Physiological Evidence for the Discrimination of L-Arginine From Structural Analogues by the Zebrafish Olfactory System

D. L. Lipschitz and W. C. Michel

Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah 84108-1270


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ABSTRACT
INTRODUCTION
METHODS
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Lipschitz, D. L. and W. C. Michel. Physiological Evidence for the Discrimination of L-Arginine From Structural Analogues by the Zebrafish Olfactory System. J. Neurophysiol. 82: 3160-3167, 1999. Although it is generally assumed that fish are capable of discriminating amino acid odorants on the basis of differences in side-chain structure, less is known about their ability to discriminate amino acids with modifications to alpha -carboxyl and alpha -amino groups. In this study, the ability of the zebrafish olfactory system to detect and presumably discriminate analogues of the basic amino acid Arg was assessed, by using cross-adaptation and activity-dependent labeling techniques. Electrophysiological recordings established that esterification (L-arginine methyl ester; AME) or deletion (agmatine or amino-4-guanidobutane; AGB) of the alpha -carboxyl group yielded odorants more potent than Arg, whereas deletion of the alpha -amino group (L-argininic acid; AA) yielded a less potent analogue. In cross-adaptation experiments, no test-competitor odorant combination yielded complete cross-adaptation, suggesting the detection of these Arg analogues by multiple odorant receptors (ORs) with partially nonoverlapping specificities. Activity-dependent immunocytochemical labeling of olfactory receptor neurons supported this conclusion. AGB, an ion-channel-permeant probe (and odorant), labeled 4.9 ± 0.4% (n = 24) of sensory epithelium, whereas the addition of Arg, 1-ethylguanidine sulfate, L-alpha -amino-beta -guanidinopropionate, or AME to AGB resulted in a significant elevation of labeling (8-14%). This study provides evidence that the olfactory system has the potential to discriminate among amino acid odorants with modified alpha -carboxyl and alpha -amino groups.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In fish, such behavior as feeding, alarm responses, predator recognition, and social and pheromonal communication are initiated by the detection of the appropriate odor (Hara 1992). Several of these responses are elicited by amino acids, which are ubiquitous and efficacious as odors in aquatic environments (Caprio 1988; Hara 1992; Suzuki and Tucker 1971). In part because of their simple molecular structures, amino acids have been used in many studies of the mechanisms underlying olfaction. Electrophysiological cross-adaptation (Caprio and Byrd 1984; Caprio et al. 1989; Michel and Derbidge 1997; Ohno et al. 1984; Sveinsson and Hara 1990), receptor binding (Cagan and Zeiger 1978), and behavioral (Holland and Teeter 1981; Valentincic et al. 1994, 1996; Zippel et al. 1993) studies indicate that the olfactory system has sufficient odorant receptors (ORs) to permit discrimination among L-alpha -amino acids on the basis of side-chain structure. Originally, these amino acid-sensitive ORs were conservatively divided into four general classes of ORs primarily involved in the detection of acidic, basic, and short- and long-chain neutral amino acids, respectively (Bruch and Rulli 1988; Caprio and Byrd 1984). Subsequent dynamic imaging (Friedrich and Korsching 1997) and cross-adaptation studies (Michel and Derbidge 1997) have indicated that there are likely to be sufficient receptors within each of the four general classes of amino acid-sensitive ORs to permit discrimination of each of the odorants used in the respective studies.

The only functionally expressed fish OR binds the basic amino acids L-arginine (Arg) and L-lysine (Lys) with high affinity, several neutral amino acids with intermediate affinity, and decarboxylated Arg and Lys analogues with low affinity (Speca et al. 1999). The ability of the goldfish (Zippel et al. 1993) to behaviorally discriminate basic amino acids Arg and Lys suggests that other ORs detecting these basic amino acids must also exist. The channel catfish is also able to discriminate Arg from Lys and from several structurally similar Arg analogues (Caprio 1978; Valentincic et al. 1994). Cross-adaptation studies (Michel and Derbidge 1997) and dynamic imaging of glomerular activity in the bulb (Friedrich and Korsching 1997) indicate that zebrafish have the potential to discriminate Arg from Lys. However, the ability of the zebrafish olfactory system to discriminate Arg from structural analogues is unknown. In the current investigation, we assessed whether the zebrafish olfactory system has the potential to discriminate Arg and analogues with modifications to the alpha -amino and alpha -carboxyl groups, using the electroolfactogram (EOG) and activity-dependent labeling techniques. It is assumed that the olfactory system must generate a unique pattern of olfactory receptor neuron (ORN) activation if these analogues of Arg are to be discriminated. To investigate this we first identified a subset of structurally distinct Arg analogues eliciting robust olfactory responses, then using cross-adaptation experiments we established the involvement of multiple ORs in their detection. Finally, an activity-dependent labeling technique (Michel et al. 1999) was used to establish that distinct subsets of ORNs are activated by these structurally related Arg analogues. These experiments suggest that the zebrafish olfactory system has the capacity to discriminate among amino acids on the basis of changes to the alpha -carboxyl and/or alpha -amino groups.


