Polyamines as olfactory stimuli in the goldfish Carassius auratus
1 Department of Biological Sciences, Louisiana State University, Life
Sciences Building Room 202, Baton Rouge, LA 70830, USA
2 Department of Fisheries, Wildlife and Conservation Biology, University of
Minnesota, 200 Hodson Hall, 980 Folwell Avenue, St Paul, MN 55108,
USA
* Author for correspondence (e-mail: srolen1{at}lsu.edu)
Accepted 27 February 2003
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Summary |
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Key words: electro-olfactogram, olfaction, receptor site, second messenger, goldfish, Carassius auratus
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Introduction |
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Olfactory stimuli in tetrapods are volatile compounds, whereas those for
fish are water-soluble. Further, in contrast to mammals
(Raming et al., 1993;
Zhao et al., 1998
;
Krautwurst et al., 1998
;
Malnic et al., 1999
;
Wetzel et al., 1999
;
Araneda et al., 2000
), the
ligand specificity for molecular olfactory receptors in any teleost is largely
unknown; the sole exception is the goldfish L-arginine/L-lysine amino acid
receptor (Speca et al., 1999
).
Activation of odorant receptors through ligand binding in vertebrates
facilitates second messenger cascades, such as the cyclic AMP (cAMP) and
IP3 signaling pathways (Bruch,
1996
; Schild and Restrepo,
1998
). In mammals, however, the prevailing evidence is for the
cAMP pathway (Belluscio et al.,
1998
; Brunet et al.,
1996
), whereas in fish it is the IP3 pathway
(Speca et al., 1999
;
Bruch, 1996
). In addition, in
both mammals (Xu et al., 2000
)
and fish (Hara and Zhang,
1996
; Nikonov and Caprio,
2001
; Friedrich and Korsching,
1997
,
1998
), ORNs expressing
receptors for different classes of odorants project their axons to the
olfactory bulb, forming a relatively precise odotopic map. Although numerous
types of volatile chemicals are known to stimulate ORNs of tetrapods, the
identification of biologically relevant classes of water-soluble odorants for
fish is limited.
Amino acids, bile salts, nucleotides, gonadal steroids and prostaglandins
have been previously identified as behaviorally relevant olfactory cues for
teleosts, and mediate behaviors ranging from feeding and predator detection to
social interactions and reproductive synchrony
(Sorensen and Caprio, 1998).
Information concerning other classes of chemicals that might also be olfactory
stimuli of biological relevance is lacking; however, an electrophysiological
survey of additional classes of water-soluble, naturally occurring chemicals
in goldfish indicated that polyamines caused large olfactory generator
potentials, which are reflected in the electro-olfactogram (EOG) recordings.
The present study investigates the electrophysiological (EOG and integrated
neural) responses of goldfish ORNs to polyamines and whether these compounds
result in changes in animal behavior.
Polyamines (putrescine, cadaverine and spermine) are naturally occurring
aliphatic polycations that are widely distributed in biological materials.
Intracellular putrescine and spermine concentrations have been reported in the
mmol l1 range for a variety of organisms (E. coli,
rat and human) (Tabor and Tabor,
1976; Ortiz et al.,
1983
), although the concentration of a specific polyamine can vary
with cell type, growth cycle phase and overall health of the cell. Putrescine,
a precursor in spermine biosynthesis, is produced by the ornithine
decarboxylase (identified in prokaryotes, fungi and mammals) and arginine
decarboxylase-agmatineureohydrolase (in prokaryotes) pathways. Putrescine and
spermine play key roles in an array of fundamental cellular processes,
including cell growth, cell division
(Tabor and Tabor, 1984
) and
ion channel modulation (see Discussion). In addition to their occurrence in
living tissues, a previous study indicated that concentrations of putrescine,
cadaverine and spermine were correlated with the degree of decomposition of
certain aquatic animals (Mietz and Karmas,
1978
). Since polyamine concentrations vary with degradation, and
polyamines are distributed ubiquitously, teleosts are likely to encounter them
in an aquatic environment. A previous investigation tested putrescine as a
possible olfactory stimulus in zebrafish, but the results were negative
(Fuss and Korsching,
2001
).
The present study, which investigates ORN responses to polyamines,
indicates that: (1) polyamines are potent olfactory stimuli to goldfish, (2)
polyamine olfactory receptor sites are relatively independent of receptor
sites for other known classes of odorants, (3) relatively independent receptor
sites exist for different polyamines, (4) polyamine odorant information is
likely to be transduced by a signaling pathway other than the classical cAMP
or IP3 cascades, and (5) polyamines are effective stimuli that
promote feeding responses, similar to the behavior exhibited in response to
L-amino acids. Preliminary results were previously reported in abstract form
(Rolen et al., 2001,
2002
).
