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1 Department of Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, New York 13210; 2 Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Submitted 12 September 2002; accepted in final form 7 August 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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; Margolis 1980). The presence of this abundant 19-kDa cytoplasmic protein in olfactory neurons of vertebrate species as diverse as fish and humans has become a hallmark of the mature olfactory neuron phenotype (Buiakova et al. 1994; Margolis 1980; Rama Krishna et al. 1992). This constellation of biological properties strongly suggested that OMP plays an important functional role in olfactory chemoreceptor neurons, a hypothesis that has received strong support as a result of recent studies on mice rendered deficient in this protein. Specifically, mice rendered deficient in OMP by targeted gene deletion have a reduced ability to respond to odor stimuli (Buiakova et al. 1996; Ivic et al. 2000). This altered physiological ability was reflected in prolonged onset and recovery kinetics after stimulation and a slower recovery from adaptation. As an additional consequence, the defects in neural activity also resulted in an altered bulbar phenotype that was consistent with apparent abnormal neuronal activity projecting to the bulb (Buiakova et al. 1996), although conduction velocity of olfactory receptor neuron axons was unaltered (Griff et al. 2000). Behaviorally, the observed physiological defects resulted in a behavioral threshold sensitivity that was 50–100 times less sensitive in knockout animals (Youngentob and Margolis 1999).
The observed sequela of gene deletion suggested that OMP is an important modulator in olfactory detection/signal transduction processing. However, the functional effects have recently been shown to extend beyond alterations in behavioral sensitivity. Using a complex five-odorant identification confusion matrix task, Youngentob et al. (2001) demonstrated that the untoward physiologic effects associated with the loss of OMP also resulted in an alteration in odorant quality perception in the null mutant. Exactly how the physiologic defects in mucosal function resulted in altered quality perception is presently a matter of conjecture that requires investigation. In this respect, one aspect of odorant quality coding that warrants consideration is the inherent differential spatiotemporal patterning of neural activity at the level of the olfactory epithelium in response to different odorants (Kauer and Moulton 1974; Kent and Mozell 1992; Kent et al. 1995, 1996; Kubie et al. 1980; MacKay-Sim and Kesteven 1994; MacKay-Sim and Kubie 1981; MacKay-Sim and Shaman 1984; MacKay-Sim et al. 1982; Moulton 1976; Scott et al. 1997; Youngentob and Kent 1995; Youngentob et al. 1995). These inherent spatiotemporal patterns, which have been shown to predict the behaviorally determined perceptual relationship among odorants (Kent et al. 1995, 2003), likely have as their underlying mechanism the summed effect of the variations in odorant receptor tuning that exist both within and across the mucosal receptor zones (Malnic et al. 1999) and the clustered expression patterns of some receptor types (Kubick et al. 1997) coupled with the heterogeneous expression patterns of others (C. L. Iwema, H. Fang, D. B. Kurtz, S. L. Youngentob, and J. E. Schwob, unpublished data).
If the mucosal representation of an odorant is, indeed, distributed such that large-scale patterns of activation serve as the first step in odorant analysis, then the observed physiologic defects associated with OMP gene deletion would be expected to affect odorant quality coding. That is, the altered responsivity of the mucosa coupled with the previously reported slowed response and recovery kinetics might be expected to degrade the odorant-induced spatial activity patterns that are characteristic of different odorants as well as alter the differential temporal activation of the epithelium. These effects, in turn, would change the organized and stereotyped spatial and temporal patterning of information that occurs at the level of the peripheral projection from the epithelium to the bulb (Mombaerts et al. 1996; Shepherd 1991; Vassar et al. 1994), thereby altering the perception of odorant quality. Therefore to test the hypothesis that odorant-induced mucosal activity patterns are altered in mice lacking the gene for OMP, we optically recorded the fluorescent changes in response to odorant stimulation from both the septum and turbinates of OMP-null mice and determined whether null mice differed from age-, sex-, and genotyped-matched controls.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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All procedures in this study were conducted in accordance with protocols approved by the Committee for the Humane Use of Animals at the SUNY Upstate Medical University, Syracuse, NY.
