Chem. Senses 26: 1145-1156,
2001
© Oxford University Press 2001
Whole-cell Response Characteristics of Ciliated and Microvillous Olfactory Receptor Neurons to Amino Acids, Pheromone Candidates and Urine in Rainbow Trout
Animal Behavior and Intelligence, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan 1 Current address: Fisheries, Neuroscience and Ecology Graduate Programs, University of Minnesota, 200 Hodson Hall, 1980 Folwell Avenue, St Paul, MN 55108-6124, USA
Correspondence to be sent to: Noriyo Suzuki, Animal Behavior and Intelligence, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. e-mail: suzuki{at}sci.hokudai.ac.jp
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Abstract |
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Olfactory lamellae of teleosts contain two morphologically different types of olfactory receptor neurons (ORNs): ciliated ORNs (cORNs) and microvillous ORNs (mORNs). However, little is known about the functional difference between these two types of ORNs in fish olfaction. We isolated cORNs and mORNs using a Ca2+-free solution method from olfactory organs of the rainbow trout and examined their response characteristics to various odorants including fish pheromone candidates by whole-cell voltage-clamp techniques. Quadruple mixture of amino acids, single amino acids, steroids (analogues of DHP; 17
, 20ß-dihydroxy-4-pregnen-3-one and ECG;
etiocholan-3-ol-17-one glucuronide), prostaglandins (PGFs) and urine
samples collected from immature and mature female fish were applied focally to
olfactory cilia or microvilli using a multi-barreled stimulation pipette with
a pressure ejection system. Inward current responses to odorants were recorded
from both cORNs and mORNs at a holding potential of -60 mV. cORNs responded to
the amino acid mixture, single amino acids, urine samples and ECG, whereas
mORNs responded specifically either to the amino acid mixture or single amino
acids. The response profiles of both cORNs and mORNs to various odorants
varied widely. None of cORNs and mORNs responded to fish pheromone candidates,
PGFs and DHPs. Androgen treatment of immature fish did not influence olfactory
sensitivity of both cORNs and mORNs to the amino acid mixture and both urine
samples. Amino acid and bile acid analyses by HPLC showed that both urine
samples contained 35 amino acids (1-40 mM) and trace amounts of taurocholic
acid and glycoursodeoxycholic acid. Our results suggest that cORNs are
`generalists' that respond to a wide variety of odorants, including
pheromones, whereas mORNs are `specialists', specific to amino acids, and also
suggest that PGFs and DHPs are not pheromones for the rainbow trout. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In amphibians, reptiles and mammals, there are two types of olfactory organs: the main olfactory organ and vomeronasal organ. The main olfactory organ contains ciliated olfactory receptor neurons (cORNs) and detects a variety of general odorants, whereas the vomeronasal organ has microvillous olfactory receptor neurons (mORNs) and detects pheromones that play important roles in the territorial defense, reproductive behavior and synchronism of sexual maturation (Farbman, 2000
; Johnston,
2000). In teleosts, which do not have vomeronasal organs, both
cORNs and mORNs are distributed in the olfactory epithelium
(Yamamoto and Ueda, 1977;
Zielinski and Hara, 1988;
Eisthen, 1992;
Zeiske et al., 1992).
Cloning of fish odorant receptor genes has been performed in the catfish
(Ngai et al., 1993),
zebrafish (Barth et al.,
1996) and goldfish (Cao et
al., 1998), and showed that the gene families of odorant
receptors in these teleosts consisted of 100-300 G-protein-coupled receptor
genes. In the goldfish, functional expression of odorant receptor genes has
been reported by Speca et al.
(Speca et al., 1999).
However, the distinct distribution of these odorant receptors between cORNs
and mORNs was not investigated. Moreover, patch-clamp studies of isolated ORNs
of channel catfish and rainbow trout
(Restrepo et al.,
1990; Miyamoto et
al., 1992; Ivanova and
Caprio, 1993; Sato and Suzuki,
2000) have recorded the responses to amino acids only from cORNs.
Another patch-clamp study on the responses of salmon ORNs to imprinted
odorants (Nevitt et al.,
1994) also did not distinguish between two types of ORNs in
respect of their functional differences.
The urine of mature females is a potent odorant in the Atlantic salmon
(Salmo salar) (Moore and Scott,
1992), and the major source of primer and releaser pheromones for
the rainbow trout (Oncorhynchus mykiss), which regulate male plasma
levels of gonadotrophin II (Scott et
al., 1994) and play important roles in male attraction
(Yambe, 2001). However, the
pheromones present in the urine of mature female rainbow trout have not yet
been identified. F-type prostaglandins (PGFs) and several steroids such as
17,20ß-dihydroxy-4-pregnen-3-one (DHP) and DHP 20-sulphate (DHP-s)
have been identified as pheromones in several species of fishes, which
regulate mate attraction and spawning synchrony
(Stacey et al., 1994;
Sorensen and Caprio, 1998).