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Materials and methods |
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Animal preparation
The procedures outlined below are in accordance with a protocol approved by
the Institutional Animal Care and Use Committee (Louisiana State University
School of Veterinary Medicine).
Each goldfish was immobilized with an initial intramuscular injection of Flaxedil (gallamine triethiodide, 0.015 mg/25 g body mass). Subsequent injections of Flaxedil were provided as needed during experimentation via a hypodermic needle embedded in the flank musculature. After immobilization, the goldfish was wrapped in a wet Kim Wipe® and secured with lateral body clamps in a custom-made Plexiglas® container; the head was stabilized with a metal mouthpiece. The gills were irrigated via a constant flow of CFTW containing the general anesthetic, MS-222 (ethyl-m-aminobenzoate methane sulfonic acid, initial concentration, 0.0005%; Sigma Chemical, St Louis, MO, USA). Minor surgery was performed to remove the skin and connective tissue superficial to the olfactory rosette, facilitating electrode placement.
Stimulus solutions and delivery
The odorants included the following L-amino acids: acidic (glutamate,
OOC-(CH2)2-CH(NH3+)-COO),
basic (arginine,
H2N-C(NH2+)-NH-(CH2)3-CH(NH3+)-COO;
lysine,
+H3N-(CH2)4-CH(NH3+)-COO
and ornithine,
+H3N-(CH2)3-CH(NH3+)-COO),
short side-chain neutral (alanine,
H3C-CH(NH3+)-COO), and long
side-chain neutral (methionine,
H3C-S-(CH2)2-CH(NH3+)-COO),
together with amines (putrescine,
H3N+-(CH2)4-NH3+;
cadaverine,
H3N+-(CH2)5-NH3+;
spermine,
H3N+-(CH2)3-NH2+-(CH2)4-NH2+-(CH2)3-NH3+;
butylamine,
H3N+-(CH2)3-CH3 and
amylamine,
H3N+-(CH2)4-CH3), bile
salts [sodium salts of taurocholic acid (TCA;
C26H44NO7SNa) and taurolithocholic acid
(TLCA; C26H44NO5SNa)], ATP
(C10H14N5O13P3Na2)
and glutaric acid
(OOC-(CH2)3-COO). All chemical
stimuli were purchased from Sigma (St Louis, MO, USA) and were of the highest
purity available. Stock solutions of amino acids, glutaric acid and amines
were prepared weekly using CFTW; bile salts and ATP were prepared using Milli
Q water (resistivity, 18.2 M cm1). All stock
solutions were pH adjusted to match control CFTW (pH 8.7) bathing the
olfactory mucosa and refrigerated when not in use. Stock solutions of ATP were
frozen (20°C) in 1 ml portions for up to one month. Stock solutions
were diluted daily to experimental concentrations
(103108 mol l1)
with CFTW. Analysis of the CFTW by the Dionex AAA-Direct Amino Acid Analysis
System (Sunnyvale, CA, USA) indicated that no free amino acids were present
(sensitivity was in the mid femtomole to low picomole range).
Stimulus delivery was via a `gravity-feed' system previously
described (Sveinson and Hara,
2000). Briefly, stimulus solutions and the CFTW used to bathe the
olfactory epithelium were delivered through separate Teflon® tubes
(diameter 0.8 mm) to the olfactory mucosa at a flow rate of 57 ml
min1. A foot switch connected to an electronic timer (Model
645, GraLab Instruments Division, Dimco-Gray Corporation, Centerville, OH,
USA) triggered a pneumatic actuator valve to introduce the stimulus for 3 s
applications. CFTW continuously perfused the olfactory mucosa to (1) prevent
the mucosa from desiccating, (2) facilitate stimulus delivery, (3) avoid the
introduction of mechanical artifacts associated with stimulus presentation and
(4) rinse the olfactory organ clear of any residual stimuli for a minimum of 2
min between stimulus applications.
Pharmacological agents
Forskolin (an adenylate cyclase activator; Sigma Chemical, St Louis, MO,
USA) and 1,9-dideoxyforskolin (inactive analog of forskolin; Calbiochem, La
Jolla, CA, USA) were dissolved in dimethyl sulfoxide (DMSO) and added to CFTW
to provide 104 mol l1 stock solutions.
Forskolin and 1,9-dideoxyforskolin were refrigerated when not in use for up to
1 week during experimental testing. U-73122, a potent inhibitor of
agonist-induced phospholipase C (PLC) activation
(Yule and Williams, 1992) and
U-73343, a weak inhibitor of agonist-induced PLC activation (purchased from
Biomol Research Laboratories, Inc., Plymouth Meeting, PA, USA) were prepared
in the same manner as forskolin and frozen at 20°C when not in use.
DMSO controls were adjusted in concentration to match those used to dissolve
the pharmacological agents.
