Optical Imaging of Odorant Representations in the Mammalian Olfactory Bulb

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Provided by Elsevier - Publisher Connector Neuron, Vol. 23, 499–511, July, 1999, Copyright 1999 by Cell Press Optical Imaging of Odorant Representations in the Mammalian Olfactory Bulb

Benjamin D. Rubin* and Lawrence C. Katz in the main olfactory bulb, while neurons expressing Howard Hughes Medical Institute different ORs converge on different glomeruli (Ressler Department of Neurobiology et al., 1994; Vassar et al., 1994; Mombaerts et al., 1996). Duke University Medical Center This organization provides an anatomical substrate for Durham, North Carolina 27710 different odors to evoke distinct spatial activity patterns in the olfactory bulb. Considerable progress has been made in understand- Summary ing the functional architecture in the olfactory systems of invertebrates, especially honeybees (Joerges et al., 1997; We adapted the technique of intrinsic signal imaging Galizia et al., 1999), and in the olfactory bulb of non- to visualize how odorant concentration and structure mammalian vertebrates, including salamanders (Kauer, are represented spatially in the rat olfactory bulb. Most 1988; Cinelli et al., 1995) and zebrafish (Friedrich and odorants activated one or more glomeruli in the im- Korsching, 1997; Friedrich and Korsching, 1998). In the aged region of the bulb; these optically imaged re- zebrafish olfactory bulb, optical imaging of calcium-sen- sponses reflected the excitation of underlying neu- sitive dyes in olfactory receptor neurons revealed that rons. Odorant-evoked patterns were similar across one class of natural odorant, amino acids, activated animals and symmetrical in the two bulbs of the same complex patterns in a portion of the bulb that were animal. The variable sensitivity of individual glomeruli distinct for each stimulus and could also encode stimu- produced distinct maps for different odorant concen- lus concentration (Friedrich and Korsching, 1997). trations. Using a series of homologous aldehydes, we In the mammalian and particularly the rodent olfactory found that glomeruli were tuned to detect particular bulb, insights into the behavior of groups of glomeruli molecular features and that maps of similar molecules have been inferred through the use of electrophysiology were highly correlated. These characterisitcs suggest (Adrian, 1950; Leveteau and MacLeod, 1966; Wilson and that odorants and their concentrations can be en- Leon, 1988; Imamura et al., 1992), 2-deoxyglucose (2-DG) coded by distinct spatial patterns of glomerular acti- autoradiography (Sharp et al., 1975, 1977; Skeen, 1977; vation. Stewart et al., 1979; Jourdan et al., 1980), and c-fos expression (Onoda, 1992; Guthrie et al., 1993; Sallaz Introduction and Jourdan, 1993) and have only recently been directly observed using functional magnetic resonance imaging Many mammalian species rely on the sense of smell for (fMRI) (Yang et al., 1998). However, these techniques behaviors that are crucial for survival and reproduction. do not have the spatial resolution to assess the re- Some olfactory behaviors, such as tracking prey, avoid- sponses of individual glomeruli to multiple odors or to ing predators, or locating a local food source require different concentrations of the same odor. Even the accurate assessment of changes in odor concentration most sophisticated maps based on 2-DG (Johnson et over space and time. Other behaviors, such as those al., 1998) can only define broadly different areas of acti- involved in determining the palatability of a food source, vation by different odorants. rely on the ability to assess odor identity (Doty, 1986). To overcome these limitations, we adapted optical Even humans, who are generally not considered mac- imaging of intrinsic signals, which has been extensively rosmates (good smellers), can accomplish impressive used to decipher the functional architecture of the mam- olfactory tasks (Vroon, 1994). For example, women can recognize their own newborn infants by smell following malian visual system (Grinvald et al., 1986; Ts’o et al., brief prior exposure (Kaitz et al., 1987). 1990; Bonhoeffer and Grinvald, 1991), to visualize the These diverse and complex perceptual tasks could patterns of activation of individual glomeruli in response rely on a spatial coding scheme in which different odors to a wide range of odorants. Intrinsic signal imaging (or different odor concentrations) evoke distinct spatial relies on activity-dependent changes in optical proper- patterns of activity in the olfactory bulb that could be ties of the tissue such as hemoglobin absorption and/or interpreted as representing different smells (Adrian, light scattering, and patterns of activity can be visualized 1950; Shepherd, 1985; Mori and Yoshihara, 1995; Buck, through the intact dura or thinned bone (Frostig et al., 1996). Recent studies indicate that olfactory receptor 1990). We first demonstrate that odorants are repre- mem- sented by distributed patterns of activated glomeruli-1000ف neurons express only a single type of the ber odorant receptor (OR) family (Buck and Axel, 1991; that are bilaterally symmetric in the same animal and Ressler et al., 1993; Vassar et al., 1993; Chess et al., similar between animals. We then show that distinct 1994; Malnic et al., 1999). This expression pattern con- patterns of glomerular activity correlate with differences fers extensive functional variety to olfactory receptor in odorant concentration and odorant identity. These neurons (Malnic et al., 1999). All neurons that express results directly demonstrate that mammalian glomeruli one type of OR project to a small number of glomeruli are tuned to detect particular molecular features, and they provide evidence that complex spatial patterns of * To whom correspondence should be addressed (e-mail: brubin@ activity are sufficient to distinguish even very similar neuro.duke.edu). odorants. Neuron 500

Figure 1. Optical Imaging of Intrinsic Signals in Response to Odorants Yields High-Resolu- tion Maps of Glomerular Activation in the Rat Olfactory Bulb The left panels (A–C) show the surface blood vessels overlying the imaged regions. (A) A strong olfactory stimulus, amyl acetate (100%), evoked widely distributed, well-isolated peaks of activation with the size and shape (100–200 ␮m in diameter) of individual glomeruli, as well as broader regions of activation. (B) A food stimulus, peanut butter, which is a mixture of numerous odorants, activated two well-defined glomeruli in the imaged region. (C and D) Some odorants, such as (ϩ)-carvone and butyric acid, evoked just a single well-defined activity peak in the imaged region. (E) An enlargement of the 500 ␮m box surrounding the activity peak elicited by butyric acid in (D) shows the extremely discrete nature of the activated region, which is only about 100 ␮m in diameter. (F) A profile plot of this peak was constructed by plotting the average pixel values along four 500 ␮m lines (horizontal, vertical, and the in- termediate diagonals) centered on the activity peak. Dashed lines indicate background and 25%, 50%, and 75% background-to- peak activity levels. Robust signal emerges from the noise in Ͻ30 ␮m. In (A) through (E), maps are oriented with anterior to the right, medial down. Scale bars, 250 ␮m.