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INTRODUCTION
METHODS
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Animal maintenance

Zebrafish, purchased from a commercial supplier, were maintained as mixed-sex populations in recirculating aquaria (40-80 l, 28°C) with fluorescent lighting (12:12 light:dark) and were used within 6 mo of arrival. Eighty percent of the water was replaced once per week with deionized water. Fish food (Tetramin) was provided daily.

Experimental procedures

To prepare a fish for recording or activity-dependent labeling, the paralytic Flaxedil (60 µg/g body weight) was administered by intramuscular injection. Immediately after immobilization, the fish was placed in a silastic-polymer (Sylgard) recording chamber. A tube inserted into the mouth provided a continuous flow of anesthetic (MS222, 20 mg/l) in artificial fresh water (AFW, see Solutions for composition) across the gills. The anesthetic was never allowed to contact the olfactory epithelium, because topical application contributes to the loss of afferent sensory activity (Spath and Schweickert 1977). If, over the course of an experiment, the fish showed any reflexive movements of the gills or the eyes, additional Flaxedil or MS222 was administered. In electrophysiological experiments, an olfactometer flow of AFW (3 ml/min) without anesthetic was provided by another tube to the left olfactory rosette. In the activity-dependent labeling experiments, fish Ringer (FR, see Solutions for composition) replaced AFW, and the olfactometer flow bathed both rosettes. Solutions were delivered by gravity-flow from polyethylene bottles. After sufficient time for the anesthetic to take effect (10 min), the flap of tissue forming the excurrent and incurrent nares and obscuring the left olfactory organ was surgically removed to facilitate placement of the recording electrode for EOG recordings. No surgical procedures were conducted in the labeling experiment. After termination of each experiment, the anesthetized fish was weighed, measured from the tip of the head to the caudal peduncle, and then decapitated. The body cavity was opened and examined under a dissecting microscope, and the presence of ovaries or testes was noted. Experimental procedures for immobilization, anesthetization, and surgery were approved by the Institutional Animal Care and Use Committee.

Electrophysiological recordings

The EOG recording procedures used in the present study to measure olfactory responses have been described previously (Michel and Derbidge 1997; Michel and Lubomudrov 1995). The EOG circuitry comprised a positive recording electrode (placed between lamellae near the raphe of the olfactory rosette, see Fig. 5A), a negative indifferent electrode (positioned on the head away from the nose), and a silver chloride ground wire (positioned underneath the fish). The positive and negative electrodes were made of glass with tip diameters of 4-10 µm and a silver-silver chloride pellet bridged to the fish through 1 M NaCl-agar (0.5%). Responses to odorants were amplified (500-1,000 × gain) and filtered (1-2 kHz) by a low-noise-differential DC amplifier. Amplified signals were digitized (100 Hz), stored, and displayed on an MS-DOS-based computer (Axotape, Axon Instruments).

Experiments began after a stable response was obtained to 100 µM L-glutamine (Gln; >0.50 mV), and no response was noted to AFW. A 50-µl bolus of Gln, introduced by a rotary-loop injector (Rheodyne) into the AFW carrier flow, reached the preparation in 4-6 s, with a peak concentration of 80% of the applied concentration shown by 8 s and decay to baseline levels shown ~2 s after reaching the peak concentration (Michel and Lubomudrov 1995). The concentrations reported have not been corrected for dilution. Odorants (at 100 µM) were tested at least twice, in random order, and preparation stability was continually monitored by retesting Gln. To minimize desensitization, 2 min were allowed to elapse between tests. For the structure-activity experiment, the relative stimulatory effectiveness of each test odorant was expressed as a percentage of the response to 100 µM Gln. Cross-adaptation procedures were used to investigate whether the more stimulatory Arg analogues interact with independent (no cross-adaptation), shared (complete cross-adaptation), or partially shared (partial cross-adaptation) ORs. Briefly, the response to a test odorant, established in AFW, was compared with the response in a competitor odorant background during a standard three-stage test series. First, the odorant was tested in AFW background; then, the odorant was tested in a competitor odor background; and finally, the odorant was retested in an AFW background. The competitor odorant (or AFW during stage 3) bathed the olfactory epithelium for at least 60 s before first application of the test odorant. Cross-adaptation was measured when the test odorant differed from the competitor odorant, and self-adaptation was measured when the test odorant was the same as the competitor odorant. No fish was tested in more than one competitor odorant background. The response to a test odorant in a competitor background is expressed as a percentage of the average of the unadapted responses obtained in stages 1 and 3. Data were pooled for males and females, because it has been shown that normalizing the responses removes a potential sex effect (Michel and Lubomudrov 1995).