Electrophysiological recording techniques
The underwater EOG, a slow DC potential change in the water above the
olfactory mucosa, is suggested to be the summed generator potentials of the
responding ORNs in response to odorant molecules
(Ottoson, 1971;
Caprio, 1995
). EOG recordings
were obtained in vivo with calomel electrodes via
Ringer's-agar-filled capillary pipettes. The pipette of the active electrode
was positioned near the midline raphe of the olfactory rosette at a location
that maximized the EOG response to 0.1 mmol l1 L-arginine;
the pipette of the reference electrode was placed against the skin adjacent to
the olfactory cavity. The fish was grounded via a hypodermic needle
inserted into the flank musculature. The EOG was amplified (Grass P-18;
Astro-Med Inc., West Warwick, RI, USA), displayed on an oscilloscope and DC
chart recorder. During the experiments, the standard (0.1 mmol
l1 L-arginine) was applied intermittently; if the responses
to the bracketed standard differed by >25%, those data were excluded from
subsequent analysis.
In vivo recordings of multiunit ORN activity were made using
metal-filled glass capillary electrodes plated with platinum (Pt) (ball
diameter, approx. 1825 µm; cross-sectional area approx.
250500 µm2; impedance, 1040 K) placed
against the sensory face of an olfactory lamella
(Gesteland et al., 1959
;
Caprio, 1995
). The electrode
was r.c.-coupled (220 pF capacitor, 20 M
resistor) to a highimpedance
probe at one input with the other input grounded via a hypodermic
needle embedded in the flank musculature of the fish. The multi-unit neural
activity was amplified (Grass P511; bandpass 30300 Hz), observed on an
oscilloscope, integrated (0.5 s) and displayed using a pen recorder.
Cross-adaptation paradigm
Electrophysiological cross-adaptation experiments to determine the relative
independence of receptors for the odorant stimuli consisted of three stages:
pre-adaptation, adaptation and post-adaptation.
During pre-adaptation, CFTW continuously bathed the olfactory mucosa for a minimum of 10 min prior to stimulus application. Initially, the concentrations of the test stimuli were adjusted to provide approximately equal EOG-response magnitude. Some cross-adaptation experiments involved mixtures of odorants, in which case the concentration of each component of a stimulus mixture was also adjusted to provide an approximately equal EOG-response magnitude when tested individually. The adjusted concentrations of the test stimuli ensured that potent and weak stimuli were approximately equipotent. CFTW served as the control during pre-adaptation.
During adaptation, the adapting solution at the previously adjusted concentration continuously bathed the olfactory mucosa for a minimum of 10 min prior to stimulus application. All stimuli tested during the adaptation paradigm were dissolved in the adapting solution. Controls were portions of the adapting solution and CFTW, respectively. Adaptation to an odorant suppressed the EOG responses to varying degrees to some test stimuli while not affecting the responses to others. Responses to test stimuli that were suppressed to the control level (complete adaptation) were considered to share the same receptor site(s) and/or the same transduction process as the adapting stimulus. Responses to test stimuli significantly greater than the control level were considered to have at least partially independent receptor site(s) and/or transduction processes from the adapting stimulus.
During post-adaptation, CFTW continuously bathed the olfactory mucosa for 10 min prior to stimulus application. Stimuli and controls were identical to those described during pre-adaptation.
Statistical analysis
Statistically significant differences between groups were determined by a
one-way analysis of variance (ANOVA) with StatMost Version 3.5 (2001; Dataxiom
Software Inc., Los Angeles, CA, USA). Means were further analyzed using the
Tukey post hoc test. P<0.05 was accepted as a
statistically significant difference. A student's t-test
(P<0.05) was utilized to determine significance between the
responses to L-arginine and those to polyamines in
Fig. 3.
|
Behavioral experiments
Two experiments were conducted. The first examined whether exposure to
polyamines stimulated changes in individual behavior similar to that observed
in response to L-amino acids, which are established feeding stimuli. The
second experiment tested whether polyamines were attractive or repulsive.
Experiment 1
The first experiment observed the behavior of groups of goldfish and
followed a well-established behavioral testing protocol (Sorensen et al.,
1988,
1989
;
DeFraipont and Sorensen,
1993
). All fish were in good condition, held under a long (16 h:8
h light:dark) photoperiod and fed ad libitum with flake food
(Chemaqua, CA, USA). Although most fish were tested only once, a small number
were tested a second time with different stimuli after a 3-week intersession
period, during which they were held in 1000 l stock tanks. The following odors
were employed for the first experiment: well water control (i.e. a sample of
the same aquarium water in which the fish were held), 102
mol l1 L-serine hydrochloride [an amino acid that is a
strong olfactory stimulant, but a poor tastant in goldfish
(Sorensen et al., 1987
; P. W.