Results isolated peaks, as well as larger, less well-defined regions (Figure 1). These peaks typically represented Optical Imaging of Odorant Responses in the Rat changes in reflected light intensity of 0.2%–0.4%. The Olfactory Bulb individual peaks, when measured at 50% of maximal In the initial set of experiments, we sought to determine activity from background, appeared as roughly circular ␮m in diameter (Figures 1D–1F). Clear 200–100ف whether optical imaging of intrinsic signals could detect regions responses to odorants in the rodent olfactory bulb. We patterns of glomerular-like activation were often seen exposed a restricted region of the dorsal surface of the after the first set of 10 s odor presentations. Some odorants, such as 100% amyl acetate, consistently activated %10ف ϫ 3 mm), which contains 1.5ف) rat olfactory bulb of the 2400 or so glomeruli (based on an average glomer- a significant proportion of the imaged bulb (Figures 1A ular diameter of 150 ␮m; Meisami and Sendera, 1993). and 5A). In other cases, the odorant produced only a To obtain images of odorant activation patterns, anes- single activity peak (Figures 1C and 1D). A few stimuli thetized animals were given multiple 10 s presentations in our set (such as apple juice) failed to activate any of of an odorant, randomly interleaved with presentation the imaged region. These patterns were very consistent: of an odorless stimulus (mineral oil). Intrinsic signals although the overall quality of the images degraded were recorded from the olfactory bulb under red (630 somewhat over time, all activity peaks remained when nm) illumination with a video camera. Differential activity an odorant was imaged at multiple time points up to 8 maps were constructed by subtracting the averaged hr apart (data not shown). In all cases, signals emerged mineral oil response from the averaged odorant re- within 3 s of odor presentation and persisted throughout sponse. the 10 s stimulus presentation. We did not observe dif- The responses of 36 olfactory bulbs (29 rats) to a ferences in the time course of signals in different regions variety of odorants were recorded, resulting in 282 odor- of the bulb. ant maps. Odorants used included single compounds Compared to optical images obtained from mamma- such as isoamyl acetate and saturated aliphatic alde- lian primary sensory cortex, the resolution of these hydes with varying hydrocarbon chain length, as well images was remarkable. The activated regions were as complex odor mixtures such as peanut butter. All extremely circumscribed, and features Ͻ100 ␮m in di- odorants were presented either as pure compounds or ameter were readily detected. (Figures 1D–1F). The sig- were diluted in mineral oil. nal rose abruptly from background noise in Ͻ30 ␮mto Presentation of many odorants elicited robust optical easily detectable levels (Figure 1F). This extraordinary patterns of activity, which consisted of one or more resolution is probably due to the unique anatomy of the Optical Imaging of the Mammalian Olfactory Bulb 501

Figure 2. The Specificity of Optically Imaged Responses Reflects the Tuning of Individual Neurons (A–D) The upper panels show the surface blood vessels (A) and responses to amyl acetate (B), 100% (ϩ)-carvone (C), and 10% propionic 4mm2. In the region indicated by the arrowhead, optically recorded activity was observed following amylف acid (D) imaged in an area of acetate stimulation (B); no activation above background at this locale is evident in response to (ϩ)-carvone (C) or propionic acid (D). A tungsten microelectrode was then used to record isolated single units in a glomerular region activated exclusively by amyl acetate (arrowhead). The lower four panels show a single raster trace of the firing pattern in response to a 15 s odorant presentation at the time indicated by the vertical dotted line; individual extracellular action potentials are indicated by the vertical ticks. The animal’s breathing rhythm, shown immediately below the raster traces, was recorded by monitoring expired CO2 concentration; the peaks correspond to exhalation and the troughs to inhalation. Poststimulus time histograms at the bottom of each panel show summed responses to three odorant presentations. This neuron (a mitral cell, based on recording depth of 488 ␮m) showed no response to mineral oil (A), (ϩ)-carvone (B), or propionic acid (D). However, consistent with the imaging findings, the neuron responded briskly to amyl acetate. Note that the response, as seen in the raster trace, was clearly synchronized to the inspiratory phase of the breathing rhythm. (E–F) Another recording in a different animal was targeted to a region of the bulb (arrowhead) that was activated by peanut butter but not by amyl acetate or (ϩ)-carvone. This neuron (a periglomerular or external tufted cell, based on a recording depth of 103 ␮m), responded selectively, as predicted, exclusively to peanut butter. Scale bars, 250 ␮m. bulb: the bulb receives inputs directly from a primary individual activity peaks. At 50% of peak activation sensory epithelium, several thousand similar afferent above background, activated regions averaged 13,190 fibers converge onto one glomerulus, and glomeruli are ␮m2, which corresponded to an average diameter of 128 located superficially. ␮m(nϭ 97 spots, 16 animals). Glomeruli in the adult ␮m (Meisami 150ف rat olfactory bulb attain a diameter of Intrinsic Signal Images Represent Patterns and Sendera, 1993). Taking into account the different of Activated Glomeruli levels of activity that would be expected at varying dis- Since the intrinsic signal is derived primarily from the tances from the glomerular center (e.g., maximal activity upper 500 ␮m of tissue (Bonhoeffer and Grinvald, 1996) is at the center, where the glomerulus is the thickest), and since this depth corresponds to the glomerular layer and assuming equal distribution of signal from an ideal- of the olfactory bulb, we expected intrinsic signals to ized spherical glomerular volume (see Experimental Pro- primarily reflect patterns of active glomeruli. Indeed, cedures), we would expect activity from a 150 ␮m glo- based on their size, shape, and odor specificity alone, merulus to measure 130 ␮m in diameter at 50% of peak it is difficult to imagine what other structure they could activity. Thus, the measured size of activity peaks in represent. Anatomical and physiological experiments imaged odor maps corresponds very closely to the ex- support this contention. We first determined the size of pected size of signals originating from single glomeruli. Neuron 502