Odorant-stimulated labeling procedures

The procedure used to stimulate and label zebrafish olfactory sensory epithelia in vivo was identical with that previously described (Michel et al. 1999). An electronically activated three-way valve was used to expose the olfactory rosettes of a fish to FR containing either 5 mM AGB alone or 5 mM AGB plus 100 µM odorant for 10 s/min for 10 min. After flushing for 5 min to wash away exogenous AGB, the fish was decapitated and the head placed in cold fixative solution (see Solutions) and stored at 4°C until required. Odorants were prepared in FR, which also bathed the olfactory epithelium during the activity-dependent labeling experiments. FR, rather than AFW, was used to decrease the amount of nonspecific labeling (Michel et al. 1999).

Fixed, isolated olfactory rosettes were dehydrated and embedded in Eponate resin and cut into 0.5-µm-thick sections. Sections were deplasticized, rinsed, and incubated overnight at room temperature with a 1:1000 dilution of polyclonal rabbit anti-AGB IgG antibody (Signature Immunologics, Salt Lake City, UT). Labeling was visualized using a nanogold-conjugated goat anti-rabbit secondary antibody (Amersham) followed by silver intensification (Marc 1999a,b).

Image analysis

AGB immunolabeling was quantified from standard eight-bit gray-scale digital images (0 = black, 255 = white). Images were captured at ×300 magnification, by using a microscope fitted with bright-field illumination and a video camera attached to a frame-grabber card in a personal computer. Quantification of activity-dependent labeling entailed calculating a pixel intensity cutoff value for distinguishing nonlabeled background from labeled cells in a region of interest (ROI) of sensory epithelium (the lower limit of the 95% confidence interval from the mean pixel intensity in an ROI of sensory epithelium devoid of labeled neurons). The percentage of labeled sensory area was calculated for three to six ROIs each from four planes of section of olfactory rosettes from a minimum of two fish.

Data analysis

The structure-activity data were analyzed using a two-way ANOVA for unbalanced data with odorant, sex, and odorant-sex interaction as the factorial model. If the ANOVA indicated a significant main effect, a post hoc Tukey's multiple comparison of means test determined whether significant differences in olfactory responses were apparent for each variable. Before the analysis, a square root transformation was performed to meet the assumptions of the ANOVA (Zar 1984). The cross-adaptation data were analyzed using a two-way ANOVA to determine the significance of the competitor odorant and test odorant main effects as well as an interaction between the two. Subsequent one-way ANOVAs determined whether responses to test odorants were significantly different within each competitor odorant. Individual differences between each test odorant (cross-adapted response) and competitor odorant (self-adapted response) were identified with a post hoc Tukey's test. In the activity-dependent labeling experiment, the percentage of labeling of sensory epithelium under the control condition (AGB only) was statistically compared with labeling with a binary mixture of AGB and Arg or another Arg analogue. A two-way ANOVA for unbalanced data determined overall significance for the odorant main effect, whereas a post hoc Tukey's multiple comparison of means test was used to investigate individual differences among the control and all binary mixtures.

Hierarchical cluster analysis was used to explore the patterns of cross-adapted responses to identify structural features of the Arg analogues that were important for determining OR specificity. A Pearson's product-moment correlation matrix was generated from the raw data and was then subjected to a hierarchical cluster analysis using the average linkage method. All statistical analyses were performed using SPSS for Windows 95 (Chicago, IL).

Solutions

The AFW solution was composed of the following (in mM): 3 NaCl, 0.2 KCl, 0.2 CaCl2, 1 HEPES, pH 7.2, in deionized water (>18 MOmega ). FR solution contained (in mM) 140 NaCl, 10 KCl, 1.8 CaCl2, 2 MgCl2, and 5 HEPES, pH 7.2. The fixative contained 1% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer (PB). PB was composed of (in mM) 64 NaH2PO4, 15 Na2HPO4, 24 CaCl2, and 167 glucose.

