate olfactory system recognize and distinguish thousands of odors? The initial step in olfactory discrimination requires the interaction of odorous ligands with specific receptors on olfactory sensory neurons. This sensory information is then transmitted to the brain where it is decoded to permit the discrimination of different odors. The mechanisms of olfactory perception will necessarily depend upon the number of individual receptors and their specificities. We recently identified a large multigene family thought to encode the odorant receptors of rat and fish (Buck and Axel, 1991; Ngai et al., 1993 [this issue of Cc//j). These receptors exhibit a putative seven transmembrane domain structure characteristic of the superfamily of G protein-coupled receptors. The repertoire of mammalian olfactory receptors is extremely large and may consist of

TPresent address: Division of Neurobiology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720.

as many as 1000 different genes (Levy et al., 1991; Parmentier et al., 1992). In the fish, the family of receptors is considerably smaller, consistent with the smaller array of perceived odorants. These data suggest that the initial step in olfactory discrimination is accomplished by the integration of signals from a large number of specific receptors, each capable of binding only a small number of odorants. The signal elicited by the interaction of odorous ligands with receptors on sensory neurons is transmitted to the brain, which then must determine which of the numerous receptors have been activated. If each sensory neuron expresses only a small subset of receptors, the problem of distinguishing which receptors have been activated reduces to a problem of identifying which neurons have been activated. At one extreme, it is possible that a given sensory neuron expresses only one type of odorant receptor. The identity of a given neuron would therefore be defined by the specific receptor it expresses. In such a model, perception of an odor encountered by the organism would result from the identification of the subset of cells responsive to that odorant. A test of this model requires the demonstration that individual sensory neurons express different complements of odorant receptors. How does the brain determine which neurons have been activated in order to decode olfactory information from peripheral sensory neurons? In other sensory systems, defined spatial patterns of sensory neurons and their projections are used to determine the position of a stimulus in the environment as well as the quality of the stimulus. In the somatosensory system, for example, the locations of receptors on the body’s surface are represented by spatially contiguous maps in the cerebral cortex (Powell and Mountcastle, 1959; Kaasetal., 1979; lwamuraetal., 1985; Rowe et al., 1985). Moreover, the different somatosensory submodalities (touch, proprioception, nociception, and thermoreception) result from the activation of distinct sensory cells that project to specific regions of the brain via topographically segregated pathways. The olfactory system integrates peripheral sensory information to define the quality of an odor, but not the position of an odorant in environmental space. The vertebrate olfactory system may therefore employ spatial segregation of sensory input to encode the identity of the odorant stimulus. What are the features of the vertebrate olfactory apparatus that might form the anatomical basis for a spatial map of olfactory information? Odorous stimuli are received from the environment by receptors on olfactory sensory neurons in the olfactory epithelium (Lancet, 1986; Reed, 1992). Each olfactory neuron projectsasingle unbranched axon; as the collection of axons emerge from the olfactory mucosal layer, they fasciculate to form the olfactory nerve. The axons of olfactory neurons synapse with dendrites of mitral and tufted neurons in the olfactory bulb, the first relay station for olfactory signaling in the brain (Mori, 1987). The dendrites of several hundred mitral and tufted

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Figure 1. Anatomy

of the Catfish Olfactory

Epithelium

(A) Dorsal view of the anterior portion of a catfish’s head (anterior tip is at the top of the figure). Arrows show the direction of water flow into the anterior naris and out of the posterior naris, as it would travel through the right olfactory pit. The skin overlying the left olfactory pit has been removed, revealing the left olfactory rosette. Bar = 0.5 cm. (B) Higher magnification of the rosette shown in (A). The anterior end of the rosette is at the left. Note the two parallel stacks of lamellae that attach to the pigment-containing medial raphe. Bar = 0.25 cm. (C) Schematic diagram of a catfish olfactory rosette. The dorsal aspect of this structure is toward the top of the figure. Thus, the views shown in (A) and (B) are looking down onto the dorsal ridges of the raphe and lamellae. The proximal faces of the nearest pair of lamellae are shown. The stippled regions, adjacent to the medial raphe, represent the areas occupied by the sensory neuroepithelium. The surrounding area comprises an indifferent, nonsensory epithelium. (D) Horizontal section of an olfactory rosette. This photomicrograph shows that each lamella consrsts of two sheets of epithelium sandwiching a central core of connective tissue. Note the presence of pigment in the raphe, which is situated medially along the anterior-posterior axis of the rosette. The darker staining of the medial portions of the lamellae with toluidine blue reflects the presence of the sensory neuroepithelium. In any given horizontal section, the sensory neuroepithelium extends at most halfway from the raphe to the lateral limits of the lamellae. Bar = 1 mm.

cells are organized into discrete units known as glomeruli. The mitral and tufted cells in turn project axons to higher cortical centers via the medial and lateral olfactory tracts. Approximately 10’ olfactory neurons project to - 1 O3 glomeruli, but each olfactory neuron axon forms synapses within only a single glomerulus. Thus, the anatomy of the olfactory system affords the opportunity for spatial segregation of afferent sensory input at all levels from the peripheral epithelium to the olfactory cortex (reviewed by Shepherd, 1991). Spatial segregation could occur in the sensory epithelium such that sensory neurons that express a given receptor, and therefore respond to a given odorant, may be topographically localized within the olfactory epithelium. This topographic localization would also be maintained in the projections of the sensory axons, such that neurons

expressing the same receptor would project to the same set of glomeruli within the olfactory bulb (Kauer, 1991; Kauer et al., 1991; Shepherd, 1991). Alternatively, sensory neurons expressing a given receptor may be randomly distributed rather than spatially segregated within the sensory epithelium. However, all neurons responsive to a given odorant would project to discrete regions (or glomeruli) within the bulb. Other models in which spatial segregation only occurs centrally within the olfactory cortex, as well as models that do not involve spatial order, have also been considered (Bower, 1991; Freeman, 1991). To determine whether neurons expressing specific odorant receptors are spatially localized within the olfactory epithelium, we analyzed the patterns of expression of odorant receptors. We chose the channel catfish as a model system for two major reasons: First, as shown in the