The detection of PGFs and DHPs by the olfactory organ of the goldfish
(Carassius auratus) (Sorensen et al.,
1988,
1991) leads to the production
of sperm (Dulka et al.,
1987) and sexual behavior (Sorensen et al.,
1988,
1989). The sensitivity of
olfactory organ to pheromones is enhanced during the fish spawning season. In
the tinfoil barb (Puntius schwanenfeldi), androgen treatment
increases the sensitivity of ORNs to 15-keto prostaglandin
F2 (15KPGF), as measured both by electro-olfactograms (EOGs)
and the frequency of courtship behavior
(Cardwell et al.,
1995). In Atlantic salmon, the sensitivity of ORNs to PGFs as
measured by EOG responses and the level of expressible milt increase as the
reproductive season progresses (Moore and
Waring, 1996). PGFs are also potent odorants for Arctic char
(Salvelinus alpinus), lake trout (Salvelinus namaycush),
brown trout (Salmo trutta) and lake whitefish (Coregonus
clupeaformis) (Hara and Zhang,
1998; Sveinsson and Hara,
2000). Similar hormonal influence on the sensitivity of isolated
ORNs to odorants by a direct application of adrenaline has been noted in the
newt (Kawai et al.,
1999).
In the present study, we isolated cORNs and mORNs from the olfactory organ
of rainbow trout with a Ca2+-free solution method and examined for
the first time their response characteristics to the quadruple mixture of
amino acids, single amino acids, pheromone candidates such as PGFs and
steroids, and urine samples collected from immature and ovulated female fish,
using whole-cell voltage-clamp techniques. We also studied the effect of
androgen treatment on the sensitivity of ORNs to the amino acid mixture and
urine samples. Amino acid and bile acid analyses of urine samples were also
performed by HPLC. Our results showed that cORNs responded to the amino acid
mixture, single amino acids, urine samples and etiocholan-3-ol-17-one
glucuronide (ECG), whereas mORNs responded specifically either to the amino
acid mixture or single amino acids.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fish and androgen treatment
Rainbow trout (fork length: 17-20 cm; weight: 60-90 g) were obtained from a
local fishery and maintained on a 12 h light/dark cycle in an Aqualex water
circulating tank (AR18-300-10, NK System, Osaka) at 
15°C. The fish
were fed daily with goldfish food pellets and used for experiments <1 month
after transportation from the local fishery.
To examine the effect of androgen treatment on the current responses of
ORNs, anaesthetized (0.02% 2-phenoxyethanol) rainbow trout were injected i.p.
with 17-methyltestosterone (MT) (231-02, Nacarai Tesque, Kyoto) or
11-keto androstendione (11KA) (28, 499-8, Aldrich Chemicals, Milwaukee, WI).
These steroids were dissolved in 50% ethanol-standard Ringer's solution [in
mM: NaCl (100), KCl (3), CaCl2 (2), MgCl2 (1), D-glucose
(10), HEPES (5) and NaOH (2.2), pH 7.4], and were injected into the test fish
at a dose of 5.0 µg/g body wt. Three days after the first injection, an
additional injection of 1.0 µg/g body wt was administered. Two days after
the second injection, body color of androgen-treated fish became obviously
darker than before and persisted for another 13-15 days. The test fish were
not fed during treatment and were used for experiments 3-21 days after the
second injection.
Procedure for isolating ORNs and whole-cell voltage-clamp recording techniques
Cell isolation procedure with Ca2+-free solution method and whole-cell voltage-clamp recordings were performed as follows. The fish were killed by quick decapitation with a knife. Olfactory rosettes were first dissected out from both olfactory organs and stored in standard Ringer's solution on ice. For one series of electrophysiological experiments, olfactory epithelial tissues were collected from olfactory lamellae isolated from one rosette and incubated in Ca2+-free Ringer's solution [in mM: NaCl (100), KCl (3), MgCl2 (1), 10 D-glucose, HEPES (5) and NaOH (2.2), pH 7.4] on ice for 1 h. After incubation, olfactory epithelial cells were dissociated by sucking them in and out of a fire-polished Pasteur pipette with a tip opening of 0.2 mm. The epithelial cell suspension was filtered using a nylon mesh (40 µm mesh size) and 0.5-0.7 ml of final cell suspension was obtained. The cell suspension was plated onto a concanavalin A (C2010 Type IV; Sigma, St Louis, MO) coated coverglass that was inserted into a perfusion chamber attached to the stage of an inverted phase-contrast microscope (Diaphot TMD, Nikon, Tokyo), and was allowed to stand for 20 min until the cells became attached to the surface of the coverglass. The standard Ringer's solution was then flown at a rate of 1.0 ml/min through the test chamber and the cells were examined microscopically before the experiment to distinguish two types of ORNs in the electrophysiological preparation. In the present study, only cORNs bearing more than four cilia, each >3 µm long and mORNs bearing clearly identifiable microvilli were selected for electrical recording. Isolated ORNs were also separately examined with trypan blue staining. A small amount of the final epithelial cell suspension (25-50 µl) was transferred to a glass tube and mixed with the same volume of 0.1% trypan blue dissolved in standard Ringer's solution. After 5 min incubation at room temperature, trypan bluestained cell suspension was transferred to a hemocytometer and examined under a Nomarski differential interference contrast microscope (BX50WI, Olympus, Tokyo).
