Evidence of a novel transduction pathway mediating detection of polyamines by the zebrafish olfactory system
Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT 84108-1297, USA
* Author for correspondence (e-mail: mike.michel{at}m.cc.utah.edu)
Accepted 17 February 2003
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Summary |
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Key words: electro-olfactogram, activity labeling, olfactory receptor neuron, odorant receptor, zebrafish, Danio rerio
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Introduction |
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Polyamines are present in all cells and their concentrations in carrion are
likely to increase due to bacterial degradation
(Morgan 1999;
Du et al., 2001
). To date, only
two polyamines have been tested as odorants in fish, and in both cases they
were tested on zebrafish Danio rerio. Putrescine failed to stimulate
pre-synaptic olfactory sensory neuron activity in the olfactory bulb
(Fuss and Korsching, 2001
).
Agmatine (AGB), a relatively rare guanidium-based diamine synthesized by
decarboxylation of L-arginine, was found to elicit large olfactory responses
and to enter a small population of microvillar olfactory sensory neurons,
which could subsequently be visualized by an anti-AGB antibody
(Michel et al., 1999
;
Lipschitz and Michel,
2002
).
Structurally, polyamines are similar to other potent aquatic odorants such as amino acids and biogenic amines. Polyamines are small molecular mass, linear aliphatic molecules that are water-soluble and have positively charged amino groups at physiological pH values, making them organic bases. The fixed spacing of their positive charges gives polyamines unique steric and cationic properties.
Cells closely regulate intracellular polyamine levels through mechanisms
controlling biosynthesis, degradation and uptake
(Morgan, 1999). Minimal levels
of intracellular polyamines are needed for the optimal growth and replication
of plant, bacterial, fungal and animal cells. Polyamines alter the
transcriptional and translational stages of protein synthesis, stabilize
membranes, modulate ion channel activity, change intracellular free calcium
levels and possibly possess important messenger functions (reviewed by
Morgan, 1999
). Additionally,
spermine, spermidine, putrescine (Lynch,
1999
; Nevin et al.,
2000
) and agmatine (Michel et
al., 1999
) have been shown to block olfactory cyclic
nucleotide-gated (CNG) channel activity.
Physiological methods have been used to explore structureactivity
relationships of many different classes of odorants in the zebrafish Danio
rerio, and electrophysiological methods
(Michel and Derbidge, 1997),
in combination with activity-dependent labeling studies
(Lipschitz and Michel, 1999b
),
identified at least partially independent receptor sites for amino acids, bile
salts and guanidine-based substances. These findings were largely confirmed
and additional sensitivity to nucleotides, saponins and sex pheromones was
identified when presynaptic olfactory sensory neuron (OSN) activity was imaged
in the olfactory bulb (Friedrich and Korsching,
1997
,
1998
;
Fuss and Korsching, 2001
). In
the present investigation, we used electro-olfactogram (EOG) recording methods
and activity-dependent labeling techniques to explore polyamine-stimulated
signaling in the zebrafish olfactory system.
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Materials and methods |
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Recording procedures
All experimental procedures have been approved by the University of Utah
Animal Care and Use Committee. Before a recording session, each zebrafish was
immobilized with an intramuscular injection of Flaxedil (60 mg
g1 body mass), secured to a custom silastic-polymer
(Sylgard) recording chamber and immediately provided with a continuous flow of
artificial fresh water (AFW; see Solutions) to the olfactory epithelium (OE)
and a separate flow of approximately 3 ml min1 of AFW
containing MS-222, a general anesthetic (20 mg l1 in AFW),
over the gills. In order to prevent the loss of afferent sensory activity
(Spath and Schweickert, 1977),
the fish were anesthetized only after immobilization, thus preventing any
contact between the anesthetic and the OE. Each zebrafish was given 10 min for
the anesthetic to fully act before the small flap of the epithelium covering
the left olfactory organ was surgically removed to expose the olfactory
lamellae. Throughout the experiment, zebrafish were monitored for any reflex
movements of the gills or eyes and, if noted, provided with additional
anesthetic by increasing the gill irrigation flow. Following each experiment,
while still anesthetized, each fish was measured (total body length) and
weighed, then killed by decapitation. The sex of the zebrafish was then
determined by the presence of ovaries or testes in the abdominal cavity as
seen under a dissecting microscope.