Ten adult mice (5 OMP-nulls on the 129S3/SvImJ background and 5 129S3/SvImJ controls) were individually housed and maintained in a temperature and humidity controlled vivarium. Following previously established procedures (Youngentob et al. 1995), mice were anesthetized and decapitated, and the extraneous tissue was dissected, retaining the region from the naris to the cribriform plate. The right nasal cavity was split into two halves along the length of the nasal cavity, exposing the septum and medial surface of the turbinates (Fig. 1). The two halves of the nasal cavity were placed in an oxygenated Ringer solution for 20 min after which the preparation was soaked for an additional 20 min in the voltage-sensitive dye di-4-ANEPPS. Unbound dye was removed by returning the preparation to Ringer solution for an additional 20 min. Prior to recording from one of the two halves of the right nasal cavity, the tissue was placed in a Delrin chamber and covered with a clear plastic plate (Kent et al. 1996). For the purpose of stimulus delivery (see following text), the Delrin chamber had an input and output port designed to be in anatomical register with the external naris and nasopharynx, respectively. Moreover, the chamber was designed to establish rapid onset and clean out of the stimulus while at the same time providing a uniform flooding of the chamber with an odorant. As such, the chamber permitted the monitoring of the inherent mucosal response [i.e., independent of any imposed chromatographic effect (Mozell and Jagodowicz 1974)].
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Optics and electronics
Except for minor modifications in the optics to accommodate the mouse preparation, and the implementation of a CCD camera (Kent et al. 2003) as the recording device, rather than a photodiode array, the recording methods were identical to those previously described (e.g., Kent and Mozell 1992; Youngentob et al. 1995) (Fig. 2). Briefly, a real fluorescence image of the mucosa stained with di-4-ANEPPS was projected onto a Dalsa (CA-D1) 12-bit, 120 x 120 digital camera. With the present configuration a 3.5 x 3.5 mm area of the mucosa was imaged onto the camera array with each detector receiving light from a 30 x 30 µm area of the preparation. Data were acquired at a rate of 40 frames/s.
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Consistency in the positioning of the recording surface images on the camera array was accomplished by use of the CCD camera, itself. The digital output of the camera was viewed directly to align each tissue with a standard preparation outline for both the septum and turbinates, respectively. Each tissue was positioned such that particular anatomical landmarks would fall on specific outline locations. Given the minor anatomical variability that existed from animal to animal, this approach allowed for maximum consistency in alignment across experiments.
Odorants and stimulus delivery
For each of five odorants (propyl acetate, 2-propanol, citral, l-carvone, and ethylacetoacetate), a concentration was chosen such that the size of their respective response were approximately equal and each fell within the dynamic range of their individual concentration-optical response relationship (Youngentob et al. 1995). The concentrations (expressed as percentage vapor saturation at 23°C) chosen were: propyl acetate, 0.75%; 2-propanol, 25%; citral, 45%; l-carvone, 25%; and ethylacetoacetate, 13% [It should be emphasized that the preceding odorant stimuli are expressed in terms of the nominal concentrations required to achieving our outlined response parameters under the artificial conditions of our odorant delivery procedure. Based on the physical parameters of the procedure, specifically, the internal volume of the Delrin chamber, the distance between the mucosal surface and the clear plate, the diameter of the input to the chamber, and the stimulus flow-rate, we estimate that the effective odorant deposition at the olfactory epithelium is equivalent to that achieved by presenting a 10–100 times smaller concentration of odorant to a behaving animal (Kent et al. 2003)]. In addition, a sixth odorant, 1% amyl acetate, served as a standard stimulus. Odorant stimuli were generated according to previously established methods, using standard flow dilution olfactometry and computer-driven electronic mass flow controllers (Teledyne Hastings Raydist, Hampton, Va) (Youngentob et al. 1991).
The odorant delivery system was identical to that previously described (Kent et al. 1996). The input port to the Delrin chamber was connected to a "T" connector through which either odorized or deodorized air flowed at a rate of 600 ml/min. To draw air through the chamber and across the mucosa, a negative pressure ample enough to produce a constant flow rate of 250 ml/min was applied to the output port. During stimulation, computer activated valves switched the flow through the T connector from deodorized to odorized air for 1 s, and data were acquired for a total of 45 s after initiation of the stimulus pulse.