A standard whole-cell voltage-clamp technique
(Hamill et al., 1981)
was used to record the current responses of cORNs and mORNs. For fabrication
of recording pipettes, thick-walled borosilicate glass tubes (1.5 mm o.d., 1.2
mm i.d.; G-1, Narishige, Tokyo) were pulled using a micropipette puller (PD-5,
Narishige, Tokyo). The pipette resistance was 8-12 M
, when filled with
K+-internal solution [in mM: KCl (93), EGTA-2K (5), HEPES (5),
ATP-2Na (1.0), GTP-Na (0.1) and KOH (2.26), pH 7.4]. The recording pipette was
connected via an Ag—AgCl wire to the headstage of a patch-clamp
amplifier (CEZ-2200, Nihon Kohden, Tokyo). The reference electrode was an
Ag—AgCl plate immersed in the bath solution. Tight seals (>1
G) were established by applying a small negative pressure to the
recording pipette after contact with the cell soma surface. Rupture of the
membrane for whole-cell mode recordings was achieved either by application of
negative pressure or zapping voltage pulses (±1.0 V, 1-10 ms) to the
recording pipette. Whole-cell recordings from ORNs usually lasted 10-20 min.
The recording was discontinued when a high seal resistance could not be
maintained properly or deterioration of the ORN responses to odorants became
evident. At each end of the recordings, voltage-gated channel currents to
stepping voltage pulses (up to ±140 mV, 25 ms) were obtained to confirm
the proper whole-cell recordings. Current signals were low-pass-filtered at 3
kHz and stored on the magnetic tape of a PCM data recorder (PCM-501ES, Sony,
Tokyo; DC-13 kHz bandwidth) for later off-line analysis. Current and voltage
data were digitized at 1 kHz sampling speed and analyzed using PowerLab (AD
Instruments, Mountain View, CA) on a Power Macintosh computer and DataSponge
(WPI, Sarasota, FL) on an IBM PC-AT compatible computer. The current data were
further processed for presentation using graphics software, Canvas 6.0
(Deneba, Miami, FL).
Application of odorants by multi-barreled stimulation pipette
Odorants were applied focally to olfactory cilia or microvilli of an ORN
using a three-, four- or seven-barreled stimulation glass pipette (each tip
opening diameter 1.0 µm). A barrel of three, four or seven glass
capillaries (G-1.2-filament; Narishige, Tokyo) fixed on both ends with
heat-shrinkable polyolefin tube (FP-301; MMM, Austin, TX) was pulled with a
programmable pipette puller (PMP-100; WPI). Each of the barreled stimulation
pipettes filled with different odorants was connected via a silicon rubber
tube to a custom-built pressure ejection system
(Sato and Suzuki, 2000). The
tip of the barreled pipettes was always positioned 20 µm away from the
cilia or microvilli of ORNs using a hydraulic micromanipulator (MB-PP2;
Narishige). In all experiments, the stimulus was delivered to ORNs at an
ejection pressure of 1.0 kgf/cm2 and a pressure pulse duration of
25 ms. To examine the whole-cell responses of cORNs and mORNs to various
odorants, experiments were performed as in the following procedure. After the
transition to the whole-cell recording configuration, a quadruple amino acid
mixture [1.0 mM: L-Glu, L-Arg, L-Ala and L-Nva; these four amino acids are
representative amino acids for the four independent amino acid receptor sites
of ORNs in the channel catfish (Caprio and
Byrd, 1984)] was applied first to an ORN, to determine the
presence of any response. Then, 2-6 different odorants were successively
applied to the ORN in a random order at 30-60 s intervals. The amino acid
mixture was frequently applied to check the deterioration of current response
during the period of odorant response recording. If the deterioration became
obvious, the recorded data were excluded from analysis.