Sorensen and T. H. Hara, unpublished results)], 102 mol
l1 L-proline hydrochloride [a potent taste stimulus, but a
poor olfactory stimulant in the goldfish
(Sorensen et al., 1987
;
Hara, 1994
)],
102 mol l1 putrescine dihydrochloride,
102 mol l1 spermine tetrahydrochloride,
102 mol l1 cadaverine dihydrochloride and
crude food odor (made by placing 40 g of flaked food into 200 ml of deionized
water for approximately 1 h, and then filtering it to remove particulates).
Odorants were prepared as needed and maintained at 4°C.
Concentrations were chosen so that when fully diluted (100010 000
times, see below) and encountered by fish, they evoked approximately the same
sized EOG responses.
For testing, groups of three fish were placed into 70 l glass aquaria, each
of which was supplied with flowing 17°C well water (100 ml
min1) and maintained on the same photoperiod as the stock
tanks. Fish were allowed to adjust to these aquaria overnight (24 h.). All
aquaria were shielded on three sides with a plastic screen, but had a clear
front with a horizontal and a vertical line drawn to assess fish swimming
rates. Gravel was used as substrate within the aquaria. An air stone was also
placed in the corner of each aquarium, with a 1.5 m (i.d.=0.76 mm) length of
Tygon flexible plastic tubing connecting the stone to a plastic 10 ml syringe
that was used to inject the odor solutions. The syringes were positioned below
the aquaria so the fish could not see them. An opaque black plastic sheet with
a small viewing hole was also stretched across the front of each aquarium so
that the fish could not see the observers who sat at a distance of 12
m. To start an experiment, each group of fish was observed for a 4 min pretest
period, after which 10 ml of test odor were then injected into the aquarium at
a moment when the fish were not near the air stone. After a 15 s period to
permit complete dilution of the odor (confirmed by dye tests), fish were
observed for a 4 min test period. The following behaviors (from
DeFraipont and Sorensen, 1993)
were noted: (1) swimming activity, i.e. the total number of times that
individual fish completely crossed either of the lines drawn across the front
of the aquaria; (2) feeding activity, i.e. the number of times that fish
rapidly opened and closed their mouths in mid-water (`snapping') or picked up
gravel off the bottom (`biting'; a characteristic behavior of goldfish when
sampling for food on the bottom); (3) social activity, i.e. the number of
times that fish physically touched each other, termed nudging behavior
(DeFraipont and Sorensen,
1993
). Activities were recorded as they occurred using a manual
counter. All stimuli were tested 10 times on 10 different groups of fish.
Because these data were ordinal and not normally distributed
(Kolmogrov-Smirnoff; Instat, San Diego, CA, USA), they were analyzed using
nonparametric tests. Briefly, starting (pre-test) values were compared across
test groups using a KruskallWallis test (Instat, San Diego, CA, USA) to
confirm that they were the same. Next, for each test odor and behavior, pre-
and test values were compared using a Wilcoxon matched pairs test.
Experiment 2
The second experiment tested for attraction using a large (1.4 m diameter,
19 cm in depth, 300 l), still-water circular maze divided into two test areas
and a neutral middle zone (Fig.
1). Gravel was placed on the bottom and the apparatus was lit by
an overhead light on a 16 h:8 h light:dark photoperiod. The same odorants as
in Experiment 1 were tested in this experiment, although they were made up and
added at a slightly higher concentration (101 mol
l1), because the greater size of the maze resulted in
greater dilution, enabling us to add odors at times when fish were not near
the stimulus port. The maze was surrounded by a dark canvas apron and had a 10
cm overhead hole through which the fish could be observed.
|
Test protocols followed those of Maniak et al.
(2000). Groups of five fish
were introduced into this maze the day before testing. The next morning, the
fish were observed for a pre-test period, after which a test stimulus was
introduced into the side that contained the fewer number of fish; a blank
control was introduced into the other side. Each test stimulus was introduced
as a 30 ml bolus, which was injected by a remote plastic syringe through 0.76
mm polyethylene tubing (Tygon) attached to an air stone and resulted in a
final concentration of 105 mol l1. Dye
tests showed that odor distribution remained restricted to the injection area
for 15 min. For each trial, the fish were continuously observed during both
the pre-test and test periods, and their positions were noted every minute for
12 min periods. All stimuli were tested at least seven times and the maze was
drained and flushed between trails for a day. Data were analyzed using
Wilcoxon matched-pairs test (Instat, San Diego, CA, USA).
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Results |
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Integrated multiunit recordings to polyamines
To determine whether (1) the EOG responses to polyamines are transduced
into action potential activity of ORNs, as are amino acids
(Fig. 4A), and (2) the large
relative EOG effectiveness of polyamines compared to amino acids is reflected
in olfactory neural (i.e. action potential) activity, integrated multiunit
responses of ORNs were also recorded in a subset of eight fish. An increase in
action potential activity was evident across stimulus concentrations in
olfactory responses to putrescine (in five of eight fish tested; no response
in three fish), cadaverine (in five of eight fish tested; no response in three
fish) and spermine [in two of eight fish tested; no response in three fish;
decline in baseline activity in three fish (inset,
Fig. 4D);
Fig. 4BD]. Neural
thresholds (based on integrated action potential activity) further indicated
that median thresholds were approximately 105 mol
l1 (Fig.