Protocol
The mucosal responses from both the mouse's septum and medial surface of the turbinates were recorded in each experimental session. The order of tissue recordings was randomly determined. For each mucosal surface, a recording session consisted of a single presentation of each of the five odorants. The inter-stimulus interval between odorant presentations was 
5 min. This interval was chosen to ensure complete recovery of the mucosal response to odorant in the null mutant (Buiakova et al. 1996; Ivic et al. 2000). The order of each odorant presentation was determined using a randomized block Latin-Square design. This design was done to minimize any bias from the order of odorant presentation across animals. In addition, the standard stimulus, amyl acetate, was presented at the beginning of a session and after the fifth stimulus. The purpose of the standard was to adjust for any gradual changes in mucosal responsivity over time (Youngentob et al. 1995). These shifts in responsivity were independent of changes in baseline drift over time (which was due to photo-bleaching of the dye) or of variations in background fluorescence on a diode-by-diode basis. Correction of the raw responses for baseline drift and level of background fluorescence were accomplished following previously established procedures (Kent and Mozell 1992).
For each stimulus presentation and each of the 14,400 contiguous pixels of the camera's array, the background fluorescence, the average height of the response and the latency of the response (i.e., both start and peak times) were calculated. As previously described (Kent and Mozell 1992), the start of the response was defined to occur when the rising phase of the response was equal to 1/e of the peak response.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Previous studies in both amphibians (Kent and Mozell 1992) and rodents (e.g., Kent et al. 1996; Youngentob et al. 1995) have demonstrated that the optically recorded response to odorant stimulation has a similar profile and response characteristics as those of the electroolfactogram (EOG) (Ottoson 1956). Thus by analogy to the EOG, the optically recorded response monitors a component of the generator potential of sensory neurons responding to odorant stimulation. Nonetheless, in considering the following results it should be recognized that the two techniques differ in a number of ways. Specifically, the present optical recording procedure monitors the DC-coupled voltage change (Kent et al. 2003) which predominantly, if not exclusively, originates from the cilia in response to odorant stimulation (Kent and Mozell 1992; Youngentob et al. 1995). By contrast, the standard EOG is an AC-coupled signal (e.g., Buiakova et al. 1996; Ivic et al. 2000) that monitors current flow through a number of cellular elements within the epithelium (Scott and Scott-Johnson 2002).
As examples of the raw responses, Fig. 3 shows one array for each mouse strain (i.e., both OMP-null and 129S3/SvImJ controls) in response to the odorant propanol delivered to the turbinate mucosa. The optically recorded response traces are shown for every fourth pixel of the 120 x 120 array. The response tracings, in turn, are superimposed on a color-scale representation of average response magnitude for all pixels of the camera array. The larger the response the brighter the pixel. As can be seen in Fig. 3A, the response of the mucosa was a typical slow monophasic potential. More importantly, however, although propanol gave responses across the entire sampled area of the two mucosas, the responsivity of the two mouse strains appears qualitatively different. Specifically, focusing on the brightness across the two panels of Fig. 3 gives the general impression that the response profile of the OMP-null animal was more subdued or muted relative to controls.
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Figure 4 represents the raw average pseudo-color topographical response profiles for the 120 x 120 array in response to each of the five odorants. In keeping with the preceding text, although the average raw patterns for each odorant were similar between control and null animals (i.e., region of maximal differential activity), a change in responsivity across the epithelium between the two mouse strains can be appreciated in the fine detail. For example, focusing on the center of each mucosal response panel for the odorant propanol, compared with controls, the response of the null animal was more evenly distributed. That is, the gradient of differential activity across the epithelium was reduced or muted.
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In extension to the preceding text, Fig. 5 highlights the effect of OMP gene deletion on the raw responses of the epithelium. For each of three parameters of the optically recorded response (i.e., average height, start time, and peak time), the panels of Fig. 5 were determined by calculating, pixel-by-pixel, the ratio of the raw control response divided by the raw response of the null animal. Thus for a ratio value >1, the response of the control animals, at any given pixel, were greater than the nulls and vice versa. As illustrated for the turbinate response to odorant stimulation, for those regions of the epithelium where the responsivity to a particular odorant was maximal (see Fig. 4), the control animals gave a relatively larger response as compared with the nulls. By contrast, for those areas of the epithelium where the control animals were least responsive to a particular odorant the null animals gave a relatively larger response. As emphasized by the scale bar provided in Fig. 5, these noted differences were rather robust across the epithelium. For example, the ratios for the average response magnitude represent a 2.0-, 2.1-, 2.3-, 2.3-, and 2.3-fold change in mucosal response for the odorants carvone, citral, ethylacetoacetate, propyl acetate, and propanol, respectively. In total, these shifts resulted in similar but muted raw response patterns for the null mutant (as seen in Fig. 4). Comparable results were observed for the septal mucosa as well.