Odorants
Eight amino acids—L-Ala, L-Arg, L-Glu, L-Nva, L-Thr, L-Met, Gly and
taurine (Nacarai Tesque)—were used for odorants. The first four of these
amino acids were used as a quadruple mixture and dissolved in standard
Ringer's solution (1 mM, pH 7.6). Other amino acids dissolved in standard
Ringer's solution (1 mM, pH 7.3-7.4) were used as single amino acid odorants.
The quadruple amino acid mixture was also used at higher concentrations (10
mM, pH 7.6) to determine the dose-dependency, current—voltage
relationship and reversal potential for responses of mORNs. The following
steroids were used for odorants as pheromone candidates: DHP (P6285, Sigma),
DHP 20-acetate (DHP-a) (P1664, Sigma), DHP-s (a gift from Dr Peter W.
Sorensen, University of Minnesota) and ECG (E 8000, Sigma). PGF and its
analogs: PGF (163-10831; Wako, Osaka), 15KPGF (K 0127; Sigma), dPGF
(39746-23-1; Cayman Chem, Ann Arbor, MI) and U-46619 (56985-40-1, Cayman Chem)
were also used as pheromone candidates. Steroids and PGFs were dissolved in
0.5% ethanol—standard Ringer's solution (U-46619 was dissolved in 0.35%
methylacetate—standard Ringer's solution) to a final concentration of
0.1 mM. Urine of immature rainbow trout was collected in February 2000 from
immature fish (GSI: 0.56 ± 0.30) without distinction of sex. A plastic
pipette tip (200 µl; Gilson, Villers, France) was directly inserted into
the bladder of each fish to collect the urine. The fish were autopsied to
check the correct placement of the pipette tip within the bladder. Urine of
mature female fish (a gift from Dr Hidenobu Yambe, Hokkaido University) was
collected in May 1999 from ovulated female rainbow trout. Urine samples of
immature and mature female fish were diluted to 20 times with standard
Ringer's solution, to a final millimolar-concentration range for their major
amino acid components. Ten-times concentrated stock solutions of amino acids
and other odorants were prepared before experiments and stored at 4C or
-80°C. The stock solutions were diluted with standard Ringer's solution to
the final concentration just prior to the experiments. Once diluted, test
solutions were stored at 4°C and subsequently used within 3 days. The
stimulus concentration at the target was estimated, by measuring liquid
junction potentials, to be diluted by at least a factor of 10 compared with
the concentration in the stimulating pipette liquid. Therefore, the actual
concentrations for amino acid stimuli at ORNs, when stimulated with 1 mM
solution in a stimulation pipette, were of the order of 0.1 mM, which was
slightly higher than the concentration ranges for amino acids to yield the
half-maximal olfactory responses in the rainbow trout
(Hara, 1982;
Evans and Hara, 1985).
Depending on the number of barreled stimulation pipettes used in different
experiments, three, four, or seven odorants were tested in each ORN. Odorants
except ECG, dPGF and U-46619 were tested for ORNs isolated from non-treated
fish. The amino acid mixture, urine samples and PGFs were tested for ORNs
isolated from MT-treated rainbow trout. The amino acid mixture, L-Met, dPGF,
U-46619, ECG and urine samples were tested for ORNs isolated from 11KA-treated
fish.
Analyses of urine samples for amino acids and bile acids
Urine samples collected from the rainbow trout were subjected to free-form amino acid and bile acid analyses. For amino acid analysis, a 150 µl of urine sample was directly loaded on an amino acid analyzer (L-8500; Hitachi, Tokyo) without pretreatment for hydrolysis and protein removal. Amino acid analysis was performed at the Center for Instrumental Analysis, Hokkaido University. For bile acid analysis, bile acids were first extracted from 700 µl of urine samples in the same volume of methanol with Bilepak II cartridge (JASCO, Tokyo). The extraction was loaded on a bile acid analysis system (LCSS-905, JASCO). Bile acid analysis was performed at the Analysis Center of JASCO Engineering Co. Ltd.
Statistical analysis
All data are expressed as mean ± SD. Differences between groups were examined for statistical significance using analysis of variance (ANOVA), two-way factorial ANOVA and Student's t-test. A P value <0.01 denoted the presence of a statistically significant difference.