4E).
|
The potency of the polyamines with respect to Arg, the amino acid standard, was noticeably less for the higher polyamine concentrations than indicated by the EOG recordings (compare Figs 4 and 3). To investigate the discrepancy between the relative magnitude of the EOG and multiunit recordings to polyamines, the net positive charge of the polyamine molecules was examined to determine whether this charge contributed to the magnitude of the EOG responses to polyamines. The amine groups of the polyamines tested in this study have pKa values of 8.911.5, which would result in a net positive charge when the pH of the polyamine test solution was adjusted to match the CFTW (pH 8.7) bathing the olfactory mucosa. To negate this excess positive charge of the polyamine molecules, the olfactory organ was adapted to 1 mmol l1 L-glutamate, a negatively charged amino acid (N=2 fish; pKa=2.2, 4.3 and 9.7) and 1 mmol l1 glutaric acid, a decarboxylated analog of glutamate (N=2 fish; pKa=4.34 and 5.22), respectively, during EOG recordings to individual applications of Arg, 10 µmol l1 putrescine, 10 µmol l1 cadaverine and 3 µmol l1 spermine. EOG responses to Arg and the polyamines persisted with only slight attenuation in the background of both 1 mmol l1 L-glutamate and 1 mmol l1 glutaric acid, respectively (Fig. 5).
|
Polyamines bind olfactory receptor sites that are at least partially
independent from those that bind other known classes of odorants
Polyamines tested during continuous presentation of either L-amino acids,
bile salts or ATP to the olfactory organ elicited responses significantly
greater than those to the adapting stimuli (one-way ANOVA; Tukey's post
hoc test, P<0.05), but of comparable magnitude to those of
odorants representing the separate odorant classes (Figs
6A-C,
7A-C). Further, adaptation to a
mixture of polyamines did not significantly attenuate the response to mixtures
of L-amino acids, bile salts or ATP to control levels (one-way ANOVA; Tukey's
post hoc test, P<0.05) (Figs
6D,
7D). The response to the
adapting stimulus, however, was reduced to control level.
|
|
Adaptation to spermine alone, but not to putrescine or cadaverine, resulted in partial cross-reactivity with L-arginine, L-lysine and L-ornithine, reducing the magnitude of the response to these compounds by 3849% of their unadapted responses (Fig. 8C); however, responses to the tested amino acids remained significantly greater than the response to the adapting solution (one-way ANOVA; Tukey's post hoc test, P<0.05).
|
Multiple olfactory receptor site types for polyamines
To determine if putrescine, cadaverine and spermine bind to a single
generic type of polyamine receptor or to different types of polyamine
receptors, EOG responses to each polyamine were recorded during adaptation to
other individual polyamines. During adaptation to each of the three tested
polyamines, EOG responses to the remaining two test polyamines were
significantly greater than that to the adapting stimulus, ranging from 42% to
72% of their unadapted responses (one-way ANOVA; Tukey's post hoc
test, P<0.05) (Fig.
8AC). Responses to the adapting polyamine in the three
paradigms were reduced to the control level.
Polyamine olfactory receptors are independent from olfactory amine
receptors
To further investigate the independence of polyamine receptor sites,
related single amine containing compounds, the deaminated analogs of
cadaverine and putrescine, amylamine and butylamine were tested during
adaptation to individual polyamines. The single amine containing compounds
elicited responses that ranged in magnitude from 68% to 79% of the unadapted
response during polyamine adaptation (Fig.
8AC). Adaptation to any one of the three polyamines did not
significantly attenuate the EOG response to either amylamine or butylamine to
control levels, suggesting that polyamine receptors do not bind single amine
compounds with high affinity (one-way ANOVA; Tukey's post hoc test,
P<0.05). In reciprocal experiments during amylamine or butylamine
adaptation performed in a single fish, EOG responses to the individual
polyamines were not eliminated and remained at 4258% of the unadapted
response (data not shown).