To confirm that optically mapped active regions reflected the activation and specificity of underlying neurons, targeted extracellular single-unit recordings were made in mapped regions of the bulb. Odor-responsive neurons (both mitral/tufted [M/T] cells and a periglomerular/external tufted cell, as determined by recording depth) were excited by the odorant predicted on the basis of the optical signals, and not to any of the odorants that did not activate the targeted region (n ϭ 4 neurons in four rats). In one animal, for example, stimulation with 100% amyl acetate activated a region in the medial region of one bulb, while two other stimuli, 10% propionic acid and 100% (ϩ)-carvone, strongly activated nonoverlapping glomeruli more laterally. A targeted recording yielded an M/T cell that clearly responded selectively to amyl acetate (Figures 2A–2D). In a second experiment, a recording targeted for a region of the bulb that responded selectively to peanut butter yielded a periglomerular or external tufted cell that was also selective for peanut butter (Figures 2E–2H) and showed no response to amyl acetate or other tested odorants. In about 50% of the targeted recordings, however, M/T cells were recorded that could not be reliably activated by any of the stimuli used. These cells may be inhibited by the anesthetic state, may be selective for odorants that were not tested, or may have weak responses below the noise level. The size and shape of the activated regions, along with electrophysiological data, strongly argue that the Figure 3. Odor Representations Are Bilaterally Symmetrical in Indi- spots of activity present in the odor maps represent vidual Animals and Stereotyped between Animals individual glomeruli. The effects of changing odorant (A) Examples of bilaterally symmetrical odor responses recorded structure and concentration on the activity maps that from olfactory bulbs within the same animal. On the left, the re- are discussed in the following sections further support sponses to heptanal (1%) were recorded simultaneously, activating the idea that the patterns of activity represent patterns two distinct, bilaterally symmetrical glomeruli in each bulb (arrows). Not all responses were symmetrical: an additional glomerulus in the of active glomeruli. left bulb (top) and diffuse lateral activity in the right bulb were not seen in the contralateral bulb. On the right, the responses to propi- Stereotypy and Heterogeneity of Maps onic acid (10%) were recorded sequentially from both bulbs of the within and between Animals same animal. Large, roughly symmetrical regions of activity were Studies in rats and mice have demonstrated that olfac- visualized in the anteromedial region of these olfactory bulbs. Ante- rior is to the right. Scale bars, 500 ␮m (left) and 250 ␮m (right). tory neurons expressing the same OR converge onto (B) Optically imaged maps of the response to propionic acid in four glomeruli that are bilaterally symmetrical between the olfactory bulbs from four different animals. In all maps (including the bulbs of the same animal and are stereotyped between two responses in [A]), propionic acid activated a sharply delineated animals (Ressler et al., 1994; Vassar et al., 1994; Mom- region in the anteromedial part of the bulb. In addition, a single baerts et al., 1996). These expression patterns are likely strongly activated glomerulus (arrowhead) was observed in roughly to provide the basis for results from previous 2-DG and half the animals in the center of the exposed region. Scale bars, 250 ␮m. c-fos studies demonstrating that patterns of glomerular activation are both bilaterally symmetrical within animals and similar between animals (Sharp et al., 1975, 1977; To determine whether functional maps of odorant re- Skeen, 1977; Guthrie et al., 1993). sponsiveness showed stereotypy between animals, we To determine whether intrinsic signal patterns dis- compared the response maps of several individuals to played the same organization, both olfactory bulbs of a variety of odorants. Some strong odorants, such as seven rats were imaged in response to the same set of 100% amyl acetate, consistently activate large numbers odorants (bulbs imaged simultaneously, n ϭ 3 animals; of glomeruli over extensive regions, whose patterns var- bulbs imaged sequentially, n ϭ 4 animals). Odorants ied considerably between animals (e.g., compare maps evoked clearly symmetrical responses in the bulb pairs. in Figures 1A and 5A). However, the maps resulting from In some instances, it was possible to identify symmetri- stimulation with other odorants (e.g., propionic acid) cally located, activated glomeruli (Figure 3A, left). In showed considerable similarity. In all animals (15/15), other cases, broad areas of activation showed clear 1%–100% propionic acid activated a large, sharply de- symmetry, but distinctly activated glomeruli in one bulb lineated region in the anteromedial portion of the bulb could not be paired with glomeruli in the other bulb (Figure 3). In roughly half the animals (7/15), a single (Figure 3A, right). Thus, odor representations between isolated glomerulus in the center of the imaged region the olfactory bulbs of the same animal show some de- was also robustly activated (Figure 3B). Similarities were gree of bilateral symmetry. evident in the responses to other odorants as well (e.g., Optical Imaging of the Mammalian Olfactory Bulb 503