Electrophysiological methods
The olfactory responses of the zebrafish were measured using EOG recording
methods as described previously (Michel
and Lubomudrov, 1995; Michel
and Derbidge, 1997
). A characteristic, negative DC voltage
potential shift was recorded in response to odors, reflecting the
extracellular ionic flux associated with the summed receptor potentials of the
activated olfactory receptor neurons. To record these responses, a reference
electrode was placed on the top of the head and a recording electrode was
placed in the left olfactory organ, between adjacent olfactory lamellae and
near the midline raphe. Both of these electrodes were made from silver/silver
chloride wire bridged to the fish by way of 3 mol l1
NaCl/agar-filled (13%) glass electrodes with tip diameters of approx.
1020 µm. A silver/silver chloride wire, placed in the AFW bath
directly beneath the body of the fish, served as the ground electrode. The
responses to the olfactory stimuli were amplified and filtered at 12
kHz by a low-noise, differential, DC amplifier, displayed on an oscilloscope
and stored digitally (100 Hz; Digidata 1200 A/D board and Axotape software,
Axon Instruments, Union City, CA, USA). Before beginning an experiment, a
stable baseline and a response of at least 0.5 mV to 100 µmol
l1 L-glutamine were required. The olfactometer and both
electrodes were adjusted before deciding to reject a fish on the basis of its
small response to glutamine. Fewer than 5% of the zebrafish test subjects were
rejected.
Odorant testing
The competitor odorant or AFW solution was selected by a six-way valve from
elevated, polyethylene bottles and delivered to the OE at a rate of 3 ml
min1. Teflon or polyethylene tubing and connectors were used
in the olfactometer construction to minimize contamination. A piece of
18-gauge stainless steel tubing directed the olfactometer output flow over the
OE. A rotary loop injector (Rheodyne, Inc., Rohnert Park, CA, USA) was used to
introduce the test odorant (50 µl) into the olfactometer flow. Fluorescent
dye calibration determined that the odor solution arrived at the OE in
approximately 8 s, achieved peak odor concentration (approximately 84% of
stock) at approx. 10 s, and then returned to baseline levels in another
1215 s. The concentrations reported have not been corrected for this
dilution. To minimize adaptation, a period of at least 2 min was allowed
between odorant tests, and ascending concentrations were tested during
doseresponse determinations. The odorants/stimulants used in these
experiments were: the amino acid L-glutamine (Gln); the polyamines agmatine
(AGB), putrescine (Put), cadaverine (Cad), spermidine (Spd) and spermine
(Spm); the monoamine histamine (His); and, in some experiments, the bile salt
taurocholic acid (TCA). The amino acid L-glutamine was used as a positive
control to establish the viability of the preparation and to allow comparison
with earlier studies. With the exception of TCA (10 µmol
l1), all of the odorants were tested at 100 µmol
l1.
Identification of odorant receptor site types
The protocol used for the cross-adaptation study was similar to the
procedures previously used to characterize olfactory receptors in zebrafish
and channel catfish (Caprio and Byrd,
1984; Michel and Derbidge,
1997
). A standard test series measured the responses to test odor
before, during, and after adaptation of the OE with a competitor odorant. The
test series was performed in three steps. First, the response to a test
odorant was measured with AFW bathing the OE. Then, the response to a test
odorant, prepared in the competitor odorant background, was measured while the
competitor odorant bathed the OE. During changes between AFW and a competitor
odorant, the competitor odorant background bathed the OE for a minimum of 60 s
before a test odorant was applied. Finally, the flow across the OE was
returned to AFW and the response to the test odorant was verified to ensure
that adaptation was reversible. During each phase of testing, each odorant was
tested in duplicate. The response to L-glutamine (100 µmol
l1) was checked between test series to assess long-term
preparation viability. The order of the odorants tested was randomized for
each zebrafish examined, and a minimum of three fish were tested in each of
the seven adapting backgrounds. All adapting odorant backgrounds were used at
a concentration of 100 µmol l1.
Transduction cascades involved in polyamine signaling
We have previously shown that adaptation with the adenylate cyclase
activator forskolin or the phospholipase C inhibitor neomycin largely
eliminates bile salt- and amino acid-evoked responses, respectively
(Ma and Michel, 1998;
Michel, 1999
), suggesting the
presence of at least two transduction cascades for odorants in zebrafish. To
explore the potential transduction cascade mediating polyamine signaling, the
responses to glutamine, taurocholic acid and the polyamines were measured
before, during and after adaptation to forskolin (10 µmol
l1) or U-73122 (1 µmol l1), a more
specific PLC inhibitor than neomycin, using the procedure described above for
identification of odorant receptor site types.