Effects of forskolin on odor evoked responses
To determine if transduction of polyamine odorant information and that for
other known odorant classes in teleosts involve the cAMP second messenger
pathway, forskolin (an adenylate cyclase activator) was continuously applied
to the olfactory mucosa on the assumption that the forskolin treatment would
either decrease the number of adenylate cyclase molecules available for
G-protein coupled receptor activation or desensitize certain components of
this pathway (e.g. cyclic nucleotide gated channels or the odorant receptors),
resulting in an attenuation of the response to odorants utilizing this
pathway. Odor-evoked responses to polyamines were slightly attenuated by the
forskolin treatment, but not to control levels (one-way ANOVA; Tukey's
post hoc test, P<0.05). During adaptation to forskolin
(5-20 µmol l1), however, the magnitude of the EOG
response to a mixture of bile salts (TCA and TLCA) was reduced to baseline
levels, while the magnitude of the EOG responses to ATP remained relatively
unaffected. During the forskolin treatment, the response to a mixture of
L-amino acids (alanine, arginine, glutamate and methionine) was reduced to
59±12% (mean ± S.D.) (Fig.
9A,C). Importantly, adaptation to the mixture of bile salts did
not attenuate the response to forskolin
(Fig. 7B). This nonreciprocal
cross-adaptation between forskolin and bile salts indicates that forskolin,
whose structure resembles that of bile salts, did not compete for the bile
salt receptors. Consistent with these data, EOG responses to bile salts were
only slightly attenuated during continuous application of 20 µmol
l1 1,9-dideoxyforskolin (an inactive analog of forskolin),
which is equivalent to the highest concentration of forskolin tested
(N=2 fish; Fig.
9B).
|
Effects of U73122 and U73343 on odor evoked responses
To determine if transduction of polyamine odorant information and that for
other known odorant classes in teleosts involve the IP3 second
messenger pathway, U-73122 [a potent inhibitor of agonist-induced
phospholipase C (PLC) activation] was continuously applied to the olfactory
mucosa. The assumption was that U-73122 treatment would inhibit G-protein
coupling with PLC resulting in an attenuation of the response to odorants
utilizing this pathway. U-73122 (1 µmol l1) did not
elicit an appreciable EOG response (<0.09 mV) when applied to the olfactory
mucosa; therefore, the concentrations of the odorant stimuli tested were
equivalent to those used during the forskolin treatment. During adaptation to
1 µmol l1 U-73122 (N=3 fish), the magnitudes of
the EOG response to a mixture of polyamines, bile salts and to ATP were
relatively unaffected (Fig.
10A,B). However, responses to a mixture of L-amino acids were
reduced to 57±12% (one-way ANOVA; Tukey's post hoc test,
P<0.05). By contrast, during adaptation to1 µmol
l1 U-73343 (a weak inhibitor of agonist-induced PLC
activation) (N=3 fish), the magnitude of the EOG response to the test
stimuli were not affected significantly
(Fig. 10C,D).
|
Behavioral experiments
Exposure to crude food odor extract stimulated large (approximately
tenfold) increases in feeding activity (P<0.01) as well as an
approximate doubling of swimming activity (P<0.01;
Fig. 11A). In contrast, adding
well water alone to tanks was without any apparent effect on any behavior, as
was the preovulatory sex pheromone. Exposure to spermine, putrescine, and
cadaverine elicited approximately threefold increases in feeding activity
(P<0.01), but had no effect on swimming or nudging (data not
shown). Similar increases in feeding behavior were elicited by exposure to the
three amino acids, L-serine, L-proline and L-arginine
(P<0.01).
|
The second behavior experiment found spermine and food odor to be highly attractive to groups of goldfish (P<0.01) (Fig. 11B). L-serine and putrescine were also attractive (P<0.05), while neither blank water control nor L-proline had any effect on fish distribution.
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Discussion |
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Peripheral recordings to polyamines
EOG doseresponse recordings indicated that polyamines at
concentrations of 1 µmol l1 to 1 mmol
l1 were considerably more effective odorants for goldfish
than L-arginine, the most stimulatory amino acid for goldfish. Integrated
multiunit neural recordings, however, did not reflect this relative magnitude
difference. Polyamines at 1 mmol l1 concentration elicited
integrated multiunit responses that were equal to or less than the multiunit
response to 0.1 mmol l1 L-arginine. Also, the
electrophysiological thresholds to polyamines estimated with neural recordings
were variable across the fish tested. A reasonable possibility to account for
these results is that the specific ORNs that responded excitedly to polyamines
are sparsely dispersed across the sensory epithelium, such that the tip of the
multiunit electrode (approx. 1825 µm platinum tip; cross-sectional
area approx. 250500 µm2) contacted fewer
polyamine-responsive ORNs relative to the number of L-arginine-responsive
ORNs. If this were the case, the neural activity of the fewer
polyamine-responsive ORNs would have to be driven by higher concentrations of
polyamines in order to generate a response with a magnitude comparable to the
response generated by the more numerous L-arginine-responsive ORNs when
presented with 0.1 mmol l1 L-arginine. The failure of
detecting calcium changes in ORN synaptic boutons of zebrafish in response to
putrescine by optical imaging (Fuss and
Korsching, 2001) is consistent with our hypothesis of a sparse
distribution of polyamine-responsive ORNs, especially since unpublished data
indicate that polyamines do evoke EOG activity in zebrafish (W. Michel,
personal communication). Further, if ORNs responding to polyamines in goldfish
are few in number, and EOG responses to polyamines are of a vastly greater
magnitude than those to the amino acid standard, 0.1 mmol l1
L-arginine, then the transduction currents associated with individual
polyamine responsive neurons are unusually large.
