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Department of Zoology, Michigan State University, East Lansing, Michigan 48824
Submitted 23 December 2002; accepted in final form 26 March 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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; Ashmore et al.
2000). Although understanding peripheral events in odorant
processing is essential to understanding olfactory system function, little is
known about signal modulation in peripheral olfactory systems. Recent studies
have found that peptides and other molecules modulate the activity of
olfactory receptor neurons (Bouvet et al.
1988; Frings 1993;
Grosmaitre et al. 2001;
Kawai et al. 1999;
Vargas and Lucero 1999).
However, because the physiological source and means of access to the olfactory
epithelium have not been established for most of these chemicals, it is not
yet clear whether or not odorant responses are modulated by endogenous
chemicals in the peripheral olfactory system.
The terminal nerve is an anterior cranial nerve that extends between the
nasal cavity and preoptic area in the ventral forebrain of most jawed
vertebrates (Wirsig-Wiechmann et al.
2002). The terminal nerve seems to play a role in reproductive
behavior, although the nature of this role has not been clearly established.
For example, in dwarf gouramis (Colisa lalia), lesions of terminal
nerve cells inhibit initial nest-building behaviors
(Yamamoto et al. 1997). In
rough-skinned newts (Taricha granulosa), gonadotropin releasing
hormone (GnRH) concentrations in the terminal nerve are higher in previously
courted females than in uncourted females
(Propper and Moore 1991). In
male hamsters (Mesocricetus auratus), lesions of the terminal nerve
decrease mating frequency, and reduce responses to female vaginal odors
(Wirsig and Leonard 1987).
Recent work suggests that the terminal nerve may play a modulatory role in
peripheral olfactory systems (Oka and
Matsushima 1993) and that this modulation may underlie its
behavioral effects. The terminal nerve contains several potentially modulatory
compounds, including acetylcholine (Wirsig
and Leonard 1986;
Wirsig-Wiechmann 1990) and
unidentified compounds that display immunoreactivity to FMRFamide and
neuropeptide Y (NPY) (Chiba
2000; Eisthen and Northcutt
1996) and tyrosine hydroxylase
(White and Meredith 1995).
One peptide that has been unambiguously identified in the terminal nerve of
many jawed vertebrates is GnRH (King and
Millar 1992; Schwanzel-Fukuda
and Silverman 1980; Sherwood
et al. 1986). GnRH-containing neurons in the terminal nerve may
release GnRH into the main olfactory and vomeronasal epithelia
(Wirsig-Wiechmann and Jennes
1993; Wirsig-Wiechmann and
Wiechmann 2001). Although the frequency of GnRH release and the
amount of peptide that reaches the olfactory epithelium are not known, these
studies suggest that GnRH released from the terminal nerve may gain access to
receptor neurons, where it may modulate odorant processing. One study suggests
that GnRH can increase the excitability of olfactory receptor neurons: GnRH
applied to olfactory neurons of mudpuppies (Necturus maculosus)
increases the magnitude of a tetrodotoxin-sensitive sodium current and alters
outward currents (Eisthen et al.
2000). In other neural systems, GnRH has been demonstrated to
modulate the excitability of neurons; for example, GnRH excites goldfish
(Carassius auratus) retinal ganglion cells
(Walker and Stell 1986) and
modulates N-type calcium channels in bullfrog (Rana catesbeiana)
sympathetic neurons (Boland and Bean
1993). To date, the direct evidence demonstrating that GnRH
modulates odorant responses in peripheral olfactory systems has not been
obtained, although one study in abstract form has reported that application of
GnRH to rodent olfactory receptor neurons can reduce and/or enhance odorant
responses, depending on the type of odorant used
(Wirsig-Wiechmann et al.
2000).
In the present study, we recorded electrical field potentials, called
electro-olfactograms (EOGs) (Ottoson
1956), from the main olfactory epithelium of axolotls,
Ambystoma mexicanum. Axolotls are essentially a subspecies of tiger
salamander (Shaffer 1993). As
nonmetamorphosing, aquatic amphibians, axolotls are excellent research animals
because they are easily maintained in the laboratory and have large,
accessible olfactory receptor neurons. To investigate whether GnRH affects
odorant responses, EOG responses were elicited by application of one of four
different amino acid odorants before, during, and after GnRH exposure. Our
data demonstrate that application of GnRH alters odorant responses in the
olfactory epithelium.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Fifty-nine adult axolotls (A. mexicanum) obtained from the Indiana
University Axolotl Colony (34 female, 15 male, 10 of undetermined sex) were
kept in aquaria (80 x 40 x 50 cm) containing 100% Holtfreter's
solution, the most commonly used medium for maintaining axolotls
(Armstrong et al. 1989;
Mattison 1982). Holtfreter's
solution contains (in mM) 60 NaCl, 2.4 NaHCO3, 0.67 KCl, 0.81
MgSO4, and 0.68 CaCl2 in deionized water (pH 7.5).