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Results |
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Morphology of cORNs and mORNs and the ratio in which they are found
Microscopic examination of isolated olfactory epithelial cell preparation for electrophysiological study confirmed that there were two types of morphologically distinct ORNs, which had either olfactory cilia or microvilli on the olfactory knob (Figure 1). Morphologically, both types of isolated ORNs were bipolar neurons with a slender dendrite (cORN, 19.0 ± 8.50 µm long, n = 20; mORN, 16.6 ± 4.24 µm long, n = 20), a cell soma (cORN, 12.5 ± 2.44 µm major axis, 7.03 ± 1.39 µm minor axis, n = 20; mORN, 13.3 ± 2.93 µm major axis, 6.44 ± 1.56 µm minor axis, n = 20) and often, an initial segment of axon (<1 µm diameter, 3-20 µm long). There were no significant differences in these dimensions between cORNs and mORNs (two-way factorial ANOVA: P > 0.1). cORNs had 2-10 cilia (3.0-10 µm long) on their knob, whereas mORNs had numerous microvilli (1.0-2.0 µm long) on their knob. mORNs were clearly distinguishable by their characteristic lump structure of short and thin microvilli from deciliated cORNs, which had short broken cilia. The trypan blue exclusion test showed that 617 000 ± 20 100 (n = 16) epithelial cells were isolated from one rosette. Of these epithelial cells, 45 000 ± 26 600 ORNs were distinguished (7.3% of total epithelial cells). Of total ORNs, the number of cORNs was 11 000 ± 4300 (24.4%; viability 97.2%), that of mORNs was 1000 ± 269 (2.2%; viability 100%), and that of unidentified ORNs was 33 000 ± 22 600 (73.4%; viability 46.6%). Thus, the ratio in which mORNs to cORNs were found in the preparations was 1:11. The majority of isolated ORNs in electrophysiological preparations became round within 3 h of continuous flow of bath solution and the morphological characteristics of the olfactory knob structures of the two types of ORNs became indistinguishable.
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Whole-cell current responses and reversal potential of mORNs to the amino acid mixture
The inward current responses to the amino acid mixture occurred in mORNs at a holding potential of -60 mV. The magnitude of the peak inward current response to the amino acid mixture increased with higher stimulus ejection pressure (Figure 2A). We determined the current peak-voltage relationship (I-V relationship) for stimulation with the amino acid mixture by varying the holding potential between -60 and +40 mV (Figure 2B). The time-course of the rising phase of the responses did not change at different voltages. However, a significant prolongation of the response decay time was noted at positive voltages. The I-V curve obtained under this condition (Figure 2C) showed a marked outward rectification. The reversal potential determined for this and other mORNs in the same condition was +6.0 ± 1.73 mV (n = 3).
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Response profiles of cORNs and mORNs to amino acids, steroids, PGFs and urine samples
Whole-cell current responses of cORNs held at -60 mV to any one of odorants tested were obtained from 44 of 175 (25.1%) cORNs (Figures 3A and 4A-D). Although the response profiles varied markedly from one cORN to another cORNs could be roughly divided into three groups based on their response characteristics to the amino acid mixture, single amino acids and urine samples: cORNs that responded only to amino acids, to either the amino mixture or single amino acids, and to either or both of them (group I in Figure 3A; Figure 4A); cORNs that responded to either or both urine samples (group II in Figure 3A; Figure 4B,C); and cORNs that responded to either or both of the amino acid mixture and single amino acid and either or both urine samples (group III in Figure 3A; Figure 4D). Since cORNs of Figure 3A, no. 1-12 were not tested with urine samples, these cORNs were excluded from this classification. The percentages of groups I-III were 21.9, 31.3 and 46.9% (n = 32) respectively. None of the cORNs responded to DHP (85/175 cORNs), DHP-a (80/175 cORNs), DHP-s (29/175 cORNs), PGF (42/175 cORNs), 15KPGF (30/175 cORNs) and taurine (27/175 cORNs).
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Whole-cell current responses of mORNs held at -60 mV were obtained from 20/70 (28.6%) mORNs (Figure 3B). Figure 4E,F show typical inward current responses of two mORNs. These mORNs responded only to the amino acid mixture or a single amino acid. None of mORNs responded to both urine samples (54/70 mORNs), DHP (33/70 mORNs), DHP-a (33/70 mORNs), DHP-s (11/70 mORNs), PGF (10/70 mORNs), 15KPGF (8/70 mORNs) and taurine (9/70 mORNs).
All current responses recorded from both types of ORNs were phasic inward currents (current peak levels: 1.4-187.0 pA) at a holding potential of -60 mV. Outward current responses were not observed in the present experiments. There was no significant difference between the mean peak inward current responses of cORNs (43.2 ± 40.7 pA, n = 32) and mORNs (48.1 ± 55.8 pA, n = 16) to the amino acid mixture at a holding potential of -60 mV (t-test: P > 0.1).