The structural formulas of the test odorants are presented in Fig. 1. L-Glutamine (100 µM) was used as the standard odorant. Compounds were prepared as 10-mM stock solutions in AFW and stored at 4°C until use. All compounds were purchased from Sigma-Aldrich (St. Louis, MO).



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Fig. 1. Structural formulas of Arg analogues tested for olfactory responsiveness in zebrafish. The test odorants included: L-arginine (Arg) and its enantiomer D-arginine (D-Arg); guanidine (GDN); aminoguanidine (AGDN); Arg analogues with alpha -amino deletions: L-argininic acid (AA), gamma -guanidinobutyrate (GBA), guanidoacetate (GDA), beta -guanidinopropionate (GPP), and guanidinosuccinate (GSC); reduction in side-chain length: L-alpha -amino-beta -guanidinopropionate (AGPA); alpha -carboxyl deletion: 1-amino-4-guanidobutane (agmatine; AGB); unblocked alpha -amino and esterified alpha -carboxyl group: L-arginine methyl ester (AME); and alpha -amino and alpha -carboxyl deletion: 1-ethylguanidine sulfate (EGS). L-Glutamine (Gln) was used as the standard odorant. The guanidinium group in each compound is shown in bold.


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DISCUSSION
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Structure-activity of Arg analogues

The 13 Arg analogues tested for their relative stimulatory effectiveness elicited olfactory responses ranging from negligible to highly stimulatory (Fig. 2). Statistical analysis indicated a significant odorant main effect (ANOVA, F = 60.322, df = 14, P < 0.001) and nonsignificant effects for comparisons between males and females (sex main effect) and for the interaction between odorant and sex. Consequently, data from male and female fish were pooled. In individual odorant comparisons, eight odorants exhibited significantly greater responses than the AFW control (Tukey's test, P < 0.05). The order of potency was AME > AGB > EGS > Gln > AGPA > Arg > aminoguanidine (AGDN) > guanidine (GDN) > AA. In evaluating structural properties conferring odorant potency, GDN was slightly stimulatory, whereas addition of an amino group (AGDN) more than doubled the potency. Addition of organic acids to GDN rendered the molecule a poor odorant (guanidoacetate [GDA], beta -guanidinopropionate [GPP], D-Arg, guanidinosuccinate [GSC], gamma -guanidinobutyrate [GBA], and AA). In contrast, the removal of the alpha -amino group in the presence of an unsubstituted alpha -carboxyl group generally rendered the analogue a weak odorant, as in AA versus Arg or GPP versus AGPA. For the more stimulatory odorants, slight differences in odor potency were noted when the number of carbons in the side chain was varied (Arg vs. AGPA). The three most stimulatory Arg analogues are characterized by a deletion (AGB) or esterification (AME) of the alpha -carboxyl group, or a deletion of both the alpha -amino and alpha -carboxyl groups (EGS).



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Fig. 2. Average (±SE) relative stimulatory effectiveness of Arg analogues (tested at 100 µM) and the artificial fresh water (AFW) control on the zebrafish olfactory organ. *Significant increase in the response to the Arg analogue compared with AFW (Tukey's post hoc multiple comparison of means, P < 0.05). The number of fish tested for each odorant is indicated above each bar.

Cross-adaptation experiment

We tested the five most potent Arg analogues and Gln in cross-adaptation experiments to determine whether they activate shared or distinct ORs. For all competitor odorants, self-adaptation was complete---i.e., competitor odorant responses in background were <10% of their unadapted responses (Fig. 3). The absence of cross-adaptation of test odorants in competitor odorant background was observed for 23% of tests (7 of 30), whereas test odorants were partially cross-adapted by competitor odorants in 77% of tests (23 of 30). No cross-adapted response was <40% of its unadapted response. Although most of the odorants exhibited approximately reciprocal cross-adaptation, nonreciprocal cross-adaptation was evidenced in four odorant pairs, Gln-Arg, AME-Arg, AME-AGPA, and AME-EGS, with the first of each odorant pair adapting the second to a greater extent (Fig. 3). The two-way ANOVA indicated significant differences for the competitor odorant main effect (F = 9.4, df = 5, P < 0.001), test odorant main effect (F = 3.0, df = 5, P < 0.05), and the interaction between them (F = 20.1, df = 25, P < 0.001). Individual one-way ANOVAs indicated significant differences among test odorant responses within each competitor odorant background (Fig. 3). In 90% of cases, the cross-adapted responses to test odorants were significantly greater than the self-adapted responses to the competitor odorant (Tukey's test, P < 0.05). There were only three instances in which cross-adaptation of test odorant responses was not significantly greater than self-adapted responses: AGPA in Arg background, and EGS and AME in AGB background (Tukey's test, P < 0.05). The absence of complete cross-adaptation among the Arg analogues tested suggests that they were detected by several ORs that in all instances have at least partially nonoverlapping ligand-binding specificities.