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Figure 5 also illustrates a change in raw temporal activity between mouse strains. That is, where the spatial pattern for the control animals was larger than the nulls, the response latency (i.e., start time) and time to peak response was also relatively faster. In contrast, for those areas of the epithelium where the control animals were least responsive to odorant stimulation the response latency and time-to-peak response of the null animals was relatively faster. Thus both changes in responsivity and kinetics were observed between mouse strains. However, in extension to previous work using EOG recording from a markedly smaller sample of the mucosa (Buiakova et al. 1996; Ivic et al. 2000), the alterations observed in the present study were location dependent. Nonetheless, taken together, these location-dependent changes resulted in a more broadly distributed and degraded raw response in the null animals.
Spatial activity patterns as a function of odorant and genotype
In summary of the preceding text, the raw response data demonstrated that the mucosal response to odorant stimulation varied both as a function of odorant and mouse strain. As previously noted, these spatiotemporal patterns of activity likely result from the collective effect of the variations in odorant receptor tuning that exist among the intermingled receptor types both within and across receptor zones, the clustered expression patterns of some receptor types, and the heterogeneous expression patterns of others. Therefore to remove common elements of broad responsivity and highlight the areas of differential odorant activity for each of the two mouse strains in response to each of the five odorants, the following procedure was used. For each animal and each presentation of odorant, the responses monitored by each pixel were equilibrated (i.e., scaled) to unity according to previously established methods (Kent and Mozell 1992). This equilibration process removed any differences in response magnitude between arrays so that magnitude differences would not be confused with pattern differences. For each odorant and each mouse strain, the equilibrated average response size was then averaged across animals on a pixel-by-pixel basis. A summary map of differential spatial activity for a given odorant and mouse strain was then produced by subtracting, pixel-by-pixel, the average height of the response in a given pixel by that same pixel's average response across all five odorants (Fig. 6). The differential response at each pixel was represented using a pseudo-color scale (Kent et al. 2003). As can be seen in this figure, and in keeping with prior work in other species, each odorant produced a unique region of increased responsivity that distinguished it from every other odorant. In this regard, although there were indeed minor variations in the shape of increased activity for each of the odorants, the unique geographic region was, for all animals in a given strain, in a qualitatively similar area of the mucosa (data not shown). In addition, the general area of increased activity appeared similar across both mouse strains, suggesting that the geographic components of the differential spatial response patterns are the same in control and null animals. Nonetheless, the data in Fig. 6 also give the impression that the two mouse strains differ with respect to the magnitude of their unique differential odorant response patterns. That is, for each of the odorants tested, there was an increasing trend toward equal responsivity across the epithelium in the null animal. Thus for example, focusing on the response to ethylacetoacetate, the region of relative maximal activity along the dorsal surface is dramatically reduced in the null animal.
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To further emphasize the apparent muting of responsivity between the two mouse strains, the data developed for the construction of Fig. 6 were again used. However, for the purpose of Fig. 7, only those pixels which demonstrated relatively greater activity were considered (those that showed decreased sensitivity relative to the average were set to 0). These data were then transformed into enhanced color scale surface plots such that the magnitude of the difference of increased relative responsivity for each pixel of the 120 x 120 array was represented by the height on the z axis. As emphasized in the panels of Fig. 7, each odorant produced a unique area of differential activity on the septal mucosa of both mouse strains that distinguished it from every other odorant. More importantly, however, relative to control animals (Fig. 7, top), the differential spatial response to an odorant appears to be degraded or muted in the null mutant (Fig. 7, bottom). Similar results were observed for the turbinate mucosa as well.