Figure 4. Individual Glomeruli Can Be Activated by Molecularly Distinct Odorants (A) Octanal (1%) (top) and amyl acetate (100%) (bottom) each activated distinct glomeruli in this imaged region. One large glomerulus (or possibly a group of adjacent or overlapping glomeruli, white arrow) was strongly activated by both of these structurally dissimilar stimuli; two other glomeruli (white arrowheads) were weakly activated by octanal but strongly activated by amyl acetate. Other glomeruli (black arrowheads) were activated by amyl acetate but not by octanal. Thus, although these two maps are distinct, they contain several glomeruli activated by both odorants. (B) In another animal, two stimuli that share the same-length hydrocarbon chain, propionic acid (10%) (top) and propanal (1%) (bottom), activate one glomerulus in common (white arrowhead) but another is activated only by propanal (black arrowhead). (C) Enantiomers of carvone activate identical regions in the imaged portion of another bulb. Both (ϩ)-carvone (100%) (top) and (Ϫ)-carvone (100%) (bottom) activated a single common glomerulus (white arrowhead). Scale bar, 250 ␮m. responses to a series of aliphatic aldehydes in two ani- This was indeed the case. Higher odorant concentra- mals are shown in Figures 6 and 7). These data indicate tions always activated a similar or larger area than the that representations for at least some odorants are ste- same odorant at 101-to105-fold lower concentrations. reotyped between individual animals. For all odorants tested, we never observed a new glomerulus being activated as concentration decreased Odorants Evoke Highly Distributed Patterns (n ϭ 29 odor maps, 7 rats; Figure 5). At the highest con- of Activity centrations, broader regions of lower activation, in addi- Odorants that give rise to different perceived smells tion to distinct glomeruli, were evident over extensive would be expected to evoke different spatial patterns regions of the bulb (Figure 5A). This may represent of glomerular activation. Even a cursory inspection of lower-level activation of multiple adjacent or overlap- the intrinsic signal response patterns confirmed that ping glomeruli or the recruitment of more diffuse popu- different odorants elicited markedly different patterns lations of neurons located deeper in the bulb, such as of activation in the same olfactory bulb. However, even M/T or granule cells. molecularly dissimilar odorants frequently activated Individual glomeruli that were responsive to an odor- common glomeruli. For example, while amyl acetate ant within the tested concentration range exhibited (100%) and octanal (10%) activated many unique re- marked differences in sensitivity (Figure 5). While some gions, a number of glomeruli were activated by both glomeruli responded only at the very highest concentra- (Figure 4A). Patterns activated by odorants sharing a tions of odorant (e.g., glomerulus 3 in Figure 5B re- distinct molecular feature, such as carbon chain length, sponded to 10%–100% amyl acetate), others were still activated, albeit weakly, at the lowest concentrations but differing in substituted group, also activated com- tested (e.g., glomerulus 1 in Figure 5B responded to mon regions (Figure 4B). Finally, a pair of stereoisomers, 0.001% amyl acetate). Though individual glomeruli dif- (ϩ)-carvone and (Ϫ)-carvone, that have similar but dis- fered in their detection thresholds, their response tinguishable odors (described as caraway and spear- curves, when plotted on a log scale, were roughly “S” mint, respectively), activated a single common glomeru- shaped. Since the signal saturated, we could not predict lus in the imaged region (Figure 4C). These data from responses at high odorant concentrations which demonstrate that odor representations in the olfactory glomeruli would remain activated as the concentration bulb are highly distributed: odorants can activate multi- decreased, although more quantitative examination of ple glomeruli, and individual glomeruli can be activated this issue is warranted. Thus, different concentrations by multiple odorants. of the same odorant, as well as the same concentrations of different odorants, elicit distinct patterns of glomeru- Increases in Odorant Concentration Recruit lar activation. Additional Glomeruli Odorant concentration plays a critical role in determin- Glomerular Receptive Ranges ing both the absolute detection threshold of an odorant If odorant identity is reflected by spatial activity patterns and its quality (e.g., many substances that smell pleas- in the olfactory bulb, even structurally similar odorants ant cause a stench at higher concentrations; Vroon, that smell differently should evoke distinct patterns of 1994). If different spatial patterns of activated glomeruli activated glomeruli. A strong test of this idea is to vary are indeed linked to distinct perceptions, we would pre- a specific chemical species along a single molecular dict that different concentrations of odorants would dimension, and determine whether the patterns of acti- yield distinct spatial maps. vated glomeruli are distinct. A corollary of this problem Neuron 504

Figure 5. Increasing Odorant Concentration Activates Increasing Numbers of Glomeruli, Resulting in Odorant Response Maps that Distinguish Odorant Concentrations (A) Effects of increasing the concentration of amyl acetate from 0.001% (top) to 100%. As odorant concentration increases, individual glomeruli are more strongly activated. At 0.001%, only a single glomerulus in the imaged region is activated above background (see [B]). From 0.01% and 5%, the pattern of activated glomeruli is very similar, although the intensity of activation of individual glomeruli increases. At the highest concentrations (10% and 100%), many new additional regions are recruited, and the maps are sub- stantially different from those at lower concentrations. However, all of the activated glomeruli seen at lower odorant concentrations are evident at the highest concentrations as well. The area outlined by the box in the blood vessel map at the bottom is enlarged in (B). Scale bar, 250 ␮m. (B) The responses of three glomeruli (from the boxed region in [A]) to increasing concentrations of amyl acetate. At the very lowest concentration (0.001%), activation above background can be detected only in glomerulus 1. Between 0.01% and 5%, glomeruli 1 and 2 are increasingly activated; at concentrations of 10% and 100% amyl acetate, a new glomerulus (3) abruptly becomes strongly activated. Scale bar, 250 ␮m. (C) By calculating the relative degree of activation of each glomerulus at different odorant concentrations, a dose–response curve for each glomerulus was constructed (odorant concentration is on a log scale). While all glomeruli are strongly activated by the highest odorant concentrations, the response of glomerulus 3 drops abruptly to background at Ͻ10% amyl acetate. Glomerulus 1 remains active throughout the tested range.

is determining the “molecular receptive range” of indi- (C4) elicited the most activity in the imaged region of vidual glomeruli to a homologous chemical series (Mori the bulb, while the largest molecules, nonanal (C9) and and Yoshihara, 1995). decanal (C10), stimulated very little of the exposed re- To investigate glomerular tuning, we used a series of gion of the bulb. The shorter-chain aldehydes (C3–C5) eight saturated aliphatic aldehydes that varied in hydro- invariably activated both distinct individual glomeruli carbon chain length from 3 to 10 carbons (Figure 6A). and broader areas consisting of numerous overlapping Consecutive members of this panel have been shown glomeruli in the anteromedial bulb. As carbon chain to activate the rat I7 OR (Zhao et al., 1998) and M/T cells length increased, activity shifted away from the anterior in the dorsomedial rabbit olfactory bulb (Imamura et al., and medial portions of the imaged area. 1992). Complete maps of the entire series were obtained Forty glomeruli that were activated by at least one from five bulbs in four animals (two examples from differ- member of the aldehyde series were analyzed (Figure ent animals are shown in Figures 6 and 7), at concentra- 6). We measured the response of each glomerulus to the tions of 1% (n ϭ 4) and 10% (n ϭ 1). In all cases, butanal eight aldehydes and expressed the level of glomerular Optical Imaging of the Mammalian Olfactory Bulb 505