;;Ecgraphy

of Odorant

Receptors

Figure 2. Low Power Dark-Field

Micrographs

of In Situ Hybridizations

Horizontal sections of catfish olfactory rosettes were hybridized to %-labeled RNA probes and subjected to autoradiography. Sections were annealed with antisense probes specific for the receptor 1 subfamily (A), receptor 32 subfamily(B), receptor 202 (C), receptor 47 subfamily(D). and olfactory cyclic nucleotide-gated channel (E). The pigmented raphe in each panel lies diagonally from the lower left corner to the upper right corner. Note the punctate array of hybridization signals with each of the receptor probes, as compared with the uniform labeling observed with the cyclic nucleotide-gated channel probe. The receptor-specific and channel-specific signals extend from the raphe to the lateral limits of the sensory neuroepithelium. (F) and (G) show the same regions of two adjacent sections hybridized to receptor 32 antisense (F) and sense (G) probes. Bar in (E) = 200 urn for (A-E). Bar in (G) = 100 urn for (F) and (G).

accompanying paper, the repertoire of putative odorant receptors in the catfish is far simpler numerically than in mammals and may consist of as few as 100 members (Ngai et al., 1993). Hence, it is possible to examine the patterns of receptor expression of a significant portion of the repertoire with a relatively small number of receptor probes. Second, the catfish olfactory epithelium is organized in a simple, regularly repeating structure, and this anatomical simplicity allows for a straightforward spatial analysis of odorant receptor expression. We have used in situ hybridization to examine the patterns of expression of receptor mRNAs in the catfish olfac-

tory epithelium. Individual olfactory neurons differ with respect to the receptors they express. Moreover, each of several distinct receptors is expressed in only 0.50/o-2% of the olfactory neurons. These observations, taken together with thesmallsizeof the receptor repertoire( - lOOgenes), suggest that individual neurons are likely to express only a small number of receptor genes. Finally, we have performed in situ hybridizations and three-dimensional reconstructionsof theolfactory epithelium to determine whether neurons expressing a given receptor define a spatial pattern within the epithelium. We find no discernible pattern of receptor expression. These data suggest that neurons

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responsive to a given odorant are randomly distributed in the olfactory epithelium of fish. If spatial segregation is employed to decode olfactory sensory information, then neuronsexpressing agiven receptor are likely to converge on discrete loci within the olfactory bulb. Results Anatomy and Organization of the Catfish Olfactory Epithelium The olfactory apparatus of the channel catfish provides a facile system in which to study the pattern of expression of individual olfactory receptors. The catfish olfactory epithelium is organized into a pair of bilateral structures known as olfactory rosettes (Figure 1A; see Caprio and Raderman-Little, 1978). In a rosette, two parallel stacks of lamellae arise from a medial raphe (Figure 1 B). The sensory neuroepithelium (the olfactory epithelium) resides medially on each face of the lamella, and is surrounded by an indifferent, nonsensory epithelium (Figure 1C). Horizontal sections of an olfactory rosette show the pigmentcontaining central raphe with the numerous lamellae cut in cross section (Figure 1 D). Each lamella consists of two sheets of epithelium sandwiching a central core of connective tissue. The medially disposed sensory neuroepithelium can be identified by histological staining; in this photomicrograph, the olfactory epithelium appears more densely stained, in part owing to the higher density of nuclei relative to the surrounding indifferent epithelium. The pseudostratified olfactory epithelium comprises three major cell types: the regenerative basal cells, which lie closest to the basal lamina; the sustentacular cells, whose cell bodies reside most apically; and the olfactory neurons, whose cell bodies occupy an intermediate position in the epithelium. Each olfactory neuron extends a single dendrite to the apical surface and an axon through the basal lamina. The olfactory neuron axons emerge from the epithelium and coalesce to form the olfactory nerve that projects to the olfactory bulb. Localization of Odorant Receptor RNAs in the Olfactory Epithelium We performed in situ hybridizations to determine the patterns of odorant receptor expression in the olfactory epithelium. In the accompanying paper, we describe the cloning and characterization of six subfamilies of odorant receptors from the catfish (Ngai et al., 1993). cDNAs from 4 of the 6 subfamilies were used as probes in in situ hybridization experiments: receptor 1 represents a subfamily of four to five members, receptor 47 represents a subfamily of three to four members, receptor 32 represents a subfamily of six members, and receptor 202 corresponds to a single gene in the catfish genome. We define a distinct receptor subfamily as those genes that cross-hybridize at high stringency. Receptors 1, 32, 47, and 202 do not cross-hybridize in blotting experiments and therefore represent distinct subfamilies. Since the RNA in situ hybridization experiments are performed under conditions of slightly higher stringency, it is likely that a given probe will only detect members within its subfamily.

