Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah 84108-1270
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ABSTRACT |
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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 -carboxyl and
-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
-carboxyl group yielded odorants more potent than Arg, whereas deletion of the
-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-
-amino-
-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
-carboxyl and
-amino groups.
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INTRODUCTION |
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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-
-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
-amino and
-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
-carboxyl and/or
-amino groups.
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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 M). 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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RESULTS |
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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], -guanidinopropionate [GPP],
D-Arg, guanidinosuccinate [GSC],
-guanidinobutyrate
[GBA], and AA). In contrast, the removal of the
-amino group in
the presence of an unsubstituted
-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
-carboxyl group,
or a deletion of both the
-amino and
-carboxyl groups (EGS).
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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
completei.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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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 -carboxyl groups (Arg, AGPA, Gln),
whereas the second cluster was characterized by odorants with deleted
-carboxyl groups (AGB and EGS), or an
-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
-carboxyl group confers properties on odorants that determine the
populations of ORs with which they will interact.
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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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DISCUSSION |
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Numerous studies have demonstrated the potential of the fish
olfactory system to discriminate among L--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
-amino and
-carboxyl groups. First,
electrophysiological cross-adaptation experiments established that Arg
analogues with substitutions to or deletions of the
-amino and
-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
-carboxyl and
-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 -carboxyl group alters Arg sufficiently for it to interact with
a relatively distinct subset of ORs. The importance of the
-carboxyl
group in the OR discrimination of Arg analogues has been demonstrated
in goldfish in which an intact
-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
-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
-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
-amino
group in EGS, but it may be due to a deletion of the
-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
-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 -amino
and
-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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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