Figure 6. Individual Glomeruli Have Restricted Receptive Ranges to Odorants Varying along a Single Molecular Dimension (A) Optical maps of the responses to a homologous series of saturated aliphatic aldehydes (names, carbon number, and structure are shown to the left of each map). The blood vessel map is shown at the top, with the boxed region indicating the area from which individual glomeruli were measured to construct molecular receptive ranges. (B) Enlarged view of odorant responses located within the boxed region shown in (A). Scale bar, 250 ␮m. (C) For each of four glomeruli in the region shown in (B) (indicated by the closed circle in the inset at right), the magnitude of the optically imaged response is plotted as a percentage of the maximal response to a given odorant. For example, the glomerulus in the top histogram responded best to butanal (C4); the responses to propanal (C3) and pentanal (C5) were roughly 80% and 60%, respectively, of this maximal response. Odorants eliciting Ͼ50% of the maximal response were considered to be effective in activating a glomerulus. These four glomeruli were activated by between one and three members of the homologous series. (D) Summary of the responses of 40 glomeruli (n ϭ 5 bulbs in four animals) to the homologous series. Molecular receptive ranges (constructed using the criteria described in [C]) are indicated by the shaded bars. With a single exception, all glomeruli responded to consecutive members of the series, and the vast majority (35/40) responded to a restricted range of 1–3 carbons.

activity in response to each odorant as a percentage of receptive ranges was insensitive to the cutoff used for the maximal response (to the most efficacious odorant). judging whether a glomerulus was activated. For exam- The molecular receptive range for a glomerulus was ple, using criteria of 33%, 50%, and 67% of maximal defined as the set of odorants that evoked half-maximal glomerular activation, the percentage of glomeruli that or greater activity (analysis of four glomeruli is shown responded to multiple aldehydes and had consecutive in Figure 6C). Of the glomeruli analyzed, 41% (16) were molecular receptive ranges was 82% (28/34), 96% (23/ activated by only one member of the aldehyde series 24), and 95% (19/20), respectively. (Figure 6D). The majority of the remaining 24 glomeruli These results are comparable to reported molecular had molecular receptive ranges of two or three odorants receptive ranges of M/T cells from the dorsomedial rab- (9 glomeruli each). In all but one case (23/24), glomeruli bit olfactory bulb (Imamura et al., 1992). Of the M/T cells that responded to more than one odorant were activated whose responses were recorded to the same homolo- by consecutive members of the aldehyde series. The gous aldehyde series at comparable concentrations, finding that most glomeruli have consecutive molecular 35% (18/51) were excited by only one member of the Neuron 506

Relationship Between Odorant Structure and Map Similarity Even from casual inspection, it was evident that the maps representing adjacent members of the aldehyde series were more similar to one another than maps of odorants that differed by Ͼ2 carbons (compare, e.g., the maps for C4 and C5 aldehydes to the maps for C7 and C8 in Figure 7A). To quantitate this initial impression, we assessed the degree of similarity between maps for different aliphatic aldehydes in the same animal (see Experimental Procedures). First, for each glomerulus, a similarity matrix was formed by calculating the difference in glomerular activation (expressed again as a percentage of maximal activation) for each pair of odorants. We then calculated a composite similarity matrix by averaging the matrices for each glomerulus within a bulb. These composite matrices express the similarity between every pair of maps based on all the glomeruli activated by one or more odorants. The analysis confirmed our initial impression of the data: in the majority of cases, odorant representations were most similar to the activity maps of odorants that differed by a single carbon. In fact, far more odorant maps were most similar to maps of adjacent aldehydes than would be expected by chance (p Ͻ 0.001, ␹2 test). Conversely, no odorant maps were most different from maps of adjacent aldehydes. In all cases, the maps of shorter-chain aldehydes (Յ5 carbons) least resembled maps of aldehydes with Ն7 or more carbons, while the longer-chain aldehyde maps (Ն6 carbons) least resembled maps of aldehydes with 3 or 4 carbons. Together, these data indicate that odorant structure similarity is correlated to odorant map similarity, and that even a slight difference in structure activates a different pattern of glomeruli.

Discussion Figure 7. The Maps of Structurally Similar Odorants Are Strongly Correlated (A) Maps of responses to a homologous series of 8 saturated ali- Using intrinsic signals to visualize responses to different phatic aldehydes, from propanal (C3, top) to decanal (C10) in a odorants, we have produced maps of functionally acti- single animal. Scale bar, 500 ␮m. vated glomeruli in a mammalian system. In many cases, (B) Correlation matrices from four animals (the bottom matrix is large regions of the olfactory bulb were active, which derived from the maps shown in Figure 6A). The closed circles we interpret as groups of responsive glomeruli. These represent the best correlation between two maps; the shaded cir- signals may originate from glomeruli that are overlap- cles, the worst correlation. For example, the top matrix shows that the map of hexanal (C6) was best correlated with the map of pentanal ping or out of the focal plane due to the curvature of the (C5) and worst correlated with the map of propanal (C3). In general, bulb. Most odorants also activated isolated glomeruli the best correlations were between maps within 1 carbon of one whose selectivity we analyzed. It is possible that in some another. The shortest-chain molecules (ϽC5) were worst correlated cases, two overlapping glomeruli may have been inter- with the longest-chain molecules (ϾC8), while the longer-chain mol- preted as a single glomerulus. However, based on the ecules (C6–C10) were worst correlated with maps of the shortest- chain molecules (ϽC4). Responses of individual glomeruli to the size, shape, and selectivity of activated areas, we be- series were used to determine the similarity between odor represen- lieve these intrinsic signals primarily originate from indi- tations (for details, see Experimental Procedures). vidual glomeruli. Extracellular single-unit recordings confirm that these signals reflect the excitation and se- series. All 33 of the M/T cells that responded to multiple lectivity of underlying neurons. aldehydes were activated by consecutive members of The activated regions originating from single glomeruli the series. In both our experiment and the physiology are among the smallest features imaged to date using study (Imamura et al., 1992), butanal (C4), pentanal (C5), intrinsic signals. At half-height, the intrinsic signals often heptanal (C7), and octanal (C8) activated the largest measure Ͻ100 ␮m in diameter, comparable to the size number of glomeruli and M/T cells, while hexanal (C6) of individual glomeruli. This implies that individual glo- and the longest-chain aldehydes (ՆC9) activated the meruli can be activated without activity spreading to fewest. neighbors, perhaps in part due to inhibitory interactions Optical Imaging of the Mammalian Olfactory Bulb 507