In situ hybridizations were carried out by annealing individual YS-labeled antisense receptor probes with multiple serial sections through the entire neuroepithelium from single olfactory rosettes. Figures 2A-2D show representative horizontal sections of olfactory rosettes following receptor probe hybridizations and autoradiography. The results with the four probes are qualitatively indistinguishable; in each case, we observe a punctate distribution of receptor RNA hybridization throughout the sensory neuroepithelium. Positive cells are only observed within the specialized zone of sensory epithelium and are never observed in the surrounding indifferent epithelium. Individual grain clusters reflect hybridization to individual neurons, and a given probe detects 0.5%-2% of the olfactory neurons (see below and Table 1). These data contrast with the hybridization patterns observed with a probe for the olfactory cyclic nucleotide-gated ion channel (Goulding et al., 1992), which demonstrates uniform labeling of the olfactory epithelium owing to the generalized expression of this channel in olfactory neurons (Figure 2E). Consecutive sections hybridized to either receptor 32 antisense (Figure 2F) or sense probes (Figure 2G) reveal no specific signal with the sense probe, whereas the adjacent section shows the characteristic punctate reactivity with the antisense probe. Analysis of multiple serial sections indicates that a given odorant receptor RNA is expressed in all lamellae and is not localized within the neuroepithelium of an individual lamella. Cells expressing individual receptors or receptor subfamilies show no segregation along anteriorposterior, dorsal-ventral, or medial-lateral axes. Are Receptors Expressed in Olfactory Stem Cells? Olfactory neurons are unique in that they are continually regenerated during the life of the organism. The in situ hybridization experiments allow us to determine whether receptors are expressed in the regenerative basal cells or whether receptor expression is restricted to mature olfactory neurons. Examination of sections of neuroepithelium at high magnification reveals that the receptor-specific grain clusters are localized over individual cells in the olfactory epithelium (Figure 3). Positive cells are found almost exclusively in the olfactory neuron cell body layer of the epithelium and have not been observed in the basal cell layer. We have also performed in situ hybridization using a nonradioactive detection method. These procedures permit the localization of RNA within individual cells and allow the identification of hybridizing cells as either olfactory neurons, basal cells, or sustenacular cells based on their morphologies and positions in the neuroepithelium. The results from an in situ hybridization using a nonradioactively labeled receptor 47 probe are shown in Figure 4. The histochemical reaction product corresponding to the hybridizing probe is found in single, isolated cells. Moreover, the morphology of these cells and their medial disposition in this pseudostratified neuroepithelium indicate that they indeed are olfactory neurons. These results suggest that olfactory receptor genes are not expressed in the mitotic stem cells, but only later in the differentiation of more mature neurons or neuron precursors. Taken together with the results shown in Figures 2 and 3, these observations demonstrate that specific receptors are ex-

Toygraphy

of Odorant

Receptors

Table 1. Ouantitation of Cells Detected with Odorant Receptor Probes by In Situ Hybridization Probe

% Positive Neurons* (mean f standard deviation)

Receptor 1 Receptor 32 Receptor 202 Receptor 47 Sumb Mixtures

0.53 1.9 1.2 1.1 4.6 4.0

+ f f f f f

0.13 0.7 0.2 0.2 0.7 0.6

a Grain clusters observed by autoradiography of in situ hybridizations were quantitated and normalized to the estimated number of total olfactory neurons (for .details see Experimental Procedures). b Sum refers to the summed percentage of neurons detected with the probes for receptor 1, receptor 32, receptor 202, and receptor 47 individually. c Mixture refers to a mixed probe consisting of receptor 1, receptor 32, receptor 202, and receptor 47 probes.

pressed in olfactory neurons dispersed throughout the neuroepithelium. Individual Neurons Express Different Complements of Receptors If the identity of a given neuron is defined by the nature of the receptor it expresses, then it is likely that the brain

Figure 3. Localization

of Odorant Receptor

Expression

in the Olfactory

discriminates odor by determining which neurons have been activated. It is therefore important to establish that individual olfactory neurons express different complements of odorant receptors. The observation that 0.5%2% of the olfactory neurons express a given receptor or receptor subfamily suggests that each cell may express only a subset of receptor genes. If we demonstrate that each of the different receptor probes anneals with distinct, nonoverlapping subpopulations of neurons, this would provide evidence that neurons differ with respect to the receptors they express. We have therefore performed the following in situ hybridizations with four receptor probes to demonstrate that each of these receptors is expressed in different olfactory neurons. Sections from a single olfactory rosette were annealed with probes specific for receptor 202 and the receptor 1,32, and 47 subfamilies. Hybridizations were carried out with the four probes individually or with a mixture of all four probes. If each receptor is expressed in a distinct, nonoverlapping subpopulation of neurons, then the sum of the cells identified with the four individual probes should equal the number of cells identified with the mixed probe. Alternatively, if the receptors are coexpressed in a significant number of cells, the sum of positive cells detected with the four individual probes would be greater than the number of cells detected with the mixed probe. Figure 5 shows a comparison of sections

Epithelium

Sections hybridized to JsS-labeled receptor probes and subjected to autoradiography were photographed using differential interference contrast optics. Sections were hybridized to receptor 1 probe (A), receptor 32 probe (B), receptor 202 probe (C), or receptor 47 probe (D). In each panel, a lamella is shown cut in cross section, so that the two layers of epithelium can be seen abutting a common connective tissue core, with the apical surfaces of each layer pointing in opposite directions. The positions of the basal cell layers, which appear somewhat more lucent than the overlying olfactory neuron cell body layers, are indicated by the closed arrows. The symmetric grain clusters appear to localize over individual cells in the olfactory neuron cell body layer. The open arrow in (C) indicates a hybridization signal over a cell that is situated just apically to the basal cell layer. Bar = 50 urn.