Response profiles of cORNs and mORNs from androgen-treated fish
Whole-cell current responses of cORNs isolated from 11KA-treated fish to any of odorants tested were obtained from 18/33 (54.5%) cORNs (Figures 3C and 5A,B). The response profiles of cORNs isolated from 11KA-treated fish varied widely, as observed in those of cORNs isolated from the control fish. However, they could be roughly divided into three groups by their response characteristics to the amino acid mixture, a single amino acid and urine samples, as classified in cORNs isolated from the control fish: cORNs that responded to either the amino mixture or a single amino acid, and or both of them (group I in Figure 3C), cORNs that responded to either or both urine samples (group II in Figure 3C) and cORNs that responded to either the amino acid mixture or a single amino acid and one or both urine samples (group III in Figure 3C). The percentages of groups I, II and III were 44.4, 27.8 and 27.8% (n = 18) respectively. None of cORNs (n = 33) isolated from 11KA-treated fish responded to dPGF or U-46619 (Figure 3C). Figure 5 shows examples of current responses of cORNs of group III to ECG. One cORN responded strongly to ECG and weakly to the amino acid mixture and urine of mature female (Figure 5A). Another cORN responded strongly to the amino acid mixture and urine of immature fish but weakly to ECG (Figure 5B).
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Whole-cell current responses of mORNs isolated from 11KA-treated fish to the amino acid mixture or L-Met were obtained from 3/5 (60%) mORNs (Figure 3D). One mORN responded specifically to the amino acid mixture and two mORNs responded specifically to L-Met. None of these mORNs (n = 5) responded to dPGF, U-46619, ECG and both urine samples.
Whole-cell current responses of cORNs isolated from MT-treated fish to any of the odorants—amino acid mixture and urine samples—were obtained from 16/40 (40%) cORNs. Only the amino acid mixture elicited inward current responses in 6/22 (27.2%) mORNs (data not shown). None of cORNs and mORNs isolated from MT-treated fish responded to PGF and 15KPGF. In androgen-treated fish, cORNs responded to multiple odorants, whereas mORNs responded specifically either to the amino acid mixture or to a single amino acid (L-Met), as observed in the control fish.
All current responses of ORNs isolated from androgen-treated fish were inward currents (current peak levels: 3.3-192.5 pA for ORNs from 11KA-treated fish, 2.2-106.2 pA for ORNs from MT-treated fish) at a holding potential of -60 mV. No outward current responses were observed.
Figure 6 compares the magnitudes of inward current responses of cORNs isolated from the control fish and androgen-treated fish to the amino acid mixture and both urine samples at a holding potential of -60 mV. Androgen treatment had no significant effect on whole-cell current responses of cORNs to the amino acid mixture and both urine samples.
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Amino acids and bile acids in urine samples
Table 1 shows the concentrations of amino acids, amino group substances and bile acids found in urine samples collected from immature and mature female fish. The composition of urinary amino acids varied between immature and mature female fish. Both urine samples contained mainly urea, taurine and ammonia (> 100 mM) together with 35 amino acids (1-40 mM). Phosphoethanolamine and carnosine were not detected in urine of immature fish but in urine of mature females. The number of amino acids and their concentrations in urine samples of mature female fish tended to be higher than those of immature fish. The results of bile acid analysis indicated that both urine samples contained a small amount of glycoursodeoxycholic acid (GUDCA) and taurocholic acid (TCA). Other bile acids (tauroursodeoxycholic acid, ursodeoxycholic acid, glycocholic acid, cholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, glycolithocholic acid, taurolithocholic acid and lithochenocholic acid) were not detected.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Functional differences between cORNs and mORNs
In the present study, we isolated two different types of ORNs—cORNs
and mORNs—from the olfactory lamellae of rainbow trout and examined for
the first time their response properties to various odorants. The previous SEM
study of the surface structure of olfactory lamellae of several salmonid
species, including rainbow trout
(Thommesen, 1983), showed that
the density of cORNs increased towards the peripheral margin of each lamella,
whereas that of mORNs increased towards the central margin and the
distribution ratio of mORNs to cORNs was 1:1.2-4.0. This distribution ratio is
2-10 times larger than the ratio of mORNs to cORNs found in our study in
epithelial cell preparations for electrophysiology. The lower ratio of mORNs
to cORNs in our study is probably due to underestimation of the number of
isolated mORNs; these olfactory knob structures might be more easily damaged
than cORNs during Ca2+-free solution cell isolation procedure,
resulting in underestimation of the number of mORNs by microscopic
examination. The present patchclamp recordings showed that the response
characteristics to the amino acid mixture for mORNs such as the
dose—response dependence and the reversal potential of
I—V relationship were essentially similar to those of cORNs, as
demonstrated in our previous study (Figures
1 and
2 in
Sato and Suzuki, 2000).
Comparison between peak values of inward current responses of cORNs and mORNs
to the amino acid mixture also showed that there was no significant difference
in responsiveness between these two types of ORNs at least to the four amino
acids, L-Glu, L-Arg, L-Ala and L-Nva in the mixture. However, cORNs responded
to several different species of odorants—amino acid mixture, other
single amino acids, urine samples and ECG—whereas mORNs responded
specifically to either the amino acid mixture or other single amino acids.