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Fig. 3. Cross-adaptation experiments suggest that several odorant receptors mediate the detection of Arg analogues. Representative electroolfactogram (EOG) recordings (top) and summary bar graphs (bottom) illustrate the effects of competitor odor background on responses to test odorants. For each panel, the competitor odorant in each experiment is indicated in the top left corner of each plot. All EOG recordings are from different preparations with unadapted responses shown in black (thick trace) and adapted responses in gray (thin trace). The bar graphs are plots of the average (±SE) percentage of unadapted responses to test odorants in competitor odorant backgrounds. Each of the five Arg analogues and Gln served as a test odorant in all trials and as the competitor odorant in three experiments with different fish. After several one-way ANOVAs, overall significance was displayed among test odorants in all competitor odorant backgrounds, Gln (F = 49.8, df = 5, P < 0.001), Arg (F = 13.8, df = 5, P < 0.001), AGPA (F = 96.6, df = 5, P < 0.001), AME (F = 10.9, df = 5, P < 0.001), AGB (F = 8.2, df = 5, P < 0.005), and EGS (F = 9.4, df = 5, P < 0.001). *Responses to the test odorant that did not differ significantly from the self-adapted response to the competitor odorant (Tukey's test, P < 0.05).

A hierarchical cluster analysis of a Pearson's correlation matrix, generated from the cross-adaptation data, should reveal the specific structural features of those odorants that are critical for OR activation. Linkage at small cluster distances indicates odorants most similar in terms of patterns of cross-adapted responses. Cluster analysis segregated the Arg analogues on the basis of their molecular structural features (Fig. 4). The first cluster contained unsubstituted alpha -carboxyl groups (Arg, AGPA, Gln), whereas the second cluster was characterized by odorants with deleted alpha -carboxyl groups (AGB and EGS), or an alpha -carboxyl group blocked by esterification (AME). The two clusters could also be distinguished by differences in odor potency, because the first cluster comprised the less potent odorants, Arg, AGPA, and Gln, and the second cluster the stronger odorants, AGB, EGS, and AME. It therefore appears that the alpha -carboxyl group confers properties on odorants that determine the populations of ORs with which they will interact.



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Fig. 4. Hierarchical cluster analysis was used to explore the patterns of cross-adapted responses to identify structural features of the Arg analogues conferring odorant quality. The results are presented as a dendrogram. Odorants linked at the smallest cluster distance evoked the most similar response patterns in the competition backgrounds. The percentage of unadapted responses to each test odorant in the competitor odorant backgrounds were used to generate a Pearson's product-moment correlation matrix. The correlation matrix was analyzed using the average linkage method of cluster analysis.

Activity-dependent labeling of ORNs

The cross-adaptation experiments suggest that several ORs with overlapping but at least partially distinct ligand-binding specificities mediate the detection of Arg analogues. If this interpretation is correct, then the intersection of the populations of ORNs stimulated by competitor-test odorant pairs may represent the cells contributing to the observed cross-adaptation. Expressed another way, a binary mixture of odorants that do not completely cross-adapt to each other should stimulate more ORNs than either odorant alone. To test this possibility, we used activity-dependent labeling techniques (Michel et al. 1999) to visualize ORNs stimulated with either AGB alone or a binary mixture of AGB and another Arg analogue. Labeling is confined to sensory epithelium located along the inner two-thirds of each lamella, as shown by taurine immunoreactivity (Fig. 5A). The activity probe AGB, as an odorant in zebrafish, stimulated and labeled 5% of the sensory epithelium when tested alone (Fig. 5B). If the other Arg analogues activated partially nonoverlapping populations of ORNs, each binary mixture was predicted to and indeed labeled more ORNs than AGB alone (Fig. 5, C-F). Most of the labeled ORNs were located in the apical portion of the olfactory epithelium, consistent with the activation of primarily microvillar receptor cells. Statistical comparisons of the amount of labeling in the olfactory epithelium after stimulation by different Arg analogues indicated a significant odorant main effect (ANOVA, F = 46.8, df = 4, P < 0.001; Fig. 6). For individual comparisons, there was a significant increase in the percentage of labeling by all binary odorant mixtures compared with AGB labeling alone (Tukey's test, P < 0.05). The percentages of labeling by AGB-AME and AGB-AGPA (13.7-14.2%) were significantly greater than those of labeling by AGB-EGS and AGB-Arg (8.2-9.2%). Nonsignificant differences in the percentage of labeling were evident between AGB-AME and AGB-AGPA and between AGB-EGS and AGB-Arg (Tukey's test, P < 0.05). The increase in activity-dependent labeling during binary odorant mixture stimulation compared with stimulation with AGB alone is consistent with the presence of ORs with nonoverlapping odorant specificities.