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Figure 4. Localization of Receptor 47 RNA to Individual Olfactory Neurons An in situ hybridization was carried out with a digoxigenin-labeled receptor 47 probe. Specific signals were detected histochemically with an alkaline phosphatase-conjugated antidigoxigeninantibodyandviewed bydifferential interference contrast microscopy. The apical dendritic knobs of olfactory neurons can be seen in the upper portion of the figure. The alkaline phosphatase reaction product is visible in the cell bodies of individual olfactory neurons. Arrow indicates the dendritic knob of the positive neuron in the lefthand panel. Bar = 10 Km.

hybridized with the individual receptor probes (Figures 5A-5D) or with the mixture of four probes (Figure 5E). The percentage of olfactory neurons detected with the mixed probe (4.0% + 0.6% [mean + standard deviation]) is significantly greater than the percentage detected with any of the individual probes alone and approximates the summed percentage of positive neurons detected with the four individual probes (4.6% & 0.7%; see Table 1). These results suggest that the four receptor subfamilies are expressed in distinct and largely nonoverlapping populations of olfactory neurons. However, given the error inherent in these experiments (standard deviation 2 15% of mean), it remains possible that a small subset of cells expresses receptors from more than one of the four subfamilies we have analyzed. Nonetheless, the present experiments clearly demonstrate that individual olfactory neurons express distinct complements of odorant receptors and that the functional identity of a neuron may be defined by the specific receptors it expresses.

Three-Dimensional Reconstruction of the Catfish Olfactory Epithelium The results from in situ hybridization experiments suggest that individual olfactory neurons express distinct subsets of receptors, and cells expressing a given receptor do not appear to be localized topologically within the olfactory rosette. However, it is possible that subtle, nonrandom patterns may have escaped our detection by mere examination of serial sections. We therefore employed computer-assisted image processing techniques to reconstruct the catfish olfactory epithelium from in situ hybridizations of serially sectioned rosettes. The difficulties in analyzing serial sections of these multilamellar structures required that we perform reconstructions on individual lamellae and generalize our conclusions to the entire rosette. Several lines of evidence suggest that the lamellae represent structurally and functionally equivalent units in the olfactory rosette. First, the structurally repeating unit of the olfactory rosette is the lamella itself, and each lamella pro-

jects independent axon tracts that together form the olfactory nerve (Hara, 1975). Second, as the catfish ages and grows in size, the surface area of the olfactory epithelium increases by the elaboration of additional lamellae in the rosette (Hara, 1975; Jakubowski and Kunysz, 1979). Finally, in our in situ hybridization experiments, we observe an even distribution of positive cells among all lamellae of the rosette, with no apparent bias in signal density from lamella to lamella (see Figure 2). We therefore reasoned that, if any pattern of receptor expression exists in the olfactory epithelium, it must occur at the level of the individual lamella or lamellar face. For the three-dimensional reconstruction of individual lamellae, serial sections were first aligned so that the borders of selected lamellae were in register and precisely overlapping one another. The outlines of lamellar sections were then traced, and the position of each specific hybridization grain cluster was scored with a dot. Dots corresponding to positive cells from each lamellar face were compiled and stored in separate files. Figures 6A-6D show a reconstruction of a single lamella from an in situ hybridization with the receptor 202-specific probe. This probe detects a unique gene in the catfish genome and therefore detects a single receptor RNA by in situ hybridization. Figure 6A shows an en face view of the reconstructed lamella with the lamellar surface shown in blue, and the individual hybridization signals from the proximal face are represented by dots. This view of the lamella is similar to the view of the olfactory rosette shown schematically in Figure lC(comparewith theright lamellaofthefrontpairin Figure 1C). A histological analysis of individual sections used in all reconstructions indicates that the limits of hybridizing cells correspond to the limits of the sensory neuroepithelium (data not shown). The array of dots thus appears to fill the sensory area of the lamellar face uniformly. Figure 6B shows the distribution of signals in the proximal face (yellow dots) in the absence of the lamellar outline; a similar view of the signals from the distal face is shown in Figure 6C (green dots). In either case, no discernible pattern of receptor expression is evident, and superimposi-

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of Odorant

Receptors

Figure 5. In Situ Hybridizations and Mixed Receptor Probes

with Individual

Sections from a single olfactory rosette were hybridized to probes for receptor 1 (A), receptor 32 (B), receptor 202 (C), or receptor 47 (D), or toa mixtureof all four probes(E). Each of these dark-field micrographs shows the same two lamellae; the refractile raphe can be seen at the leftmost edge of each panel and was included as an anatomical reference. The number of cells detected with the mixed probe is clearly greater than the number detected with any of the probes individually and approximates the sum of positive cells found with the individual probes (see text). Bar = 100 urn.

tion of these two reconstructed images reveals no point-topoint correspondence between the arrays of the two lamellar faces (Figure 6D). Reconstructed images from the lamella adjacent to the one shown in Figures 6A-6D are shown in Figures 6E-6H. A comparison of the distributions of positive cells in these two lamellae fails to reveal a common topography of receptor expression, demon-

strating that there is no predictable pattern of receptor 202 expression in the olfactory epithelium. A reconstruction of a lamella from a rosette hybridized with the receptor 1 subfamily-specific probe is shown in Figure 7. As we found for the expression of receptor 202, there is no obvious pattern of cells expressing members of the receptor 1 gene subfamily (Figures 7A and 78) and