Activity-dependent labeling
Amino acids, but not the bile salt taurocholic acid (or the adenylate
cyclase activator, forskolin), were shown to stimulate transduction pathways
linked to ion channels within OSNs permissive to permeation by the guanidinium
analog, agmatine (AGB) (Michel et al.,
1999; Lipschitz and Michel,
1999b
; Lipschitz and Michel,
2002
). Methods reported in detail in these earlier studies were
used to determine if polyamines were capable of stimulating activity-dependent
labeling of OSNs with AGB. Briefly, a fish was immobilized and placed in the
recording chamber as described above. The OE was bathed with fish Ringer and
once a minute for 10 min a 10 s bolus of odorant and 5 mmol
l1 AGB in fish Ringer was introduced into the olfactometer
flow to stimulate the OSNs. Presentation of 5 mmol l1 AGB in
fish Ringer served as a control. After a brief rinse in fish Ringer, the
olfactory rosettes were fixed overnight (see Solutions), embedded in Eponate
plastic and sectioned at 500 nm using a diamond knife (Delaware Diamond Knife,
Wilmington, DE, USA) and an ultramicrotome (Leica Ultracut UCT, Leica
Microsystems, Bannockburn, IL, USA). After deplasticization with 25% sodium
ethoxide in ethanol, the sections were incubated in anti-AGB antibody (1:100
in 0.1 mol l1 phosphate buffer, 1% goat serum) overnight and
visualized with a goat anti-rabbit, nanogold-conjugated, secondary antibody
and silver intensification (Michel et al.,
1999
). After placing coverslips on top, eight-bit digital
bright-field images were captured on an Axioplan 2 microscope equipped with an
Axiocam digital camera and Axiovision software (Carl Zeiss Inc., Thornwood,
NY, USA).
To quantify the proportion of labeled epithelium in each image, an area of interest (AOI) was drawn to include only sensory epithelium. Labeled pixels within this region were defined as those having intensity values less than 2 standard deviations (S.D.) below the mean pixel intensity value of a second AOI (drawn within the larger AOI) that contained no labeled neurons. The percentage of labeled epithelium was calculated by dividing the number of labeled pixels by the total number of pixels and multiplying by 100. For each olfactory rosette, at least 26 areas of interest were examined on each of 3 (or more) planes of section separated by a minimum of 10 µm, to ensure that the same OSNs were not repeatedly sampled.
Data analysis
Relative stimulatory effectiveness and concentrationresponse
relationships were calculated by measuring the peak response (in mV) for
replicate odorant presentations for each fish from a minimum of three fish per
odorant. One-way analysis of variance (ANOVA; SPSS ver.11, SPSS Inc., Chicago,
IL, USA) established overall significance of polyamine specificity and
concentration-response functions. A Dunnett's post-hoc t-test was
used to determine which polyamines (or polyamine concentrations) elicited
responses that were significantly greater than the responses to the AFW
controls. For cross-adaptation experiments, the average response to a test
odorant in the adapting background was normalized to the average response to
the same test odorant measured in the AFW background immediately before
adaptation. A one-way ANOVA established the overall significance of an
adapting background and a Dunnett's post-hoc t-test was used to
determine responses significantly greater than the self-adapted response. A
paired t-test was used to determine if forskolin or U-73122
significantly affected the responses to the test odorants. Data are presented
as means ± standard error of the mean (S.E.M.) unless otherwise
indicated.
Solutions
All of the odorant solutions used were prepared in deionized water with a
resistivity of >18 M cm1. The composition of the
AFW was (in mmol l1): NaCl, 3; KCl, 0.2; CaCl2,
0.2; Hepes, 1; pH 7.2. The composition of fish Ringer was (in mmol
l1): 140 NaCl, 10 KCl, 1.8 CaCl2, 2
MgCl2, 5 Hepes; pH 7.2. Fixative contained 2.5% glutaraldehyde, 1%
paraformaldehyde, 3% sucrose and 0.01% CaCl2 in 0.1 mol
l1 phosphate buffer, adjusted to pH 7.4. Odorants were
prepared every other week as 10 mmol l1 stocks in AFW (pH
7.007.15) and refrigerated at 4°C. The working solutions for each
odorant were prepared daily in fresh AFW. For cross-adaptation experiments,
test odorants were prepared in AFW and in the competitor odorant for use
during background adaptation. All of the odorants were tested at a
concentration of 100 µmol l1 except for TCA, which was
tested at 10 µmol l1. These concentrations allowed
reproducible responses to be obtained over the lengthy experimental procedure
time (often up to 3 h). All chemicals were obtained from Sigma Chemical
Co.