between glomeruli. In contrast, intrinsic signals are typi- odorant molecule with different affinities. Glomeruli were cally much broader in visual, somatosensory, and audi- activated by a restricted range of odorants that varied tory cortex, even when weak stimuli are used. For exam- along a particular molecular dimension (carbon chain ple, deflection of a single whisker in rats results in the length). Selectivity for this feature, also reflected in the activation of a region of barrel cortex that at half height responses of receptor neurons and M/T cells (Imamura is Ͼ10 times the size of the corresponding anatomically et al., 1992; Katoh et al., 1993; Sato et al., 1994; Mori defined cortical barrel (Masino and Frostig, 1996). Unlike and Yoshihara, 1995), is likely due to the ability of ORs cortex, the olfactory bulb directly receives afferents from to bind multiple ligands with similar structures. Multiple primary sensory neurons. Tens of thousands of olfactory glomeruli were activated by the same odorant, just as receptor neurons converge upon single glomeruli, which neurons expressing different ORs can respond to the are solely composed of neuropil and are wrapped in same odorant. Finally, patterns of active glomeruli were glial sheaths. Based on anatomical, physiological, and roughly bilaterally symmetrical within animals and ste- metabolic mapping studies, glomeruli are widely consid- reotyped between animals, as has been observed in the ered to be functional units of olfactory processing (Scott projection patterns of receptor neurons expressing the et al., 1993). Furthermore, glomeruli and their inputs are same OR. located superficially, which reduces blurring due to light Though responses clearly showed stereotypy and bi- scatter and also reduces signal attenuation from overly- lateral symmetry, it was equally clear that the patterns ing tissue. The combination of these unique features of varied to a considerable degree. This is not surprising; glomeruli may account for the remarkable high spatial variability in olfactory bulb response patterns within and resolution we observe in the olfactory bulb maps. between animals has been reported in studies using The ability to noninvasively visualize the functional 2-DG (Sharp et al., 1975, 1977; Skeen, 1977; Stewart et architecture of the bulb in living animals paves the way al., 1979; Jourdan et al., 1980; Johnson et al., 1998) and for numerous experimental approaches that have been fMRI (Yang et al., 1998). In particular, many of these used with great success in the visual system, such as studies (e.g., Stewart et al., 1979) show that overall levels repeated imaging during development or following be- of activity can differ between the bulbs in an individual havioral training, targeted injections of tracers to deci- animal, as appears to be the case in some of our re- pher intrinsic circuitry, and combining molecular mark- cordings (e.g., Figure 3A). The absence of strict stereo- ers with functional maps. Given the great similarity typy and symmetry in the activity patterns could be due between rat and mouse olfactory bulbs, it is highly likely to a number of factors, including inherited variability that images of similar resolution can be obtained in both (e.g., OR expression levels, OR expression patterns, or normal and genetically modified mice, which will assist receptor neuron projections), experience-dependent in correlating genotypic and functional alterations. plasticity (e.g., Coopersmith and Leon, 1984; Leon, 1992), or experimental variability (e.g., differences in an- Functional Anatomy Underlying Spatial esthetic state, blockage of nasal canals, background Representations of Odors odors, or the placement of the imaging window). Within the last decade, the cloning and analysis of ro- The optically imaged patterns of activity in response dent ORs has greatly increased understanding of how to changes in odorant concentration and structure that the mammalian olfactory system is organized (Buck and we visualized were consistent with odor representations Axel, 1991; reviewed by Mombaerts, 1999). These find- visualized in rats using other techniques. Increases in ings provide a framework for understanding how the odorant concentration result in larger regions of high responses of receptor neurons could be translated into 2-DG uptake (Stewart et al., 1979) and c-fos expression glomerular activity. Each olfactory receptor neuron is (Guthrie and Gall, 1995). Consensus regions of the bulb believed to express only a single type of OR (Ressler et are activated by odorants that share particular chemical al., 1993; Vassar et al., 1993; Chess et al., 1994; Malnic features (Johnson et al., 1998). However, since 2-DG is et al., 1999). ORs bind molecules with similar structures limited in that the responses to only a single odorant can (Zhao et al., 1998), endowing olfactory receptor neurons be mapped in each animal, the responses of individual with the ability to respond to odorants with shared mo- glomeruli to varying odorant concentration and struc- lecular features (Sato et al., 1994; Malnic, et al., 1999). ture that we have described have been inferred but not Thus, a receptor neuron expressing one OR can be acti- directly observed from these studies. vated by many different odorants. Conversely, neurons Odor representations have also been studied with expressing different ORs can be activated by the same voltage- and calcium-sensitive dyes in nonmammalian odorant (Malnic et al., 1999). Receptor neurons that ex- vertebrates (salamander, Cinelli et al., 1995; zebrafish, press a particular OR converge upon only a few of the Friedrich and Korsching, 1997, 1998) and invertebrates .(glomeruli in the rodent bulb (Ressler et al., 1994; (honeybee, Joerges et al., 1997; Galizia et al., 1999 2000ف Vassar et al., 1994; Mombaerts et al., 1996). These pro- Along with our results, these studies demonstrate that jections are bilaterally symmetrical within an animal and odor representations in distantly related species share stereotyped between animals. a number of characteristics, including distributed glo- The odor responses that were visualized are consis- merular responses, bilateral symmetry, and stereotypy. tent with these properties of ORs and receptor neurons. These general features indicate that olfactory systems Glomeruli responded to different concentrations of the across phyla share common organization principles (Hil- same odorant, with different glomeruli displaying widely debrand and Shepherd, 1997), whether they are the varying detection thresholds (Figure 5). This most likely product of convergent evolution or have been preserved reflects the ability of multiple ORs to bind the same from common ancestors. Neuron 508