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As emphasized in Figs. 6 and 7, the general physical pattern of differential spatial activity in response to different odorants appear to be generally similar in both the OMP-null and control animals, while the distinctiveness of the patterns appeared altered as a function of genotype. To formally evaluate these observations, we first examined whether the five odorants, do indeed, produce differential patterns of response and whether these patterns varied as a function of genotype. To accomplish this, the following procedure was used. First, for the present and all subsequent analyses, the septal and turbinate mucosas were considered as one complete expanse of tissue. Second, for each animal and each presentation of odorant, the responses monitored by each pixel were equilibrated, as previously noted in the preceding text. Third, the differential spatial activity pattern for a given odorant, in a given animal, was produced by subtracting, pixel-by-pixel, the average height of the response in a given pixel by that same pixel's average response across all five odorants. Finally, the fourth step was to calculate the degree of dissimilarity between the differential response arrays for each of the five odorants tested both within an animal and between every possible pair of animals (i.e., both OMP-nulls and controls). The degree of dissimilarity between any two differential response arrays was calculated by summing the absolute values, on a pixel-by-pixel basis, of the difference between the two paired response arrays and then dividing that summation by the total number of pixels in the array. As a result, this measure served as an indication of the degree of response dissimilarity both as a function of odorant presented and genotype. These comparisons, in turn, yielded a 50 x 50 dissimilarity matrix for the entire sampled mucosal surface. The 50 x 50 dissimilarity matrix was subjected to multidimensional scaling analysis (MDS) (Schiffman et al. 1984) with the objective of placing an animal's response to the presentation of an odorant in an "odorant/mouse space" according to the degree of dissimilarity in odorant-induced spatial activity patterns (Loo et al. 1996). The MDS analysis yielded a two-dimensional solution, thereby providing a set of coordinates which located each mouse's (both OMP-null and control) response to a particular odorant within the MDS space. To evaluate formally whether the odorant-induced spatial activity patterns varied as a function of odorant and/or genotype, a multivariate ANOVA (MANOVA) was performed, using the MDS coordinates as the dependent variables and odorant and genotype as the independent variables. The results of these analyses demonstrated highly significant effects of odorants [F(8,86) = 9.604; P = nil], thereby giving further support to the existence of different spatial activity patterns across the mucosa for different odorants. In contrast, however, there was no effect of genotype [F(2,43) = 0.0017; P = 0.998], suggesting consistency in the location of the observed patterns of greater and lesser relative responsivity for each odorant across the two mouse strains. Taken together, these results confirmed our subjective impression that although different odorants produced different inherent activity patterns across the two mucosal surfaces sampled, these patterns were similar in the two mouse strains.
To evaluate the apparent degrading or muting of the spatial response pattern in the null animals (Fig. 7), the distinctiveness of an odorant's differential mucosal response was quantitatively expressed as the average percentage difference (APD) (Kent and Mozell 1992; Loo et al. 1996). For each of the animals (both OMP-null and control), the APD for a given odorant was calculated by subtracting, pixel-by-pixel, the equilibrated individual odorant pattern of interest by the equilibrated average response pattern of all five odorants; calculating the sum of the absolute value of the differences across the pixels; dividing by the number of contributing pixels; and multiplying by 100. This APD expression, therefore quantified the average, pixel-by-pixel, percentage difference between the odorant of interest and the average of all five odorants in a particular animal. As can be seen in Table 1, for each of the five odorants, the APD decreased markedly as a function of genotype. On average, the patterns of differential responsivity were 23.5% less distinct or muted in the null mutant with the response to the odorant propyl acetate showing the least muting (i.e., 9.95%) and that to propanol showing the greatest (i.e., 34.67%).
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To test formally the significance of the apparent genotype-related shifts, we evaluated the null hypothesis that the distinctiveness of the odorant patterns, as expressed by the APD, did not change with genotype. The data from the individual animals were subjected to an ANOVA with the principle analysis of interest being the effect of genotype on the APD. The results of this analysis gave strong support to the hypothesis that a muting or degradation of the odorant-induced spatial patterns occurred as a function of OMP gene deletion [F(1,44) = 12.313; P = 0.0011].