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Figure 6. Three-Dimensronal

Reconstruction

of Two Adjacent

Lamellae from a Rosette Hybridized

to the Receptor 202 Probe

Lamellae were reconstructed from serial sections following hybridization with the receptor 202 probe and autoradiography. En face views of one lamella are shown in (A-D); similar views of the adjacent lamella are shown in (E-H). In each reconstruction, the lamella attaches to the medial raphe to the left, and the dorsal ridge is toward the top of each panel. (A and E) The reconstructed lamellae are shown in blue, with hybridization signals from the proximal face represented by dots (each corresponding to a single cell). The dots uniformly fill the medially disposed sensory neuroepithelium region of the lamella in an apparently random array (see Figure IC). (6 and F) Hybridization signals from the proximal faces are shown in isolation with yellow dots. (C and G) Hybridization signals from the distal faces are shown with green dots. (D and H) Hybridization signals from both the proximal and distal faces of the respective lamellae are displayed together, demonstrating the absence of any coincident pattern between cells expressing this receptor on opposite faces of the same lamella. A comparison of the patterns in the two lamellae (A-D versus E-H) similarly fails to reveal a reproducible pattern from lamella to lamella.

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of Odorant

Receptors

Figure 7. Three-Dimensional

Reconstruction

of a Single Lamella Following Hybridization

to the Receptor

1 Subfamily

Probe

Hybridization signals detected with the receptor 1 subfamily probe are shown in the absence of the lamellar backround; only the area immediately surrounding the sensory neuroepithelium is shown. The signals from the proximal face ([A], yellow dots) and the signals from the distal face ([B], green dots) fill the sensory neuroepithelium region and show no discernible pattern or overlap ([Cl, yellow and green dots). The left border of the sensory epithelium attaches to the raphe, whereas the upper border lies dorsally.

the array in the proximal face shows no precise or direct overlap with the array in the distal face (Figure 7C). Similar results were obtained in a reconstruction of a lamella following hybridization with the receptor 32 subfamilyspecific probe (data not shown). At the present time, we cannot discern whether all members of a receptor subfamily are expressed in the same neuron, or, alternatively, whether individual subfamily members are expressed in different cells. This ambiguity complicates our analysis of in situ hybridizations using receptor subfamily probes, since it is possible that individual subfamily members in fact are specifically localized within the epithelium, but their expression patterns may be obscured by the expression of other subfamily members by different cells in nonoverlapping patterns. However, from our datawe can conclude that the receptor 1 and receptor 32 subfamilies are not expressed in any discernible or predictable pattern in the olfactory epithelium. Taken together, our in situ hybridizations and three-dimensional reconstructions indicate that neurons expressing a given receptor are not topographically localized. Rather, individual neurons expressing a given receptor appear to be randomly distributed throughout the catfish olfactory epithelium. Discussion Higher vertebrates are able to recognize and discriminate thousands of discrete odors. Elucidation of the logic underlying olfactory sensory perception will require an understanding of the diversity of odorant receptors along with an analysis of the patterns of receptor expression within the olfactory epithelium. We previously identified an extremely large multigene family of putative odorant receptors in mammals (Buck and Axel, 1991). In the accompanying paper, we have characterized this family of receptors in the catfish and have shown that the size of this family of receptors reflects a more limited repertoire

of perceived odors (Ngai et al., 1993). These observations suggest that the recognition of odors is accomplished by a large number of different receptors, each capable of interacting with a relatively small number of odorants. Discrimination among the vast array of odors requires that the brain discern which of the numerous receptors have been activated. If individual neurons express only a subset of the odorant receptor repertoire, then the nature of a given odorant can be discerned by the identity of the cells that it activates. In the simplest form of this model, each olfactory neuron would express only one type of receptor, but more complex models can be envisaged in which a single neuron expresses multiple receptors, requiring more complex integration at the level of the olfactory bulb and cortex. The numerical simplicity of the odorant receptor repertoire in the fish allows an analysis of the diversity of receptor expression in a significant fraction of individual olfactory neurons. We have performed in situ hybridization experiments in which probes for four distinct subfamilies are annealed either singly or in combination to adjacent sections of olfactory epithelium. The number of cells detected by a mix of four probes approximates the sum of the cells detected by the four probes individually, indicating that the four receptor subfamilies are expressed in different and largely nonoverlapping subsets of neurons. In this and the accompanying paper, we demonstrate that each of the different receptor probes anneals with 0.50/o2% of the olfactory neurons (Ngai et al., 1993). These data suggest that each neuron expresses a limited subset of receptors. If we assume that the catfish genome encodes as few as 100 receptor genes, then each neuron expresses one or a few receptors. Our interpretations must be tempered by several considerations. First, our receptor probes do not distinguish among the different subfamily members. Thus, we cannot determine whether an individual cell expresses only one or several members of a given subfamily. Second, our conclusions are based upon stud-