Polyamine olfactory receptors are independent from those for known
odorant classes and related compounds
The previously identified, biologically relevant odorants to teleosts
(amino acids, bile salts, nucleotides, sex steroids and prostaglandins) are
initially recognized and discriminated by different molecular olfactory
receptors (Sorensen and Caprio,
1998). These odorants bind to seven transmembrane domain G-protein
coupled receptors located within ciliary and/or microvillar membranes of ORNs
(Buck and Axel, 1991
;
Cao et al., 1998
;
Speca et al., 1999
;
Mombaerts, 1999
). The present
cross-adaptation experiments suggest that olfactory receptor sites for
polyamines are relatively independent from olfactory receptor binding sites
for these other known classes of odorant stimuli.
In the present experiments, EOG responses to a mixture of polyamines were
not attenuated significantly by adaptation to L-amino acids, bile salts or ATP
and, conversely, adaptation to a mixture of polyamines did not attenuate
significantly the EOG responses to these other classes of odorants. Further,
during adaptation to cadaverine and putrescine, responses to the amino acids
L-lysine and L-ornithine remained unaffected. Thus, the addition of an
-carboxylic acid group to putrescine and cadaverine (resulting in
L-ornithine and L-lysine, respectively) results in the binding of these
compounds to different (i.e. amino acid) receptor sites. Consistent with these
data, putrescine and cadaverine were shown to bind with low affinity to the
L-arginine/L-lysine amino acid olfactory receptor in goldfish
(Speca et al., 1999
). Further,
EOG responses to single amine containing compounds in the present study were
not eliminated during adaptation to polyamines, nor were responses to
polyamines eliminated during adaptation to amines. These data suggest that the
removal of a single amine group from putrescine and cadaverine (resulting in
butylamine and amylamine, respectively) decreases the affinity of these
molecules for polyamine receptors. Therefore, the persistence of the EOG
responses to single amine containing compounds suggests that goldfish probably
possess molecular olfactory receptors with the ability to discriminate
polyamines from single amine containing compounds. Single amine compounds were
previously shown to be olfactory stimuli for sharks
(Hodgson and Mathewson, 1978
),
but a recent study failed to visualize calcium influx into ORN synaptic
terminals in the zebrafish olfactory bulb in response to amines
(Fuss and Korsching, 2001
).
Although the present study suggests that the teleost olfactory system responds
to single amines, future studies should reinvestigate single amine compounds
as olfactory stimuli for fish.
Previous investigations in teleosts indicated that ORNs possessing
receptors for different classes of compounds project axons to specific
sub-regions of the olfactory bulb (Hara
and Zhang, 1996; Nikonov and
Caprio, 2001
; Friedrich and Korsching,
1997
,
1998
). An independence of
olfactory receptor sites for polyamines, distinct from other known classes of
biologically relevant stimuli, suggests the possibility for differential
processing of polyamine odorant information within the olfactory bulb of the
goldfish and possibly for other teleost species. In addition, the
identification of independent olfactory receptors for polyamines may possibly
aid research efforts into the molecular and biochemical characterization of
teleost orphan olfactory receptors.
Relatively independent receptor sites for different polyamines
In addition to receptor sites for polyamines being independent of those for
other known biologically relevant odorants for fish, the present results
indicate the relatively independent olfactory receptor sites among the
polyamines themselves. Although the partial reduction of the EOG response to
other polyamines during adaptation to a single polyamine suggests that some
receptor sites possibly accommodate a number of polyamines, the
cross-adaptation results indicated the existence of polyamine receptor sites
that are specific for each of the different tested polyamines. These results
suggest the possibility that goldfish can behaviorally discriminate among the
polyamines; however, further behavioral testing is required to assess this
suggestion.
Second messenger pathways in polyamine odorant transduction
Subsequent to receptor activation, cytosolic second messengers (cAMP and/or
IP3) increase via heterotrimeric G-protein modulation of
enzymatic activity resulting in odorant-induced sensory transduction currents
(Bruch, 1996;
Schild and Restrepo, 1998
).
The present results showed that the response to polyamines persisted with only
a slight reduction from control levels during forskolin adaptation, suggesting
that polyamine transduction is relatively independent of the cAMP second
messenger pathway. The current study also found no evidence linking the
IP3 pathway to polyamine odorant transduction. Continuous
application of U-73122 (an agonist-induced PLC inhibitor) to the olfactory
mucosa failed to affect the EOG response to polyamines (or to bile salts or
ATP), while reducing the EOG response to L-amino acids to 57% of the unadapted
response. That the IP3 signaling cascade is involved in the
transduction of L-amino acid odorant information in teleosts is also
consistent with molecular investigations
(Bruch, 1996
).