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Fig. 5. Odorant-activated labeling of zebrafish olfactory organs. A: taurine immunoreactivity (IR) of an olfactory rosette illustrates that the taurine-rich sensory epithelium extends from the raphe (r) approximately two-thirds of the way along each lamella (l). Scale bar, 100 µm. B-F: higher magnification views of labeling of ORNs stimulated with 5 mM AGB alone (B) or 5 mM AGB with the addition of one of the following 100 µM Arg analogues (C) Arg, (D) AGPA, (E) EGS, or (F) AME. Scale bars, 10 µm. The average percentage of labeling for each image was AGB control, 5%; binary mixtures of AGB and Arg, AGPA, EGS, or AME, 9.2%, 13.7%, 8.2%, and 14.2%, respectively. B-F: "l" is positioned to delineate the boundary between sensory and nonsensory regions. A: (*) approximate placement of the recording electrode during the EOG experiments.



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Fig. 6. Quantification of odor-stimulated immunostaining. Average (±SE) percentage of activity-dependent labeling of zebrafish olfactory organs after stimulation by 5 mM AGB or binary mixtures of AGB + 100 µM Arg analogues are plotted. For individual comparisons, different letters indicate significant differences in percentage of labeling among Arg and analogues (Tukey's test, P < 0.05). Numbers above histograms are the number of planes of sections analyzed, each comprising three to six regions of interest.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Numerous studies have demonstrated the potential of the fish olfactory system to discriminate among L-alpha -amino acids on the basis of differences in side-chain structure (Bruch and Rulli 1988; Cagan and Zeiger 1978; Caprio and Byrd 1984; Caprio et al. 1989; Holland and Teeter 1981; Little 1977; Michel and Derbidge 1997; Ohno et al. 1984; Sveinsson and Hara 1990; Valentincic et al. 1994, 1996; Zippel et al. 1993). In this study we demonstrated that the olfactory system has the potential to discriminate Arg analogues with identical side chains but modified alpha -amino and alpha -carboxyl groups. First, electrophysiological cross-adaptation experiments established that Arg analogues with substitutions to or deletions of the alpha -amino and alpha -carboxyl groups each activate at least a subset of ORs that are distinct from those activated by Arg. Then, activity-dependent labeling experiments confirmed that at least a portion of these ORs are distributed on distinct populations of ORNs. Consequently, the criteria necessary for odorant discrimination of multiple ORs with somewhat differing specificities that are expressed on at least partially nonoverlapping populations of ORNs have been met for these Arg analogues. Collectively, these studies suggest that the zebrafish olfactory system is adapted to detect and potentially discriminate among amino acid analogues with modifications to either the parent structure or the side chain. Whether zebrafish actually behaviorally discriminate among these odorants as has been established in the channel catfish for Arg, D-Arg, AME, and AGPA (Valentincic et al. 1994) is unknown.

The cross-adaptation experiments provided the first clue that the detection of these Arg analogues is mediated by multiple ORs. In such an experiment, the degree of competition among odorants is thought to reflect the extent of interaction with shared ORs (Caprio and Byrd 1984; Michel and Derbidge 1997). Had complete and reciprocal cross-adaptation been observed, we would have concluded that 1) a single OR, or 2) more than one OR with virtually indistinguishable ligand-binding properties mediated the detection of these Arg analogues. On the other hand, had no cross-adaptation been observed, we would have concluded that these odorants interact with distinct ORs. However, an intermediate level of partial and variable cross-adaptation was observed, leading to the conclusion that each of the Arg analogues tested must interact with several ORs having at least partially nonoverlapping specificities. Alternatively, ORs with relatively distinct ligand-binding properties might be subject to partial and variable cross-adaptation if they were coexpressed in a subset of ORNs. In this scenario, adaptation with one odorant would partially depolarize the cell and potentially desensitize other expressed ORs.

