Evidence of a novel transduction pathway mediating detection of polyamines by the zebrafish olfactory system
W. C. Michel*,
M. J. Sanderson,
J. K. Olson and
D. L. Lipschitz
Department of Physiology, University of Utah School of Medicine, Salt
Lake City, UT 84108-1297, USA
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Fig. 1. Typical electro-olfactogram (EOG) responses elicited by the monoamine and
polyamine odorants and the amino acid standard L-glutamine. The chemical
structure, common name and abbreviation (in parentheses) of each odorant is
shown below the response. The peak response was used to quantify response
magnitude. All responses were obtained from the same fish. Odorants were
tested at the concentration of 100 µmol l–1.
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Fig. 2. The stimulatory effectiveness of monoamine and polyamine odorants compared
to L-glutamine, the amino acid standard. All odorants were tested at 100
µmol l–1. The average electro-olfactogram (EOG) response
(mV) for each odorant was calculated from data obtained from three fish tested
with only 100 µmol l–1 concentrations of each of the test
odorant and from the responses obtained to each stimulus in the pre-adapted
state during the cross-adaptation experiments (N=20). Asterisks
designate responses significantly greater than the response to AFW (one-way
ANOVA, Dunnett's post hoc t-test; P<0.05). Values are
means ± S.E.M. Abbreviations as in
Fig. 1.
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Fig. 3. The concentration–response relationships for spermine, spermidine and
agmatine reveal detection thresholds for each of these stimuli in the 1
µmol l–1 range. For spermine and spermidine, data from a
total of three fish were pooled; for agmatine, data from 20 fish was analyzed.
Each fish was used for only one concentration–response series. Odorants
were tested as an ascending concentration series. The average responses to the
100 µmol l–1 L-glutamine and AFW controls are shown as
single points and as a dotted line (AFW only). Values are means ±
S.E.M. Responses to spermine, spermidine and agmatine that are significantly
greater than the response to the AFW control are designated with a, b and c,
respectively (one-way ANOVA; Dunnett's post hoc t-test;
P<0.05). Abbreviations as in
Fig. 1.
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Fig. 4. Examples from two cross-adaptation experiments demonstrating that the amino
acid L-glutamine (Gln) and the polyamine spermine (Spm) interact with
different odorant receptors. Electro-olfactogram (EOG) responses to 100
µmol l–1 glutamine and 100 µmol l–1
spermine were selectively attenuated when the background bathing the olfactory
epithelium was switched from AFW to (A) 100 µmol l–1
glutamine and (B) 100 µmol l–1 spermine, respectively.
Responses to glutamine (upper traces) or spermine (lower traces) before (Pre),
during (x-adapt) and after (Post) exposure to a background odorant are shown
for each adapting odorant. Data plotted in A and B were obtained from two
different fish.
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Fig. 5. Cross-adaptation experiments suggest the presence of relatively independent
receptor sites for each of the polyamine odorants tested. The
electro-olfactogram (EOG) responses to each test odorant in (A) putrescine
(Put), (B) cadaverine (Cad), (C) histamine (His), (D) spermidine (Spd), (E)
spermine (Spm), (F) agmatine (AGB) and (G) L-glutamine (Gln) competitor
odorant backgrounds are expressed as a percentage of the response obtained to
the odorant prior to switching into the competitor odorant background. Each
competitor odorant was tested on three fish. The dotted line indicates the
level of response predicted if no cross-adaptation is occurring. Asterisks
designate responses significantly greater than the self-adapted response to
the background odorant (one-way ANOVA, Dunnett's post hoc t-test;
P<0.05). Values are means ± S.E.M.
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Fig. 6. Application of the adenylate cyclase activator forskolin has little effect
on most polyamine-evoked electro-olfactogram (EOG) responses, suggesting that
the adenylate cyclase activation is not critical during the initial
transduction of a polyamine stimulus. (A) Example responses elicited by
glutamine, spermidine and TCA in an AFW background (left traces) or in the
presence of 10 µmol l–1 forskolin (right traces). (B)
Summary changes in odor-evoked responses in the presence of forskolin
normalized to the pre-drug treatment level. The magnitude of the taurocholic
acid-evoked response was significantly reduced (t-test;
P<0.05) but the L-glutamine-evoked response, while reduced, was
not significantly smaller than the pre-forskolin exposure response level
(t-test; P>0.05). Asterisks designate responses
significantly reduced compared to the pre-forskolin exposure responses (paired
t-test; P<0.05). Each odorant was tested on three
preparations and values are means ± S.E.M. ns, non-significant.
Abbreviations as in Fig. 1.
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Fig. 7. Application of the phospholipase C inhibitor U-73122 (1 µmol
l–1) differentially affected odorant-evoked
electro-olfactogram (EOG) responses. (A) Example responses elicited by
glutamine, spermidine and TCA in an AFW background (left traces) or in the
presence of U-73122 (right traces). (B) Summary changes in odor-evoked
responses in the presence of U-73122 normalized to the pre-drug treatment
level. U-73122 had little effect on taurocholic acid-, AGB- and
spermidine-evoked responses, suggesting that the phospholipase C activation is
not critical during the initial transduction of bile salt or polyamine input.
Amino acid-evoked responses were significantly reduced to 20–50% of
their pre U-73122 levels. Asterisks designate responses significantly reduced
compared to the paired pre U-73122 responses (paired t-test,
P<0.05). Values are means ± S.E.M. Abbreviations as in
Fig. 1.
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Fig. 8. L-glutamine stimulates a significant increase in activity-dependent
labeling of the zebrafish olfactory epithelium compared to agmatine (AGB)
control preparations, but the polyamine stimuli do not. The olfactory
epithelium was stimulated with (A) 5 mmol l–1 AGB, (B) 5 mmol
l–1 AGB + 100 µmol l–1 spermine, (C) 5
mmol l–1 AGB + 100 µmol l–1 putrescine or
(D) 5 mmol l–1 AGB + 100 µmol l–1
L-glutamine. In all preparations the labeling is largely restricted to cells
within the sensory epithelium. In all panels the midline raphe (R) is located
on the right and non-sensory epithelium can be seen on the left end of the
lamellae (L). Scale bar, 10 µm.
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Fig. 9. Polyamine stimulation of the olfactory epithelium resulted in a modest,
non-significant, increase in activity-dependent labeling of OSNs compared to
the level of labeling noted during control stimulation with AGB alone or the
robust stimulation elicited by glutamine. Each odorant was tested on a minimum
of two olfactory rosettes from two different fish. A minimum of six areas of
interest from three planes of section were sampled for each fish. Asterisks
designate significantly greater labeled epithelium than in the AGB control
preparations (one way ANOVA, post hoc t-test; P<0.05).
Values are means ± S.E.M. Abbreviations as in
Fig. 1.
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© The Company of Biologists Ltd 2003