In contrast to polyamines, the EOG response to bile salts was reduced by
forskolin to control levels, while responses to L-amino acids were attenuated
by approx. 41%. These data suggest that the cAMP pathway is utilized by ORNs
responding to bile salt odorants, while at least some ORNs responding to
L-amino acids also utilize this pathway. The forskolin results are consistent
with data from similar experiments obtained in zebrafish
(Michel, 1999); however,
species differences may also occur as the IP3 pathway was reported
to be involved in the transduction of bile salt odorant information in the
Atlantic salmon Salmo salar (Lo
et al., 1994
).
The elimination of ORN responses to bile salts by forskolin might have
occurred via multiple mechanisms. Direct activation of adenylate
cyclase by forskolin could have effectively saturated enzymatic activity,
reducing the number of enzymes available for receptor activation.
Alternatively, protein kinase A activation by elevated cAMP concentrations
could have desensitized the bile salt receptors
(Boekhoff and Breer, 1992;
Schleicher et al., 1993
).
Also, increased cytosolic cAMP concentrations could have opened cyclic
nucleotide-gated channels, elevating intracellular calcium concentrations and
leading to increased channel susceptibility to intracellular calcium block
(Frings et al., 1992
;
Balasubramanian, 1996) and decreased affinity of the cyclic nucleotide-gated
channel for cAMP (Kramer and Siegelbaum,
1992
). It is also possible that forskolin might have competed for
bile salt receptors; however, continuous application of bile salts to the
olfactory mucosa did not reduce the EOG response to forskolin. In addition,
continuous application of 1,9-dideoxyforskolin (an inactive analog of
forskolin) did not attenuate the EOG response to bile salts. Irrespective of
which of these possible mechanisms were operating, our collective data suggest
that bile salt odorant information is transduced in goldfish via the
cAMP second messenger pathway.
Spermine attenuation of responses to amino acids and spontaneous
activity
Although the polyamines spermine, cadaverine and putrescine were each used
as adapting stimuli in the present study, only spermine as an adapting
stimulus attenuated EOG responses to amino acids; also, the response to
spermine in some multiunit preparations recorded with the microelectrode only
caused a reduction in baseline spontaneous activity. Possibly both of these
effects were a direct result of spermine block of specific ORN ion channels.
Polyamines have been indicated to modulate a variety of ion channels,
including Kir channels (Fakler
et al., 1994; Lopatin et al.,
1994
; Ficker et al.,
1994
; Pellegrini-Giampietro,
2003
), glutamate receptor channels
(Bowie and Mayer, 1995
;
Donevan and Rogawski, 1995
;
Kamboj et al., 1995
),
voltage-gated calcium and potassium channels
(Droiun and Hermann, 1994
),
KATP channels (Niu and Meech,
1998
), calcium-activated potassium channels
(Weiger et al., 1998
), nAchR
channels (Haghighi and Cooper,
1998
) and retinal rod cyclic nucleotide-gated channels
(Lu and Ding, 1999
).
Intracellular putrescine and intra- and extracellular spermine were also
indicated to attenuate the conductance of the rat olfactory cyclic
nucleotide-gated (CNG) channel (Lynch,
1999
; Nevin et al.,
2000
). Consistent with the possibility that spermine blocks CNG
channels of ORNs, forskolin experiments in zebrafish
(Michel, 1999
) and goldfish
(present study) indicated that the cAMP signaling pathway, and therefore the
CNG channel, is involved in L-amino acid odorant transduction. In the present
experiments, both forskolin and spermine treatments reduced the EOG responses
to L-amino acids by comparable margins, 41% and 3849%,
respectively.
Behavioral responses to polyamines
Both behavioral experiments strongly suggest that polyamines function as
feeding cues in the goldfish. Both putrescine and spermine elicited high
levels of spontaneous feeding behavior that were similar in nature and
magnitude to the two amino acids, L-serine and L-proline. Further, no changes
in social behavior or swimming rates were seen to any of these cues. It was
reasonable that food odor alone was a stronger feeding stimulus than any of
the synthetic cues, since naturally occurring foods are a mixture of many
different compounds. Interestingly, the polyamines, and spermine in
particular, were strong attractants, perhaps stronger than L-serine and
equivalent to food odor itself. In contrast, L-proline, a strong tastant, but
a poor odorant in this species (Hara,
1994; P. W. Sorensen and T. J. Hara, unpublished results), failed
to cause the fish to spend more time within that odor than the control,
suggesting that olfactory cues may have a greater role in attraction in this
species than gustation, and that polyamines exert the bulk of their behavioral
activity through the olfactory system.
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