Emerging evidence suggests that detection of amino acids by fish may be more analogous to pheromone detection by the mammalian vomeronasal system than to general odorant detection by the main olfactory epithelium. In mammals, general odorant input is received by the main olfactory bulb from the ciliated receptor cells located in the main olfactory epithelium. Pheromonal input is received from microvillar receptor cells in the vomeronasal organ by the accessory olfactory bulb (Halpern 1987). Although fish have no vomeronasal organ or accessory olfactory bulb, a mixed population of ciliated and microvillar receptor cells, located in the main olfactory epithelium, are capable of detecting both general odorants and pheromones. Both OR and V2R-like receptors are expressed in fish olfactory receptor neurons (Barth et al. 1996, 1997; Byrd et al. 1996; Cao et al. 1998; Naito et al. 1998; Ngai et al. 1993a-c; Speca et al. 1999; Weth et al. 1996). The recently characterized goldfish Arg-Lys OR shares sequence homology with the mammalian V2R vomeronasal receptor family but not with the odorant receptor family expressed in the main olfactory epithelium (Speca et al. 1999). The apical position of V2R receptor labeling (Cao et al. 1998; Speca et al. 1999) and of Arg analogue-stimulated ORN labeling (current study) is consistent with microvillar receptor cell activation. Other similarities between the processing of amino acid input by fish and pheromones by mammals are evident at the level of the olfactory bulb. In the main olfactory bulb, individual odorants activate several glomeruli (Greer et al. 1981; Guthrie and Gall 1995; Guthrie et al. 1993; Johnson et al. 1998; Lancet et al. 1982), and all ORNs expressing a particular OR terminate in a single pair of glomeruli (Mombaerts et al. 1996; Ressler et al. 1994; Vassar et al. 1994), leading to the conclusion that most odorants are capable of activating several ORs. In the accessory olfactory bulb, ORs project to many small, and often poorly delineated, glomeruli (Belluscio et al. 1999; Rodriguez et al. 1999). Glomeruli in the accessory olfactory bulb may receive input from more than one type of receptor (Belluscio et al. 1999). In fish, amino acid input projects to a lateral chain of ~120 small and poorly defined glomerular modules (Friedrich and Korsching 1997). Each amino acid activates from 11 to 62% of the glomerular modules but it remains to be determined whether the axons of ORNs expressing the same OR innervate one or more glomeruli, whether glomeruli receive input from different ORs, or whether ORNs express multiple ORs. The latter remains a distinct possibility, because the goldfish Arg-Lys OR and several other members of the same receptor family, although not yet characterized (Cao et al. 1998; Speca et al. 1999), are expressed in sufficient numbers of ORNs to allow overlapping distributions to exist.

Significant increases in the proportion of activity-labeled ORNs for each of the Arg analogues confirm that at least a portion of the ORs activated by these odorants are distributed on nonoverlapping populations of ORNs. Owing to limited data on the functional properties of individual ORs and their expression patterns, only approximate estimates of the number of receptors activated by the Arg analogues can be made. It is likely that the labeling we observed reflects activation of members of the recently identified amino acid-sensitive OR family (Speca et al. 1999). The distribution of the V2R-like receptors has not been thoroughly characterized, but several members of this receptor family, including the Arg-Lys-sensitive OR, are expressed in a large proportion of the ORNs, whereas other uncharacterized members of this family are expressed in a relatively small number of the ORNs (Cao et al. 1998; Hirai et al. 1996; Speca et al. 1999). If activity-dependent labeling by the Arg analogues activates only those ORs expressed in the more restricted subsets of ORNs (1-5%) (Speca et al. 1999), as few as 1-3 to >9-10 ORs may be involved. Because Arg did not stimulate labeling of a large proportion of the ORNs, zebrafish may not express an Arg-sensitive OR similar to the goldfish Arg-Lys OR (Speca et al. 1999). Alternately, our labeling method may not be sensitive enough to detect ORNs expressing only a few copies of a particular OR.

There are only 20 amino acids commonly incorporated into proteins, but more than 150 other amino acids are known to occur biologically. In zebrafish, each of the common amino acid odorants activates an average of 33% of the ~120 glomerular modules in the amino acid-sensitive macroglomerular complex of the lateral chain of the olfactory bulb (range, 11% for L-aspartate to 62% for L-methionine) (Friedrich and Korsching 1997). However, through the use of a combinatorial coding scheme, these glomerular modules are presumed to be able to distinguish the common amino acids. Our findings indicate that the zebrafish olfactory system has the potential to distinguish Arg analogues (and probably analogues of other amino acids as well) with nonbiologic substitutions to or deletions of the alpha -carboxyl and alpha -amino groups.