Temporal activity patterns as a function of odorant and genotype
In addition to differing spatial patterns of mucosal activity for different odorants, there also existed differing temporal characteristics of the responses. We first evaluated, using the procedures outlined in the preceding text, whether the five odorants produced different temporal patterns of response latency (i.e., start time) and whether these temporal patterns varied as a function of genotype. Second, using our APD measure, we evaluated the apparent muting of the temporal response pattern as a function of OMP gene deletion. The results of these analyses demonstrated a highly significant effect of odorant [F(8,86) = 5.548; P = nil] and no effect of genotype [F(2,43) = 0.317; P = 0.968] on determining the differential temporal odorant response of the epithelium and a significant effect of genotype on the APD of the temporal response pattern [F(1,44) = 16.318; P = 0.0002]. With regard to the later formal result, Table 2 illustrates the change in average APD data as a function of genotype. For each evaluated odorant, the APD decreased in the null animal. On average, the distinctiveness of the temporal response pattern decreased 26.66% in the null mutant with the smallest change attributable to the ethylacetoacetate response (16.47%) and the largest change occurring in the propanol comparison. Therefore as a whole, the data suggest that while different odorants produced different temporal patterns of activity across the epithelium, these temporal patterns were similar in the two mouse strains for any given odorant. Nonetheless, the results support the supposition that a muting or degradation of the temporal activation pattern occurred as a function of OMP gene deletion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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). This observation coupled with the known neurophysiological defects at the level of the epithelium (Buiakova et al. 1996; Ivic et al. 2000) led to the suggestion that one possible mechanism underlying the change in quality perception in OMP-null mice might be an alteration in the spatiotemporal response to odorants across the mucosa. Thus the major objective of the present investigation was to evaluate whether, and to what degree, the large-scale spatiotemporal response of the epithelium to different odorants was altered, relative to controls, in the null mutant. With regard to the preceding proposition, the qualitative and quantitative data of the present study addressed two fundamental issues relevant to the spatiotemporal differentiation of odorants in the two mouse strains. Specifically, we evaluated whether the odorant-induced spatiotemporal activity patterns varied as a function of odorant and/or genotype and whether the distinctiveness of the differential patterns were muted or degraded as a function of OMP gene deletion.
In addressing the first question, the present study confirmed for both mouse strains the existence of different spatiotemporal activity patterns in response to different odorants. As such, the data extend to yet another species the prior observations obtained in both rats (Kent et al. 1995, 1996, 2003; MacKay-Sim and Kesteven 1994; Scott et al. 1997; Youngentob and Kent 1995; Youngentob et al. 1995) and two amphibian species (Kauer and Moulton 1974; Kent and Mozell 1992; Kubie et al. 1980; MacKay-Sim and Kubie 1981; MacKay-Sim and Shaman 1984; MacKay-Sim et al. 1982; Moulton 1976) that non-homogeneous patterns of sensitivity to different odorants exist along the olfactory epithelium. In addition, the application of the CCD camera to the optical recording technique (with its 14,400 pixels) provided a more fine-grained method [over the previous 10 x 10 photodiode array (e.g., Kent et al. 1992, 1996; Youngentob et al. 1995)] for the evaluation of the epithelial response to odorants. In this regard, a secondary observation in this study was a band-like configuration of mucosal activity patterns (in both mouse strains) reminiscent of the zonal distribution of odorant receptors (Dear et al. 1991; Nef et al. 1992; Ressler et al. 1993; Strotmann et al. 1992; Vassar et al. 1993). This finding was consistent with those of Kent et al. (2003) (recording from rats), who emphasized that while the neurophysiologic data appeared consistent with the conclusion that receptors are arranged in zones, they were not consistent with the notion that like receptor types are necessarily distributed homogeneously within a zone. Clearly, in the present study, the response areas both within and across reported receptor zones (Figs. 4, raw responses, and 6, differential odorant pattern responses) are far from homogeneous. This lack of homogeneity in responsivity was consistent with the observations of several molecular studies that some receptor types (e.g., OR37 and OR-Z6) have a clustered distribution (Breer et al. 1994; Kubick et al. 1997; Pyrski et al. 2001; Strotmann et al. 1994) and that odorant receptors previously held to be randomly distributed within a given zone are instead distributed heterogeneously with an aggregation of receptors in a particular region (C. L. Iwema, H. Fang, D. B. Kurtz, S. L. Youngentob, and J. E. Schwob, unpublished results).