This specific response of mORNs to amino acids in the present study is
consistent with the previous finding of the higher amplitude of EOG
(electro-olfactogram) response to amino acids in the central margin of the
lamella, where mORN distribution density is higher than in the peripheral
margin (Thommesen, 1983).
Similar specific responses of mORNs to amino acids have been noted in the
functional expression study of goldfish odorant receptors
(Speca et al., 1999)
and in the labeling study of zebrafish ORNs with activity-dependent
ion-channel-permeant probes (Lipschitz and
Michel, 1999). The small population (25-29%) of ORNs responded to
any one odorant tested in the present study. Since the trypan blue exclusion
test showed that most of isolated ORNs were viable (viability 97-100%), the
rest of the population of ORNs (71-75%), which did not respond to any of
odorants we tested, might have responded to other odorants we did not test.
Thus, the basic difference of response characteristics between cORNs and mORNs
was the response selectivity to different species of odorants. Therefore,
cORNs of the rainbow trout might be termed `generalists', which respond to
various species of odorants, whereas mORNs might be called `specialists',
which respond specifically to amino acids.
Natural olfactory stimuli for fish, such as odorants from river water and
urine, are themselves odorant cocktails. Masu salmon can discriminate the
composition difference of amino acids dissolved in different river waters
(Shoji et al., 2000).
Analyses of amino acids and bile acids of urine samples of immature and mature
female rainbow trout in the present study showed that these samples contained
many amino acids together with trace amounts of two bile acids. Some steroids
and their metabolites have also been identified in the urine of mature female
rainbow trout (Scott and Liley,
1994). Urine from ovulated female Atlantic salmon also contained
large quantities (18 ng/ml) of immunoreactive PGFs
(Moore and Waring, 1996).
Thus, the wide variety of cORNs and mORNs which have not only different
responsiveness to different species of odorants but also many different types
of amino acid receptor sites (see following Discussion) may help fish to
detect a particular odorant of biological significance among many natural
odorants at the peripheral level of the olfactory system.
Amino acid receptor sites and odorant receptors
Amino acid receptor sites in channel catfish ORNs have been classified into
four independent receptor sites: the sites for acidic amino acids, basic amino
acids, short-chained neutral amino acids and long-chained neutral amino acids
(Caprio and Bryd, 1984). The amino acid mixture used in the present study
contained L-Glu, L-Arg, L-Ala and L-Nva, each of which is the representative
for these four independent receptor sites. In our study, we identified various
types of cORNs and mORNs with regard to amino acid receptor sites. One type of
cORN (Figure 3A, no. 39), for
example, responded to L-Met, one of the long-chained neutral amino acids, but
not to an amino acid mixture containing L-Nva for this site. Another cORN
(Figure 3A, no. 19) responded
to L-Thr, one of the short-chained neutral amino acids, but not to the amino
acid mixture that contained L-Ala for this site. Other cORNs
(Figure 3A, nos. 15-18)
responded not only to the amino acid mixture but also to other single amino
acids common for the four amino acid receptor sites. On the other hand, some
mORNs (Figure 3B, nos. 13-16)
responded only to the amino acid mixture for the four amino acid receptor
sites. Other mORNs responded to a single amino acid, Gly
(Figure 3B, no. 17), L-Thr
(Figure 3B, no. 18) and L-Met
(Figure 3B, no. 19-20), but not
to the amino acid mixture. Thus, the response profiles of cORNs and mORNs to
different amino acids for four independent amino acid receptor sites varied
greatly from one ORN to another. Although amino acid receptor sites for
different groups of amino acids are not always rigidly determined when
stimulated by rather high concentrations of amino acid stimuli
(Hara, 1976;
Hara, 1982), it seems that
individual ORNs of the rainbow trout may have much more complex combination of
multiple amino acid receptor sites than those of the channel catfish
(Kang and Caprio, 1995).
Gene cloning studies of G-protein-coupled odorant receptor have shown that
individual ORNs express only one of a family of 100-300 genes in the channel
catfish, zebrafish and goldfish (Ngai
et al., 1993; Barth
et al., 1996; Cao
et al., 1998) and 1000 genes in the rat
(Buck and Axel, 1991).
Furthermore, recent studies of functional expression of mouse odorant receptor
(Malnic et al., 1999;
Touhara et al., 1999)
have shown that one odorant receptor detects multiple species of odorants,
and, conversely, one odorant is detected by multiple odorant receptors. The
present results showed that individual cORNs responded to different species of
odorants, amino acids, ECG and urine samples, and that individual cORNs and
mORNs responded to different amino acids for different amino acid receptor
sites. Therefore, one odorant receptor of an individual ORN in the rainbow
trout could detect multiple odorants of different species as in the rat and
mouse. In addition, since electrophysiological properties, such as
I-V relationship and reversal potential for the amino acid mixture,
were in this study essentially similar both in cORNs and mORNs, different
types of odorant receptors might be equipped within the transduction machinery
of individual cORNs and mORNs, which have similar efferent ion channels for
the transduction machinery.