In general, Friedrich and Korsching (1997) found that the extent of overlap of glomerular module activation increased with the structural similarity of odorants. The extent of the overlap of glomerular activation patterns could be predictive of the magnitude of cross-adaptation, but this comparison is impossible for the current study, because only Arg was common to both studies. However, comparison of data from an earlier cross-adaptation study (Michel and Derbidge 1997) in which six of the amino acids tested were also tested in the glomerular activation study of Friedrich and Korsching (1997) revealed only a weak correlation between these two parameters. As expected, activation of more shared glomeruli by two amino acids results in greater cross-adaptation, whereas activation of few or no shared glomeruli results in less cross-adaptation. Noteworthy is the correspondence between the basic amino acids, Arg and Lys, in their highly correlated patterns of glomerular activation (0.69) and greater reciprocal cross-adaptation (Arg = 35% unadapted in a Lys background, Lys = 31% unadapted response in a Arg background). The presence of an Arg-Lys OR similar to the goldfish OR (Speca et al. 1999) in a high proportion of zebrafish ORNs may account for this correlation. If amino acid-sensitive ORs innervated several glomerular modules and/or several ORs innervate the same glomerular module, then glomerular activation patterns would not simply reflect OR activation, and highly correlated patterns of glomerular activation might not be reflected in cross-adaptation studies. Surprisingly, cross-adaptation was observed among some stimuli with little or no correlation in their patterns of glomerular activation. For example, no correlation in patterns of glomerular activation was observed between L-glutamate (an acidic amino acid) and Arg, but there was some cross-adaptation (Arg = 81% unadapted response in an L-glutamate background; L-glutamate = 66% unadapted response in an Arg background). Although as yet unidentified glomeruli activated by both of these stimuli may account for the observed cross-adaptation, it may also reflect antagonistic receptor interactions (Cromarty and Derby 1998; Olson and Derby 1995). In vivo recordings from channel catfish of single ORN activity reveal that nonstimulatory amino acid odorants antagonize the responses of excitatory and suppressive odorants 66% of the time (Kang and Caprio 1997).

Cluster analysis has indicated that substitution in, or deletion of, the alpha -carboxyl group alters Arg sufficiently for it to interact with a relatively distinct subset of ORs. The importance of the alpha -carboxyl group in the OR discrimination of Arg analogues has been demonstrated in goldfish in which an intact alpha -carboxyl group is essential for high-affinity odorant binding to the expressed Arg-Lys OR (Speca et al. 1999). Further examination of the structural features of the other stimulatory and nonstimulatory Arg analogues provides additional insight into the structural aspects conferring odorant potency. Removal of the alpha -amino group renders the analogue a weak odorant (Caprio 1978), as in AA versus Arg or GPP versus AGPA in the present study. A similar loss of odor potency was noted after alpha -amino group modification to L-alanine in the channel catfish olfactory system (Caprio 1978). It is unknown why odor efficacy is maintained after deletion of the alpha -amino group in EGS, but it may be due to a deletion of the alpha -carboxyl group. The ligand-binding properties of amino acid-sensitive ORs are likely to vary across fish, because deletion (AGB) or esterification (AME) of the alpha -carboxyl group of Arg increased potency more than threefold for zebrafish, whereas esterification only slightly increased potency for channel catfish (Caprio 1978).

The relative stimulatory effectiveness of the Arg analogues established electrophysiologically does not appear to be in full agreement with the results of the activity-dependent labeling experiment. The magnitude of an odorant-evoked response measured electrophysiologically reflects the sum of ion flux through all ORNs sensitive to that odorant; however the same relationship may not exist during activity-dependent labeling experiments. Until all pathways mediating olfactory transduction in zebrafish are identified and the permeation properties of AGB through all odor-stimulated ion channels are defined, the relationship between electrophysiological response and AGB permeation cannot be fully established.

Our finding that the zebrafish olfactory system has ORs capable of detecting Arg analogues with structural modifications to the alpha -amino and alpha -carboxyl groups indicates that the olfactory system is adapted to detect both common amino acids and structural analogues that may not occur naturally. Whether this constellation of amino acid-sensitive ORs is used by the zebrafish in the detection of novel odorants or serves in the specific recognition of odorants not considered in the current investigation is unknown. Future studies, focusing on other synthetic analogues unlikely to be encountered by these fish, will undoubtedly reveal the fish olfactory system to have additional discriminatory potential.


    ACKNOWLEDGMENTS

We thank Signature Immunologics for the gift of the anti-AGB and anti-taurine antibodies, and F. Sahlolbei, D. Elkin, and J. Edwards for assistance.

This research was supported by National Institutes of Health Grants 2RO-DC-01418 and 5PO1-NS-07938.


    FOOTNOTES

Address reprint requests to D. L. Lipschitz.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 June 1999; accepted in final form 17 August 1999.


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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society