With regard to the principal focus of the study, the results clearly demonstrated that the epithelium of OMP-null mutants responded to odorant stimuli with both spatial and temporal activity patterns that resembled the patterns observed in control animals. That is, as seen in the raw responses illustrated in Fig. 4 and the differential responses of Figs. 6 and 7 and confirmed quantitatively, the relative areas of spatiotemporal activity for each odorant did not appreciably shift to different locations within the sampled area as a function of OMP gene status. Accordingly, this component of the neurophysiological results suggests that, at least at the level of the differential organization of the odorant receptors both within and across receptor zones (Dear et al. 1991; Kubick et al. 1997; Nef et al. 1992; Ressler et al. 1993; Vassar et al. 1993; C. L. Iwema, H. Fang, D. B. Kurtz, S. L. Youngentob, and J. E. Schwob, unpublished results), odorant coding as a function of the differential response of different receptors (Malnic et al. 1999) appears well maintained despite the loss of OMP.
The quantitative and qualitative results of the second question gave strong support to the hypothesis that while the general spatiotemporal patterns of activity did not shift as a function of gene deletion, the patterns became muted or degraded (Figs. 4, 6, and 7). In other words, for both the spatial and temporal response to an odorant, there was a significant increase toward equal responsivity across the epithelium in the null mutant. On average, the raw spatial and temporal response patterns were, relative to controls, degraded by a factor of 2.2-fold. Moreover, when the distinctiveness of the differential spatial and temporal mucosal response to different odorants was emphasized, the responses of the null animals were muted by 23.5 and 26.66%, respectively. In considering the implications of these results, the dichotomy between the distinctiveness data and the pattern results requires some comment. Specifically, one might question how the muting or degrading of the pattern would not manifest itself in an alteration of pattern, per se. The answer to this question lies in the analyses themselves. On the one hand, using our measure of pattern dissimilarity, the MDS analysis focused, on a pixel-by-pixel basis, on the physical location of differential mucosal activity in response to the various odorants. By comparison, recall that the APD measured the absolute differences between an individual odorant pattern of interest and the average pattern of all five odorants without regard to exactly where on the epithelium the area of differential activity occurred. That is, for any comparison, we calculated, on a pixel-by-pixel basis, the absolute difference in equilibrated responses between corresponding pixels of the paired arrays. Consequently, only in the case where a change in the characteristic maximal area of differential odorant responsivity occurred (i.e., equal responsivity relative to the average) would we expect to see a "shift" in the pattern coincide with an apparent muting.
In summary of the preceding text, response differences between mouse strains were clearly observed in the raw data illustrated in Figs. 4 and 5, as well as in the differential odorant response data of Figs. 6 and 7. Further, quantitative analysis demonstrated that the underlying differential patterns of response to different odorants were not altered (i.e., whole-sale shifts in spatial location) between mouse strains, but rather the patterns were muted or degraded (i.e., latter more broadly distributed) in the null mutant. Whether the latter observation, in fact, constitutes a "shift" or difference in differential odorant response pattern, per se, is partly philosophical. Nonetheless, taken together, the data demonstrate an alteration in the epithelial response to different odorants in the OMP-null animal.
The two sets of results (i.e., similar but degraded patterns) were not entirely surprising given the present working hypothesis regarding OMP's role in odor processing. The current body of evidence suggests that OMP plays a novel modulatory role in the odor detection/signal transduction process (Buiakova et al. 1996; Ivic et al. 2000; Margolis et al. 1997; Youngentob and Margolis 1999). Neurophysiologically, loss of the protein has previously been shown by others to result in a reduction in peak response magnitude in paired-pulse experiments and a slowing of onset and recovery kinetics as measured by the EOG (Buiakova et al. 1996; Ivic et al. 2000). Thus a priori, there was no expectation that the lack of OMP would alter the stereotyped expression pattern of odorant receptors across the olfactory epithelium (Dear et al. 1991; Kubik et al. 1997; Nef et al. 1992; Ressler et al. 1993; Strotmann et al. 1992; Vassar et al. 1993; C. L. Iwema, H. Fang, D. B. Kurtz, S. L. Youngentob, and J. E. Schwob, unpublished data). Indeed, the results outlined in the preceding text confirm this proposition. Rather the present results are in keeping with the notion that gene deletion alters the responsivity of the epithelium to an odorant as well as the kinetics of the response. However, in extension of prior observations (Buiakova et al. 1996; Ivic et al. 2000), the present data (under our setting of monitoring large-scale patterns of activation) would further suggest that the relative effect of gene deletion on the differential response of the epithelium to an odorant was location dependent. This, in turn, resulted in a significant increase toward equal responsivity across the epithelium (Figs. 4 and 5).