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ies with four receptor subfamilies. It is possible that individual neurons express multiple receptors from subfamilies that we have not yet identified or tested. Nevertheless, our in situ hybridization experiments provide strong evidence that individual olfactory neurons express different complements of odorant receptors and are therefore functionally distinct. How then does the brain discern which of the functionally distinct neurons have been activated by a specific odorant? We consider two models that invoke the spatial segregation of either olfactory neurons in the epithelium or their projections in the olfactory bulb to identify those neurons activated by a given odorant (Kauer, 1991; Kauer et al., 1991; Shepherd, 1991). In one model, neurons expressing a given receptor are spatially localized within the epithelium and project to one or a subset of spatially segregated glomeruli within the olfactory bulb. In a second model, neurons expressing a given receptor may exhibit no spatial order and may be randomly distributed throughout the epithelium, whereas their axons project to one or a subset of glomeruli within spatially defined loci in the bulb. In either instance, exposure to a given odorant would result in the stimulation of a spatially restricted set of glomeruli, such that individual odorants would be associated with specific topographic patterns of activity within the olfactory bulb. Distinguishing among these models requires an examination of the pattern of receptor expression in the neurons of the olfactory epithelium and the pattern of projections these neurons extend to the olfactory bulb. Topography and Olfactory Coding in the Fish Several independent studies have demonstrated topographically defined patterns of activity in response to specific odorants in the fish olfactory bulb. Electrophysiological recordings from single olfactory bulb neurons in salmonid fish have demonstrated that individual glomerular units respond differentially to different odorant stimuli (MacLeod, 1976). In addition, the medial portion of the olfactory bulb shows enhanced activity in response to bile acid odorants, whereas the lateral portion shows enhanced activity in response to amino acid odorants (Doving et al., 1980). Similarly, in the goldfish, pheromoneinduced male courtship behaviors and sperm release are mediated by olfactory activity that projects from the olfactory bulb to the cortex via the medial olfactory tract (Stacey and Kyle, 1983; Demski and Dulka, 1984). Since the medial olfactory tract is largely composed of output fibers from the medial portions of the olfactory bulb (DuboisDauphin et al., 1980; Satou et al., 1979, 1983; Satou, 1990) the segregation of pheromone-induced activity to the medial olfactory tract probably reflects the spatial segregation of pheromone-responsive glomeruli. These studies demonstrate the spatial segregation of axonal projections in the olfactory bulb, but is this pattern reflective of the segregation of functional classes of neurons in the olfactory epithelium? Electrophysiological studies have failed to demonstrate any clear-cut segregation of odorant-induced activity in the fish olfactory epithelium (Thommesen, 1983). Axon

tracing experiments in salmonid fish show that individual glomeruli receive inputs from olfactory neurons dispersed throughout the olfactory epithelium (Riddle and Oakley, 1991). Injection of dye into individual glomeruli results in the retrograde labeling of neurons in an apparently random fashion in all parts of the epithelium. Moreover, injection of two distinguishable dyes into widely separate glomeruli results in the appearance of different dyes in adjacent olfactory neurons. These data demonstrate that neurons intermingled in the epithelium project to distinct and separate regions of the bulb (Riddle and Oakley, 1991). Thus, the fish olfactory bulb exhibits spatially organized units of activity, but this spatial order probably does not reflect the localization of functional classes of neurons in the epithelium. It should be noted, however, that in the carp, a topographic segregation of axons from specific regions of the epithelium to defined regions of the bulb is observed (Sheldon, 1912; Satou et al., 1983). The isolation of a large fraction of the genes encoding putative odorant receptors from the catfish has allowed us to determine directly whether neurons expressing a given receptor are topologically segregated within the olfactory epithelium. The numerical simplicity of the catfish olfactory receptor repertoire (Ngai et al., 1993), coupled with the anatomical simplicity of the olfactory apparatus, facilitates a direct analysis of the pattern of expression of the individual odorant receptors by RNA in situ hybridization. We have used probes representative of four distinct receptor subfamilies encompassing about 15 different genes and find no evidence for the restricted spatial localization of neurons expressing specific odorant receptors within the catfish olfactory epithelium. Rather, individual receptors or receptor subfamilies are found in cells distributed throughout the olfactory epithelium, with no segregation along anterior-posterior, dorsal-ventral, or medial-lateral axes. Furthermore, three-dimensional reconstruction techniquesfail to demonstrate more subtle patterns of receptor expression. Neurons expressing specific receptors therefore appear to be randomly distributed within the olfactory epithelium. These data, taken together with electrophysiological and axon tracing experiments, are consistent with a model in which functionally discrete classes of neurons expressing specific odorant receptors are not spatially ordered within the epithelium in the fish. Specific odors are therefore unlikely to elicit defined spatial patterns of activity within the olfactory epithelium. Dispersed olfactory neurons with common receptors are therefore likely to converge on common glomeruli in the olfactory bulb. Experiments combining in situ hybridization with specific receptor probes and axon tracing to label the projections of individual sensory neurons expressing a specific odorant receptor should now permit a more direct test of this model. Regulation of Receptor Expression in Individual Neurons The suggestion that an individual olfactory neuron expresses only one or a small number of receptors has interesting implications for the regulation of receptor gene ex-