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Results |
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Glutamine and all of the other odorants elicited responses that were
significantly greater than the response to the AFW control
(Fig. 2). The average response
to the glutamine of 0.98±0.08 mV was similar to that recorded in our
earlier studies (Michel and Lubomudrov,
1995). The least potent odorants, histamine, putrescine and
cadaverine, elicited responses that were smaller than the glutamine standard.
In contrast, responses to spermidine and AGB were nearly fourfold larger than
the glutamine standard and spermine elicited the largest olfactory response we
have observed in zebrafish (at 100 µmol l1), nearly
eightfold larger than the response to the glutamine standard.
|
Doseresponse relationship
The large responses to spermine, spermidine and AGB might be an indication
of heightened olfactory sensitivity to these stimuli. To assess this
possibility we determined their threshold concentrations and
concentrationresponse characteristics
(Fig. 3). The detection
threshold for each of these polyamines was approximately 1 µmol
l1 (one way ANOVA; P<0.05), similar to the range
of detection thresholds previously noted for amino acid and bile salt stimuli
in this species (Michel and Lubomudrov,
1995). Olfactory responses to the highest concentrations tested
(100 µmol l1) had not saturated.
|
Receptor site characterization
To determine if the polyamine odorants bound to unique odorant binding
sites, we continuously exposed the OE to a background odorant to desensitize
the OE to that odorant, then tested the responsiveness of the desensitized OE
to other odors. When the response to a test odorant is abolished in an
odorant-adapting background, it was assumed that the test and adapting odorant
interact at some level of the transduction cascade, probably at the odorant
binding site (the odorant receptor). The response to each of the seven
odorants in its own adapting background was eliminated (Figs
4,
5), indicating that
self-adaptation was complete. In contrast, the responses to the test odorants
were sometimes partially, but never fully, cross-adapted.
|
|
Adaptation to putrescine (Fig. 5A) and cadaverine (Fig. 5B) reduced the response to histamine by about 50%, but otherwise had little effect on the responses to polyamines or to glutamine. Histamine adaptation (Fig. 5C) reduced to the greatest extent the responses to putrescine and cadaverine, but responses to all of the test odors remained significantly larger than the self-adapted response to histamine. Adaptation to spermidine (Fig. 5D) and spermine (Fig. 5E) generally reduced the responses to the other test odorants, but affected the responses to each other and to AGB to a greater extent than the responses to the less potent odorants. During spermine adaptation, the responses to the test odorants in different fish were variable. As a result of this variability, the cross-adapted responses to histamine, cadaverine, AGB and spermidine were not significantly greater than the self-adapted response to spermine. Similar variability in test odorant response magnitude was noted when AGB was used as the adapting background (Fig. 5F). In the AGB background, the responses to putrescine and glutamine were largely unaffected, while responses to cadaverine, histamine, spermidine and spermine were not significantly greater than the self-adapted response to AGB. Interestingly, the response to glutamine was not significantly affected by any adapting background and was actually significantly larger than its unadapted response in the spermidine background. During adaptation to glutamine (Fig. 5G), the responses to the test odorant were all significantly larger than the self-adapted response to glutamine.
Signaling cascade mediating polyamine responsiveness
Adenylate cyclase- and phospholipase C-mediated signaling cascades were
implicated in the transduction of bile salt
(Michel, 1999) and amino acid
odorants (Ma and Michel,
1998
), respectively. To implicate one of these cascades in
polyamine detection, we tested the effects of the adenylate cyclase activator
forskolin and the phospholipase C inhibitor U-73122 on polyamine-evoked
responses.