Based on the foregoing, it is worth considering how the data obtained in the present study relate specifically to those of previous investigators (Buiakova et al. 1996; Ivic et al. 2000). As noted, prior studies could be interpreted as suggesting that an overall change in both the time course and magnitude of the odorant response occurs in the null animal, whereas the current study suggests that these alterations, relative to controls, varied with location. In this regard, there are a number of methodologic differences that could account for this disparity. First, by contrast to previous studies using the EOG, which sampled a very limited punctate area of the epithelium, the present study applied optical recording methods (in conjunction with a voltage-sensitive dye) to study the large-scale activation patterns across the extent of the epithelium in a fine spatial matrix. Therefore the current method, by providing a more comprehensive and accurate representation of the mucosal response to an odorant in both space and time, may have been more sensitive to potential variations in the epithelial response of the OMP-null animal. Second, the stimulation parameters used in the current study contrast sharply with those previously used. As noted in METHODS, the inter-stimulus interval between odorant presentations was 5 min. Based on the prior EOG studies (Buiakova et al. 1996; Ivic et al. 2000), this interval was chosen to ensure complete recovery of the mucosal response to odorant stimulation in null animals. Thus while the earlier work emphasized stimulation paradigms (i.e., paired-pulse stimulation and short inter-stimulus intervals) designed to exacerbate the consequences of gene deletion on the neuronal response, the current study was directed toward examining the large-scale spatial response patterns under unperturbed conditions. As such, the stimulus conditions of the current study may have been more appropriate to uncover subtle response differences across the extent of the epithelium. Finally, there are distinct technical differences between the EOG and optical recording techniques. Although both methods have similar profiles and response characteristics, the two likely monitor different components of the generator potential. That is, the present optical recording procedure monitored the DC-coupled voltage change of the epithelium in response to odorant stimulation. Moreover, this response predominantly, if not exclusively, originated from the cilia. By contrast, the EOG method applied in prior studies on OMP-null animals was a low-frequency filtered AC-coupled signal that monitored current flow through a number of cellular elements within the epithelium. Therefore it is highly likely that the two methods monitored correlated yet different components of the neural response potentially relating to the differences observed in the current study.
Given the present findings, it is important to consider exactly how a muting of the spatial and temporal response of the epithelium might translate into an alteration in odorant quality perception. At the level of the olfactory bulb, there is ample evidence for both a spatial and temporal component to the encoding of odorant quality (Chaput et al. 1992; Cinelli et al. 1995; Freidrich and Korsching 1997; Imamura et al. 1992; Johnson et al. 1998, 1999; Johnson and Leon 2000; Kauer and Cinelli 1993; Laurent 1999; Laurent et al. 1996; Macrides and Chorover 1972). We propose therefore that the alterations in neural function at the periphery were translated to the bulb. In terms of spatial coding, it has been suggested that the bulb is an array of functional modules (i.e., clusters of glomeruli) that serve to spatially segregate and tune responses to the molecular features of an odorant (e.g., Johnson and Leon 2000; Johnson et al. 1998, 1999). The information carried by individual glomeruli therefore are a reflection of the periphery by virtue of the organized and stereotyped patterning of information that occurs from the epithelial projection to the bulb (Mombaerts et al. 1996; Shepherd 1991; Vassar et al. 1994). Consequently, it is reasonable to consider that the degrading of peripheral information would be translated to the bulb. That is, for the active glomeruli in response to an odorant there would be a significant increase toward equal responsivity like that seen on the mucosa, thereby altering the code. In addition, the spatial muting effect might further impact the coding process by interfering with the lateral inhibitory mechanisms, which subsequently fine tune the response of mitral and tufted cells to odorant features. Finally, one could further envision how a muting of the temporal sequence of odorant activation would also effect the reshaping of odor codes, resulting from the internal connectivity of early olfactory circuits. In total, therefore the degraded or disrupted information at the level of the bulb would be expected to alter the perception of odorant quality.
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* S. L. Youngentob and P. F. Kent contributed equally to this work. 
Address for reprint requests and other correspondence: S. L. Youngentob, Dept. of Neuroscience and Physiology, SUNY Upstate Medical University, 750 E. Adams St. Syracuse, NY 13210 (E-mail:youngens{at}mail.upstate.edu).
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