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pression. The random distribution of neurons expressing a given receptor within the olfactory epithelium suggests that the choice of receptor expression is not governed by positional information within the epithelium, but rather is stochastic. Alternatively, it is possible that immature neurons not yet expressing receptor randomly project axons to any one of the multiple glomeruli within the olfactory bulb. Contact with individual glomeruli may then elicit a retrograde signal that directs the expression of specific receptor genes. In this manner, receptor gene expression may not be regulated by the position of the neuronal cell body within the epithelium, but rather by the ultimate position of its axon terminal within the olfactory bulb. Whatever the mechanisms governing the choice of receptor, if a given olfactory neuron expresses only one or a small number of receptors, this control mechanism must also allow for the expression of only 1 or a few of the - 100 genes within the fish odorant receptor repertoire. Although it is unlikely from our analysis of the genes encoding either the rat or fish odorant receptors that DNA rearrangement is responsible for the generation of structural diversity among the putative odorant receptors, it remains possible that DNA rearrangements may be involved in the regulation of expression of this gene family. Topography and Olfactory Coding in Higher Vertebrates Higher vertebrates have evolved more complex olfactory strategies than fish: they are thought to recognize a far greater repertoire of odorants, and odorants elicit a far greater diversity of responses. This complexity may reflect differences in the organization of the olfactory systems between fish and higher vertebrates. In both fish and mammals, specific odorants appear to elicit spatially defined patterns of glomerular activity in the olfactory bulb (Adrian, 1950; Pinching and Doving, 1974; Stewart et al., 1979; Doving et al., 1980; Jourdan et al., 1980; Teicher et al., 1980; Jourdan, 1982; Lancet et al., 1982; Royet et al., 1987; Mori et al., 1992). In contrast with fish, however, electrophysiological studies in higher vertebrates have shown that neurons in different regions of the olfactory epithelium exhibit different levels of responsiveness to specific odorants (Kauer and Moulton, 1974; Thommesen and Doving, 1977; Mackay-Sim et al., 1982). The presence of odorant-specific spatial patterns in both the olfactory epithelium and olfactory bulb suggests a spatially ordered pattern of projection of axons from the epithelium to the bulb. In support of this suggestion, axon tracing in amphibians and mammals demonstrates that defined subregions of the olfactory epithelium project to restricted areas in the olfactory bulb (Le Gros Clark, 1951; Land, 1973; Land and Shepherd, 1974; Costanzo and Mozell, 1976; DuboisDauphin et al., 1981; Greer et al., 1981; Jastreboff et al., 1984; Mackay-Sim and Nathan, 1984; Fujita et al., 1985; Mori et al., 1985; Astic and Saucier, 1986; Saucier and Astic, 1986; Astic et al., 1987; Astic and Saucier, 1988; Duncan et al., 1990; Schwarting and Crandall, 1991). However, the projection from the olfactory epithelium to the olfactory bulb lacks the point-to-point precision found in

other sensory systems. Rather, specific glomeruli receive afferent inputs from neurons dispersed in fairly broad, but nevertheless circumscribed, areas of the epithelial surface (Jastreboff et al., 1984; Asticet al., 1987). The observation that only a dispersed subset of neurons projects to a specific glomerulus suggests that neighboring neurons within the same circumscribed region project to different glomeruli. The Relationship between the Fish Nose and the Mammalian Nose How can we reconcile the apparent differences in spatial localization of receptor expression between the fish and mammalian olfactory systems? In the fish olfactory system, sensory neurons expressing a given receptor appear to be randomly distributed throughout the epithelium. In situ hybridizations reveal a dispersed and punctate pattern of receptor expression, such that the probability of a specific receptor being expressed is constant in all regions of the epithelium. In contrast, the data from higher vertebrates suggest that functionally distinct classesof neurons are segregated in broad but nonetheless discrete regions of the olfactory epithelium. Olfactory neurons from spatially defined areas of the epithelium in turn project to glomeruli within circumscribed regions of the bulb. These patterns are consistent with current in situ hybridization studies in mammals that demonstrate that the expression of specific receptors or receptor subfamilies is restricted to broad, but anatomically discrete, domains in the olfactory epithelium (Ft. J. Vassar, J. N., and R. A., unpublished data; L. Buck, personal communication). However, within a given domain, the pattern of individual receptor expression appears to be random. Thus, the properties of an individual domain within the mammalian olfactory epithelium are reminiscent of the properties of theentireolfactory epithelium of the fish. The more complex mammalian olfactory system may therefore represent a multiplicity of distinct domains, such that each domain may express only a subset of the entire receptor repertoire. In such a model, the diversity of receptor expression in a given domain would be significantly smaller than in the entire epithelium. The complexity of a domain of the mammalian epithelium may therefore approximate the complexity of the entire fish olfactory epithelium. Compartmentalization of the epithelium into anatomically and functionally discrete units of lesser complexity reduces the problems inherent in regulating the expression of specific receptors from among - 1000 genes, and may facilitate the guidance of a complex array of axonal projections from the epithelium to the bulb. Experimental Procedures In Situ Hybridizations with 3SS-Labeied Probes in situ hybridizations using YYabeled RNA probes were performed as described (Wilkinson et al.. 1967a, 1967b; Gouiding et al., 1992). ?3abeled RNA probes were synthesized with SP6, T3, or T7 phage RNA polymerases (Melton et al., 1964) from linearized DNA templates constructed in the RNA expression plasmids, pBluescript KS II(+) (Stratagene) or pcDNA-I (invitrogen). Clones encoding full-length cDNAs (with insert sizes ranging from 1.5 kb to 2 kb) were used as

Cell 676

templates to synthesize probes from receptor 1, receptor 32, and receptor 202 sequences (Ngai et al., 1993). For the detection of receptor 47 and receptor 47 subfamily sequences, we used a clone (pBB7) containing a PCR product corresponding to amino acid codons 122293 of receptor 47; pB67 shows 96% nucleotide identity with the receptor 47 sequence (Ngai et al., 1993). A probe for the olfactory-specific cyclic nucleotide-gated channel was synthesized from a partial-length cDNA (pFNCl6-3) as described (Goulding et al., 1992). Olfactory rosettes were dissected from adult catfish (Ictalurus punctatus) and fixed overnight in paraformaldehyde. Following dehydration, the rosettes were embedded in paraffin, oriented for horizontal sections, and sectioned to 10 urn thickness. To facilitate the threedimensional reconstruction of the olfactory epithelium, serial sections were collected from individual olfactory rosettes. Each series included the entire sensory neuroepithelium. Slides were hybridized to - 10 ngl ml sense or antisense RNA probes (approximate specific activity = 1.3 x IO9 dpmlug), washed at high stringency, dehydrated, and exposed toKodak NTB9emulsionfor3-4weeks(Wilkinsonetal., 1967a, 1987b). After developing, tissue sections were stained with toluidine blue 0 and mounted in Permount. This histological staining allows the discrimination of the sensory neuroepithelium (olfactory epithelium) from the surrounding indifferent, nonsensory epithelium.