Responses of ORNs to pheromone candidates and urine samples
PGFs and DHPs have been identified as pheromones for several fish species
(Stacey et al.,
1994). However, neither cORNs nor mORNs of rainbow trout responded
to these pheromone candidates. U-46619 and dPGF which elicited the bulbar
responses of Arctic char (Sveinsson and
Hara, 2000) were also ineffective on the rainbow trout. In rainbow
trout (Scott et al.,
1994) and Atlantic salmon
(Waring et al.,
1996), DHP-s had only a small priming effect on their steroid
level. An EOG study of rainbow trout has shown that the olfactory epithelium
does not respond to PGFs (Hara and Zhang,
1998). Together with these previous results, it could be concluded
that PGFs and DHPs are not pheromones for the rainbow trout. In the African
catfish (Clarias gariepinus), ECG was identified as a pheromone by
behavioral observation and recordings of EOG and olfactory tract activities
(Resink et al.,
1989). Small EOG responses to ECG have also been recorded in
several salmonid species (Hara and Zhang,
1998). In the present study, ECG induced inward current responses
in two cORNs. However, this substance may not be a pheromone for the rainbow
trout, because cORNs responsive to ECG did not show a specific response to
this substance; ECG-responsive cORNs also responded to the amino acid mixture
and both urine samples. These cORNs did not show a specific response similar
to that of mORNs in the mouse vomeronasal organ, which showed specific
responses to putative pheromones
(Leinders-Zufall et al.,
2000). Moreover, putative pheromones induced responses in only a
particular subset of mORNs (0.2-3% of total mORNs) in the epithelium of
vomeronasal organ (Leinders-Zufall,
2000). If there is a similar situation for the specific responses
of a small population of ORNs, the number of ORNs recorded in our study might
not be sufficient to detect the responses of many ORNs to pheromones. On the
other hand, we recorded specific current responses to urine of mature female,
which should contain unidentified substances as pheromones
(Scott et al., 1994;
Yambe 2001), from two cORNs
(Figures 3A, no. 26;
3C, no. 11;
4C). As mentioned above, urine
from mature females contained many amino acids and trace amounts of two bile
acids. Since we diluted urine samples 20-fold for stimulation, the
concentration ranges of bile acids in urine samples should be close to the
rainbow trout's olfactory threshold
(Døving et al.,
1980), whereas those of amino acids in urine samples were similar
to those of amino acid mixture and single amino acids tested in our study.
Therefore, amino acids in urine samples should be the main contributors to
inward current responses of cORNs to urine samples. However, these cORNs
responded to only urine of mature female. The specific components for urine of
mature female, phosphoethanolamine and carnosine, might also contribute to the
specific responses of cORNs to urine of mature female. In any case, the
present results suggest that cORNs may serve as pheromone reception neurons in
the rainbow trout, unlike in the rat and mouse, where mORNs serve as pheromone
reception neurons.
Effect of androgen treatment on ORN sensitivity
The magnitudes of EOG responses to PGFs increase as the male reproductive
season progresses in S. salar
(Moore and Waring, 1996) and
this phenomenon was reproduced by androgen treatment in P.
schwanenfeldi (Cardwell et
al., 1995). In the present study, however, PGFs did not
induce any current responses in ORNs and androgen treatment did not influence
inward current responses of cORNs to the amino acid mixture and urine samples.
In our previous study (Sato et
al., 2000), olfactory nerve responses of lacustrine sockeye
salmon (Oncorhynchus nerka) to river water did not change
significantly with different degrees of maturation. PGFs did not induce
responses in olfactory nerves of sockeye salmon and masu salmon
(Oncorhynchus masou) (Sato and Shoji, unpublished data). Therefore,
olfactory response properties of ORNs of Oncorhynchus salmonids to
different odorants may be different from those of ORNs of Salmo
salmonids during maturation and also in pheromone detection. Many fish species
of non-ostariophysi, including salmonids, do not show any EOG responses to
PGFs and DHPs (Stacey et al.,
1994).
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Acknowledgments |
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We thank Mr Kiyokatsu Hibi (Analysis Center of JASCO Engineering Co. Ltd, Tokyo) for bile acid analysis, and Dr Yukichi Abe (Center for Instrumental Analysis, Hokkaido University) for amino acid analysis of rainbow trout urine samples. We also thank Dr Peter W. Sorensen (University of Minnesota) for the valuable comments on the manuscript.
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Accepted July 11, 2001
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