A response of 6.0±3.3 mV (N=6) to 10 µmol
l1 forskolin confirmed our previous observation that
forskolin elicited a large EOG response
(Michel, 1999). An inactive
forskolin analog, 1,9 dideoxyforskolin (10100 µmol
l1), used as a negative control for adenylate cyclase
activation (Doi et al., 1990
),
was non-stimulatory (0.09±0.05 mV, N=5). To implicate
adenylate cyclase in the polyamine signaling cascade, we compared
polyamine-elicited responses obtained in AFW with responses measured when the
OE was bathed with 10 µmol l1 forskolin to desensitize
OSNs that transduce olfactory input via the CNG pathway
(Fig. 6). In the forskolin
background, the response to forskolin was eliminated, indicating that
self-adaptation was complete (not shown). In the forskolin background, the
responses to TCA and cadaverine were significantly reduced to about
3546% of their unadapted levels (paired t-test;
P<0.05). The responses to spermine, spermidine, putrescine, AGB,
cadaverine and glutamine were not significantly affected by forskolin
adaptation, suggesting that adenylate cyclase activation is not required for
transduction of these polyamines.
|
Application of the phospholipase C inhibitor U-73122 (1 µmol l1) did not elicit an olfactory response, but the drug did affect the responses elicited by some odorants (Fig. 7). U-73122 significantly reduced responses to amino acid odorants from 53% (glutamine) to 67% (arginine) and significantly reduced the response to spermine by 30% (paired t-test; P<0.05). Inhibition of PLC had no effect on taurocholic acid or AGB-evoked responses.
|
Polyamine-stimulated labeling of ORNs
In view of the robust EOG responses obtained to the more potent polyamine
stimuli and the failure of the manipulation of either adenylate cyclase or
phospholipase C signaling to affect polyamine responses, we were interested in
determining the cell type involved in polyamine signaling. We previously
showed that amino acids, but not bile salts or forskolin, stimulated
activity-dependent labeling (Michel et
al., 1999; Michel,
1999
; Lipschitz and Michel,
1999a
) of primarily microvillar OSNs
(Lipschitz and Michel, 2002
).
Using identical procedures, polyamine stimulation failed to significantly
increase activity-dependent labeling of the OE, whereas L-glutamine-stimulated
preparations resulted in a significantly higher proportion of labeled OE (Figs
8,
9). Stimulation with putrescine
resulted in slightly more labeled epithelium than did stimulation with either
spermine or spermidine. Qualitatively, the intensity of labeling during
spermine stimulation was lighter than in either the AGB control condition or
the L-glutamine-stimulated test condition.
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Discussion |
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Although zebrafish have been subjects for numerous physiological
characterizations of olfactory sensitivity
(Michel and Lubomudrov, 1995;
Friedrich and Korsching, 1997
,
1998
;
Michel and Derbidge, 1997
;
Lipschitz and Michel, 1999b
;
Friedrich and Laurent, 2001
;
Fuss and Korsching, 2001
),
only two of the odorants investigated in the present study had previously been
examined. AGB was shown to be a potent odorant with the unique property of
being ion channel-permeant, thus allowing activity-dependent labeling of some
odor-stimulated olfactory sensory neurons
(Michel et al., 1999
;
Lipschitz and Michel, 1999b
).
Putrescine, a weak stimulus in the present investigation, failed to elicit a
measurable bulbar response in experiments using optical imaging methods
(Fuss and Korsching, 2001
).
Explanations for this apparent discrepancy include the possibility that
putrescine input might be communicated along extrabulbar pathways
(Hofmann and Meyer, 1995
), or
that the optical imaging method employed in the bulb might not have been
sufficient to detect a weak stimulus, particularly if the glomerular field
activated was not routinely examined.
The potency of the polyamines is correlated with the structure of the
molecule. Decarboxylation of the basic amino acids lysine and ornithine
produces cadaverine and putrescine, respectively. These 4- and 5-C aliphatic
diamines were relatively weak stimuli, as was lysine
(Michel and Lubomudrov, 1995).
Addition of an amino propyl group to putrescine produces the triamine
spermidine, a significantly more potent odorant. Addition of another amino
propyl group to spermidine produces the tetra-amine spermine. At 100 µmol
l1, spermine elicited the largest EOG response so far
recorded for any olfactory stimulus in the zebrafish. AGB, a structurally
distinct diamine produced by the decarboxylation of arginine, was
approximately as potent as spermidine. Histamine, the only monoamine tested,
was the least potent odorant tested in this study, but it did elicit a
response that was significantly greater than the AFW control.