In Situ Hybridizations wlth a Nonradioactively Labeled Probe A nonradioactively labeled antisense RNA probe was synthesized from linearized pBE7 (receptor 47) template plasmid with T7 RNA polymerase in the presence of digoxigenin-UTP (specific activity = 33% digoxigenin-UTP; Boehringer Mannheim Biochemicals). Tissue section preparation, hybridization, and high stringency washes were carried out according to established procedures for in situ hybridizations with “S-labeled probes (Wilkinson et al., 1987a, 1987b), except that 0.1% CHAPS was included in the hybridization buffer and the first wash buffer. Digoxigenin-labeled probe was hybridized at a final concentration of - 5 ug/ml. After the final high stringency wash, slides were washed successively in Tris-buffered saline (TBS) and TBS plus 0.2% Triton X-100, blocked in 1% heat-inactivated normal goat serum, and reacted with alkaline phosphatase-conjugated antidigoxigenin F(ab) (1 :lOOO dilution; Boehringer Mannheim Biochemicals) overnight at 4oC. The slides were then washed in TBS plus 0.2% Triton X-100, TBS, and alkaline phosphatase buffer (100 m M Tris-HCI [pH 9.5), 100 m M NaCI. 50 m M MgCI,, 2 m M levamisole). The calorimetric reaction product was developed at room temperature for 8-12 hr with 185 ugl ml 5-bromo-4~chloro-3indoyl-phosphate and 330 uglml nitro-blue tetrazolium in alkaline phosphatase buffer. Following development, slides were washed in phosphate-buffered saline, fixed briefly in formaldehyde, dehydrated successively in ethanol and xylenes, and mounted in Permount. Mlxed Probe In Sltu Hybrldlzatlons ?S-labeled antisense RNA probes for receptor 1, receptor 32, receptor 202, and receptor 47 were prepared and hybridized to slides carrying sections from the same rosette. Hybridizations were performed with each of the four probes individually or with a mixture of all four probes. Following autoradiography, the number of positive cells per section detected with a given probe or the probe mixture was determined. For each of the hybridizations with an individual probe, three sections were used for quantitation (n = 3). whereas five sections were scored for the mixed probe hybridization (n = 5). The number of positive cells was then normalized to the amount of sensory neuroepithelium per section, as estimated by measuring the contour length of epithelium hybridizing to the olfactory cyclic nucleotide-gated channel probe in per unit length of olfactory epithelium was derived by quantitating the number of cell nuclei (including those in the basal cell layer) and assuming that olfactory neurons make up roughly one-third of the cells (Cancalon, 1982). From these manipulations, it is possible to estimate the percentage of olfactory neurons that hybridize to a given probe. Three-Dimensional Reconstructlons of lndlvldual Olfactory Lamellae In situ hybridizations with Y&labeled probes were carried out on sets of serial horizontal sections from individual olfactory rosettes. The

resulting arrays of hybridization signalsover these tissue sectionswere photographed at low power using dark-field optics. Micrographs of individual sections were then aligned sequentially to one another and digitized using a high resolution CCD camera (Macagno et al., 1979; Allen and Levinthal, 1990). Because of the fragility of the sectioned tissue, which resulted in some relative displacement of groups of lamellae from section to section, alignment in each series was optimized for a selected region comprising a few lamellae. The medial raphe was used as a reference point to align the lamellae whenever it was present in a section. In general, most of the sensory neuroepithelium is found at or below the dorsal ridge of the raphe (see Figure 1C). Thus, the region for which a precise alignment is most critical contains a welldefined internal point of reference. Approximately 100 sections (individual section thickness = 10 pm) were digitized for each series. The outlines of individual lamellae were traced using the CARTOS II system in manual mode (Allen and Levinthal, 1990). This operation was repeated for all the digitized sections in a series, and the data for each lamella were stored in aseparate file. The location of each labeled neuron in a lamella was then scored with a dot in the center of the corresponding grain cluster; no bias was given for variations in signal intensity. For each series, separate files were established for the traced lamellar outlines and for the hybridization signals from each lamellar face. Thus, signals from each face could be examined individually or superimposed together. The images shown in Figures 6 and 7 were obtained by rotating the stored files to give en face views of the reconstructed lamellae. Acknowledgments We thank B. Allen and H. Wang for their assistance with the computerized three-dimensional reconstructions, L. Eoyang and T. Livelli for technical assistance, J. Sliney and staff for aquaculture, and P. J. Kisloff for assistance in preparing the manuscript. We also thank Dr. T. M. Jesse11 for suggestions and for critically reading the manuscript. This work was supported by the Howard Hughes Medical Institute and by the National Institutes of Health (ROl-20336). J. N. was also a Hughes Fellow of the Life Sciences Research Foundation for a portion of this study. M. M. D. was partially supported by a grant from the National Institutes of Health (T-32 GM07367). Received

December

10, 1992; revised January 8, 1993.

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