The particular locations of active neurons and glomer- processing perspective. The convergence of thousands uli evoked by specific odorants suggest a preserved of functionally homogeneous receptor axons onto single functional organization within and between species. For glomeruli could increase the ability of the olfactory sys- example, responses to propionic acid have been studied tem to discriminate signal from noise (Laurent, 1997). using 2-DG (Bell et al., 1987; Slotnick et al., 1989) and Moreover, if the spatial arrangement of glomeruli is c-fos (Onoda, 1992; Sallaz and Jourdan, 1993; Guthrie chemotopic (as is the case in a portion of the zebrafish and Gall, 1995) in different strains of rats. In all cases, olfactory bulb; Friedrich and Korsching, 1997), the tun- including our study, the largest responses to propionic ing of M/T cells could be sharpened via lateral inhibitory acid are localized to the dorsomedial bulb. Aliphatic interactions between neighboring cells with overlapping aldehydes, which we found activated the dorsomedial molecular receptive ranges (Yokoi et al., 1995) while olfactory bulb in rats, also excite M/T cells from this minimizing arbor length. The combinatorial nature of the region of the rabbit olfactory bulb (Imamura et al., 1992). glomerular code could allow many more odorants to be These results imply a conservation in the evolution of discriminated than would be possible using a limited the olfactory system, at least between rodents and lago- (though large) number of receptors alone (Buck, 1996). morphs. It is also possible that the precise projections of receptor neurons to the olfactory bulb is important for the devel- Coding in the Olfactory System opment and maintenance of the olfactory system, by Initially, olfactory information is encoded by the identity allowing a constantly regenerating population of recep- of receptor neurons that express ORs with an affinity tor neurons to “plug in” to the correct neural circuit. for the odorant. This code is combinatorial: a receptor The location or identity of active neurons or glomeruli neuron can respond to many odorants (Gesteland, 1965; is not the only information available to make determina- Sicard and Holley, 1984; Sato et al., 1994), and an odor- tions about the nature of a stimulus. The importance of ant can activate receptor neurons that express different spike timing has been demonstrated in a series of stud- ORs (Malnic et al., 1999). The combinatorial receptor ies focusing on local field potential oscillations, which code is translated into a combinatorial glomerular code are driven by synchronized spiking projection neurons as functionally similar receptor neurons converge onto (analogous to M/T cells in vertebrates), in the locust a small number of glomeruli in the olfactory bulb. We and honeybee antennal lobes. While the phase of spike observed that changing the concentration of an odorant timing relative to the population does not convey infor- by as little as 2-fold alters the pattern of activated glo- mation about the odorant, the timing of spikes in these meruli (Figure 5). Even structurally similar odorants, dif- neurons relative to particular cycles of the local field fering by the addition of a single carbon atom, can be potential is odorant specific (Laurent and Davidowitz, discriminated on the basis of this code (Figure 7). Simi- 1994; Laurent et al., 1996; Wehr and Laurent, 1996; larly, activity in glomerular modules found in a distinct Laurent, 1997). When the synchronization of projection region of the zebrafish olfactory bulb can discriminate neurons is abolished by picrotoxin while sparing their amino acids that vary in both concentration and struc- general odor selectivity, the ability to discriminate simi- ture (Friedrich and Korsching, 1997). lar odorants is impaired (Stopfer et al., 1997). Since different odorants evoke spatially distinct activ- These experiments suggest that odor perception re- ity patterns in the olfactory bulb, it has been hypothe- lies on both spatial and temporal patterns of activity, sized that higher-order brain regions use this spatial particularly for discriminating related odorants. In our information to discriminate and identify odors. This spa- experiments, however, it was possible to discriminate tial coding hypothesis has been tested behaviorally, but very similar odorants based solely on the pattern of has not yet received any direct supporting evidence active glomeruli, despite the fact that we recorded from (Slotnick, 1990; Holley, 1991). Lesioning the modified a relatively small percentage of the bulb surface. In one glomerular complex, a site of focal high 2-DG uptake in example, this was not the case: the stereoisomers (ϩ)- suckling rats (Teicher et al., 1980; Greer et al., 1982), did and (Ϫ)-carvone both activated a single glomerulus in not produce deficits in nipple attachment or suckling, the imaged area of the bulb (Figure 4C). It is possible as would be expected if this region of the bulb was that the spatial representations of these odorants did necessary for suckling (Hudson and Distel, 1987; Risser differ over regions of the bulb that were not examined. and Slotnick, 1987). Similarly, the removal of the dor- Alternatively, (ϩ)- and (Ϫ)-carvone may be distinguished somedial bulb, which includes the major region acti- by temporal rather than spatial activity patterns. Of vated by propionic acid, had no effect on the detection, course, both spatial and temporal information may be discrimination, sensitivity, or recognition of propionic used for odor discrimination, a possibility that awaits acid (Slotnick et al., 1987, 1997; Lu and Slotnick, 1994). experimental investigation. However, this type of bulb lesion spares propionic acid– responsive glomeruli (Guthrie et al., 1995). Despite the Conclusions robustness of the system, it is possible that the lesions Odorants evoke patterns of activity in the rat olfactory did in fact cause subtle deficits in perception that could bulb that are consistent with studies of rodent ORs and not be detected by the behavioral assays that were studies of spatial activity patterns in the olfactory bulb used. These studies imply that while discrete regions of that relied on other techniques. We have demonstrated the bulb may contribute to the perception of specific that similarity in odorant concentration and structure are odors, they are not necessary. correlated to similarity in patterns of activated glomeruli. A spatial code of glomerular activity in the olfactory Although the relationship between chemical structure bulb is potentially advantageous from an information and perceived smell is exceedingly complex, a number Optical Imaging of the Mammalian Olfactory Bulb 509