Fish OSNs express odorant receptors, which are either members of the
classical odorant receptor (Ngai et al.,
1993a,b
;
Barth et al., 1996
) or V2R
receptor (Naito et al., 1998
;
Speca et al., 1999
) families.
Odor-stimulated activation of these receptors has been shown to activate
either cyclic nucleotide-gated or IP3-mediated signaling pathways
through g-protein coupled mechanisms (reviewed by
Schild and Restrepo, 1998
).
Although the molecular structure of ORs activated by polyamines remains to be
determined, physiological and pharmacological data from zebrafish suggest that
polyamine odorants activate receptors linked to a transduction cascade
differing from those used by either amino acid or bile salt odorants. The
polyamines putrescine, spermidine and spermine activate a pathway that is
seemingly impermeable to AGB uptake and largely unaffected by adenylate
cyclase activation or phospholipase C inhibition (present study). The bile
salt taurocholic acid activated a pathway that is also impermeable to AGB
uptake and unaffected by PLC inhibition, but is significantly affected by
adaptation to forskolin (Michel et al.,
1999
; Michel,
1999
), which implicates adenylate cyclase signaling; however
inositol phosphate signaling has been implicated in bile salt detection by the
Atlantic salmon (Lo et al.,
1994
). Amino acids activate a pathway in microvillar cells
(Lipschitz and Michel, 2002
)
that is permeable to AGB uptake. Amino acid-evoked responses are partially
reduced by either adenylate cyclase activation or phospholipase C inhibition
(Michel et al., 1999
;
Michel, 1999
). Adenylate
cyclase activation reduced the responses to amino acid odorants to
5070% of their unadapted response levels
(Michel, 1999
). Phospholipase
C inhibition reduced responses to the same amino acids to 2050% of
their unadapted levels. Thus, both signaling pathways may be involved in amino
acid transduction.
In the present study, adenylate cyclase activation reduced the response to cadaverine to less than 50% of its unadapted response, while the responses to histamine, putrescine, agmatine, spermidine and spermine were not significantly affected. These results suggest that cadaverine, a 4-C diamine, may be linked to a different transduction pathway from the other polyamines, perhaps interacting with a receptor for the basic amino acid lysine. Phospholipase C inhibition had little effect on evoked responses to spermine and AGB. The failure of both forskolin and U-73122 to significantly affect evoked responses to AGB and spermine indicates that these odorants are unlikely to use either adenylate cyclase-mediated or phospholipase C-mediated transduction cascades. The signaling cascade activated by polyamines warrants further investigation.
Polyamines reportedly play many roles in cell growth and biosynthesis
through interactions with nucleic acids, proteins and membranes
(Morgan, 1999). Particularly
relevant to the present study is the involvement of polyamines in ion channel
regulation. Ion channels known to be affected by polyamines include ionotropic
glutamate receptor/channels, potassium channels and the olfactory CNG channel
(Williams, 1997
).
Physiological concentrations of intracellular spermine, spermidine and
putrescine rectify the olfactory CNG channels
(Lynch, 1999
). More
importantly, extracellular spermine (0.11 mmol l1)
also blocks olfactory CNG channel-mediated currents
(Nevin et al., 2000
). Our
observations of a reduction in response magnitude noted during spermine and
spermidine cross-adaptation and a reduction in the intensity of
activity-dependent labeling in spermine stimulated preparations may be an
indication of an extracellular polyamine-mediated block of ion channels
involved in odor transduction.
Spermine and spermidine were originally isolated from seminal fluid and are
likely to be present in elevated concentrations in the chromatin-enriched
spawning fluids of fish; however, their extracellular concentrations remain to
be determined. As their names imply, putrescine and cadaverine are olfactory
stimuli originally associated with decaying or rotting tissue
(Goldberg et al., 1994;
Greenstein et al., 1997
) and
with the decomposition of food products by bacteria
(Morgan, 1999
), but were
subsequently found to be present in all tissues. Increasing levels of
putrescine or cadaverine stimulate rats to bury conspecifics 2448 h
after death and anesthetized rats sprinkled with either putrescine or
cadaverine are also buried (Pinel et al.,
1981
). Although the behavioral responses elicited by polyamines in
zebrafish remain to be determined, the behavioral observations of polyamine
detection by mammals indicate that this class of stimuli are of general
significance to many vertebrates.
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Acknowledgments |
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