Aquaria were equipped with a recirculating filter system in which Holtfreter's
solution from groups of tanks passed through mechanical and biological filters
and an ultraviolet sterilizer before being returned to tanks.
To minimize stress, no more than six same-sex individuals were housed in each tank. Axolotls were fed commercial salmon chow (Rangen, Buhl, ID) twice each week. The temperature of the tanks ranged between 18 and 22°C, and the photoperiod was altered monthly to match that of the animals' native habitat in Mexico City.
Before surgery, axolotls were anesthetized with pH-corrected 0.1% MS 222 (tricaine methanesulfonate, Sigma Chemical, St. Louis, MO, pH 7.5) in Holtfreter's solution, and immobilized with an intramuscular injection of gallamine triethiodide dissolved in amphibian Ringer solution (Flaxedil, Sigma Chemical; 0.1–0.3 mg/100 g body weight, pH 7.6). Supplemental doses of MS 222 were delivered to the gills, and additional Flaxedil injected intramuscularly as necessary throughout the experiment.
The original experiments described in this paper were conducted in accordance with guidelines established by the Society for Neuroscience, and were approved by the Michigan State University animal care and use committee.
EOG recording
The main olfactory epithelium was exposed by removing the tissue dorsal to
the nasal capsule. To record electrical field potentials, a glass capillary
electrode (100- to 200-µm tip diameter) was filled with 1% agar in Ringer
solution bridged to a chloride-coated silver wire. An Ag-AgCl reference
electrode was placed under the skin on the head and isolated from both the
Holtfreter's and odorant solutions with petroleum jelly
(Park et al. 2001). Electrodes
were coupled to a differential amplifier (DP-301, Warner Instruments, Hamden,
CT). Signals were digitized via an ITC-18 interface (Instrutech, Great Neck,
NY), and recorded and analyzed on a Macintosh computer using AxoGraph software
(v. 4.4, Axon Instruments, Foster City, CA).
The magnitude of the EOG response was measured as the maximal height of phasic displacement from the baseline level. Absolute response values in millivolts were obtained by comparison with the deflection elicited by a known calibration voltage.
Stimulus compounds and delivery
To determine whether the effects of GnRH are odorant-specific, we selected
stimuli to represent four broad categories of amino acids, with minimum cross
reactivity among odorants, as described by Caprio and Byrd
(1984). Odorant stimuli
consisted of 1 mM solutions containing one of four amino acids (Sigma
Chemical): L-lysine (Lys, a basic amino acid),
L-methionine (Met, a neutral amino acid with a long side chain),
L-cysteine (CysH, a neutral amino acid with a short side chain), or
L-glutamic acid (Glu, an acidic amino acid). Stock solutions
containing 10 mM odorant dissolved in Holtfreter's solution were prepared
weekly, stored at 4°C, and diluted in Holtfreter's solution before each
experiment. The pH of each solution was adjusted to 7.5–7.6 using 1 N
HCl or 1 M Tris base to match the Holtfreter's solution in which axolotls were
maintained and that bathed the olfactory mucosa during experiments.
During each trial, a continuous flow (3.5–4 ml/min) of Holtfreter's
solution bathed the olfactory mucosa. For each EOG recording, 
70 µl of
a 1 mM stimulus solution at room temperature (23–25°C) was injected
into the flow of the Holtfreter's solution from a 1 ml syringe connected to a
pressure injector (Picospritzer II, General Valve, Fairfield, NJ). The time of
arrival of the stimulus at the olfactory mucosa was measured by adding a dye
solution (fast green) to the odorant solution on some trials. Using this
method, we found that the odorant arrived at the epithelium 10 s after
injection into the carrier stream and remained on the epithelium
2–3 s.
Experimental protocol
The mammalian form of GnRH (also called mGnRH or LHRH; Peninsula Labs,
Belmont, CA), which is the form present in the terminal nerve of amphibians
(Sherwood et al. 1986), was
dissolved in dH2O and stored in 100 µl aliquots at
–20°C. At the beginning of each day of recording, one aliquot of
GnRH was dissolved in Holtfreter's solution at a final concentration of 10
µM. This concentration of GnRH was selected to match that used in a
previous study in which we found that 10 µM GnRH increased
voltage-dependent inward currents in salamander olfactory cells
(Eisthen et al. 2000). The pH
was then adjusted to 7.5–7.6 using small amounts of 1 N HCl or 1 M Tris
base that were not sufficient to substantially alter the concentration of the
solution.
To determine whether GnRH affects EOG responses, we recorded EOG responses
before, during, and after GnRH application; the experimental protocol is
illustrated schematically in Fig.