of studies suggest that some odorants that are structur- filter in order to reduce high-frequency noise while maintaining spa- ally similar are perceived as having similar odors (Braun tial information. To analyze activated glomeruli, oval regions of inter- and Marcus, 1969; Ohloff, 1986). To test whether similar est between 75 and 200 ␮m in diameter were drawn around the activity peaks, and the mean pixel value within these areas was odor representations are related to similar perceived measured. In order to measure background activity levels, boxes odors, it will be necessary to make psychophysical mea- 150–300 ␮m on each side were drawn around the oval regions and surements for multiple odorants as well as detect the placed to exclude intact bone, other activity peaks, and major blood responses of populations of glomeruli in single animals. vessels. The mean value within these boxes (excluding the ovals) This approach is now possible. In addition, since intrin- was subtracted from the mean value within the ovals to obtain a sic signal imaging can be repeated at multiple time measure of activation above baseline. For display, images were cropped and normalized (IPLab and Adobe Photoshop). points in the same animal (Grinvald et al., 1991; Chap- To calculate the expected size of signal originating from a glomer- man et al., 1996), it will be possible to determine whether ulus, we assumed equal distribution of signal throughout an ideal- learning, which leads to changes in the meaning of an ized spherical glomerular volume as well as equal signal integration odor, correlates with changes in how the odor is repre- from the entire glomerulus. A three-dimensional glomerulus measur- sented in the olfactory bulb. ing 150 ␮m in diameter would project to a 150 ␮m diameter spot on the glomerular surface, with the greatest amount of activity in the center since the glomerulus is thickest at this point. At half- Experimental Procedures maximal activity, the width of this projected glomerular activity (x) ␮m for 130ف ϫ diameter (x2 ϭ 12–0.52), which is 0.866ف would be Animal Preparation a glomerulus 150 ␮m in diameter. Experiments were performed on 29 female Long Evans rats between To quantitate the degree of similarity between maps for different postnatal days 50 and 102. Rats were initially anesthetized with a aliphatic aldehydes in the same animal, we first constructed a simi- mixture of ketamine (80 mg/kg i.p.) and xylazine (10 mg/kg i.p.). In larity matrix for each glomerulus. This was accomplished by calcu- initial experiments (n ϭ 13), a double tracheotomy was performed, lating the difference in glomerular activation (expressed again as a and rats were artificially ventilated while anesthetic state was main- percentage of maximal activation) for each pair of odorants. For tained with isofluorane (1%–3%). In these experiments, stimulus example, if propanal activated a glomerulus maximally (100%), and delivery was controlled by periodic application of negative pressure the response to butanal was 70% of the maximal response, the to the tracheotomy tube leading to the nasal cavity, to draw odorous difference between the two odorants for this glomerulus was 30%. air into the nose. The remainder of the rats were allowed to breathe This calculation was repeated for every possible pair of odorants, freely, and intraoperative anesthesia was maintained with thiopental yielding a similarity matrix for each glomerulus. We then calculated (15–20 mg/kg i.p.). Similar results were obtained with both prepara- a composite similarity matrix by averaging the matrices for each tions. glomerulus within an animal. These composite matrices express the Once anesthetized, rats were placed in a stereotaxic apparatus, similarity between every pair of maps based on all the glomeruli and the bone overlying the dorsal surface of one or both olfactory activated by one or more odorants. In these matrices, large scores mm anteroposterior ϫ 1.5 mm mediolateral) was removed represent large differences in how two odorants activated the set 3ف) bulbs or thinned. The use of a closed imaging chamber was difficult due of glomeruli analyzed. to the limited width of the skull overlying the olfactory bulbs. How- ever, brain pulsation was very limited or nonexistent, due to the Electrophysiology intact dura (and thinned bone in some cases) and the relatively small Following four imaging experiments, 2–10 M⍀ tungsten electrodes size of the craniotomy. Therefore, open chambers using coverglass were used to record neurons in imaged regions of the olfactory and bone wax were constructed and filled with saline for imaging. bulb. Electrode penetrations were targeted toward regions of the bulb that were selectively activated by one of the imaged odorants. Odorant delivery was performed as described in the previous sec- Intrinsic Signal Imaging tion. Data were digitized and recorded at a rate of 30 kHz and Intrinsic signals were recorded using a commercially available im- then analyzed using Spike2 software (Cambridge Electronic Design aging system (Imager 2001 system, Optical Imaging). Prior to each Limited). experiment, the surface blood vessel pattern under green illumination (546 nm) was acquired using a video camera. Stimuli were delivered by placing a test tube filled with 1 ml of an odorant (Sigma Acknowledgments and Fluka, at highest purity available) within 1 mm of the rat’s nose for the 10 s data acquisition period. Odorants were presented either We would like to thank J. Crowley for assistance with early aspects in pure liquid form or diluted in mineral oil. Trials using an odorant of this project; members of the Katz lab for discussion; and D. and mineral oil were randomly interleaved and repeated 6–12 times Coppola, H. Wilson Horch, D. Katz, and S. Simon for helpful com- with a 50 s interstimulus interval in order to minimize adaptation ments on the manuscript. and habituation. This is a small number of trials compared to imaging studies of visual cortex but was necessary due to the relatively long Received June 17, 1999; revised June 30, 1999. interstimulus interval and was found to be sufficient due to the robustness and reproducibility of the intrinsic signal. Prior to data acquisition, the camera was focused just beneath References the surface of the olfactory bulb in order to blur artifacts caused by surface blood vessels. Video frames acquired under red (630 nm) Adrian, E.D. (1950). 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