1. The interval between consecutive odorant presentations was 4
min and did not produce any indication of odorant adaptation during baseline
recordings of EOG responses during initial trials. To determine the baseline
response level, we recorded at least two to four EOG responses to the stimulus
odorant before GnRH application. Once the EOG responses were relatively
consistent (<10% difference in EOG magnitude for 
2 consecutive
recordings), 10 µM GnRH prepared in Holtfreter's solution was delivered to
the olfactory epithelium continuously for 12 min. This time frame was selected
because previous work indicated that the effects of GnRH on single olfactory
receptor neurons are significant beginning 10–15 min after initial
GnRH exposure (Eisthen et al.
2000). Three EOG responses to the stimulus odorant were recorded
during GnRH application. During the period after GnRH was applied
("wash"), we recorded another six EOG responses to the stimulus
odorant while bathing the olfactory epithelium in running Holtfreter's
solution. To investigate the effects of consecutive exposures to GnRH, we
repeated this procedure for another two trials for each animal with a 60- to
80-min interval between trials. To optimize the signal, the recording
electrode was sometimes relocated at the beginning of a trial. Thirty-three
animals were used in these experiments: 9 for Lys, 10 for Met, 7 for CysH, and
7 for Glu. Seven animals were used in a control experiment in which plain
Holtfreter's solution was substituted for the GnRH solution, and 19 additional
animals were used in experiments to examine the effects of 1 and 5 µM
GnRH.
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Data analysis
For purposes of statistical analysis and data display, we designated the mean magnitude of the two to four EOG responses recorded before the GnRH application as 100% and normalized all other data collected in each trial relative to this mean level. EOG responses from each trial were grouped into blocks of three categories, as follows: baseline, the average magnitude of two to four EOG responses recorded before GnRH exposure; treatment, the average magnitude of three EOG responses recorded during GnRH exposure; and wash, the average magnitude of six consecutive EOG responses recorded during the wash period.
To determine whether EOG responses differed before, during, and after
application of GnRH, we used one-way ANOVA tests to compare data within
trials. To determine whether the effect of GnRH on EOG responses differed
among the three trials, we used a two-way ANOVA (effect of GnRH application
x trial number). Two-way ANOVAs were also used to determine whether EOG
responses elicited by the four odorant stimuli differed (effect of GnRH
application x odorant) and whether the three different concentrations of
GnRH produced different effects (effect of GnRH application x GnRH
concentration). In cases in which an ANOVA indicated a significant difference,
we used Tukey's post hoc test to perform two-point comparisons. To determine
whether the percent of trials resulting in reduction of the EOG response
during GnRH application differs among odorants or among the three trials for
each individual, 
2 tests were used. For some two-point
comparisons, Student's t-test was used.
Because of the relatively long time required to complete a trial, 1 h,
we were concerned about obtaining spurious results due to changes in the
animal's state. In addition, we did not want to include data from trials in
which the GnRH might have been ineffectively removed during the wash period.
We therefore excluded from analysis data from any trial in which the magnitude
of the EOG response during the wash period did not recover to 90% of
baseline. Using this criterion, 125 of 147 trials (85.0%) conducted with 59
animals produced analyzable data. The percent of trials producing analyzable
data did not vary among odorants (2, P = 0.52,
n = 98 trials), among trials (2, P = 0.43,
n = 98 trials) or among concentrations of GnRH (2,
P = 0.38, n = 70 trials) nor between the sexes
(2, P = 0.56, n = 147 trials). Our
observations indicate that the few trials excluded from analysis involved
technical problems such as difficulties with solution delivery or changes in
the level of anesthesia. Thus we did not discard all data from an animal for
which one trial was problematic.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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10% of the baseline
magnitude in control experiments. During the period in which Holtfreter's
solution was applied instead of GnRH, small increases or decreases in the
magnitude of the EOG response were observed. In the majority of trials (13 of
19), EOG responses were slightly reduced during the wash period, although in
the other six trials, a small enhancement in EOG responses was observed.
Because EOG responses did not differ significantly across three trials
[F(4,57) = 0.49, P = 0.74], we pooled data and then
determined whether EOG responses were different before, during, and after
Holtfreter's solution application. As indicated in
Fig. 3, no significant changes
were observed [F(2,18) = 1.09, P = 0.36].
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Effects of GnRH are similar across odorants
EOG responses to each odorant are illustrated in Fig. 2, B–E. In experiments in which 10 µM GnRH was applied to the olfactory epithelium, odorant responses were generally reduced during GnRH application. During the wash period, the response recovered to approximately the initial magnitude, and was enhanced relative to baseline in some trials.
The pattern of EOG responses during a trial did not differ significantly across trials [F(4,63) = 0.55, P = 0.70 for Lys, F(4,72) = 0.28, P = 0.89 for Met, F(4,46) = 0.06, P = 0.99 for CysH, and F(4,54) = 0.10, P = 0.98 for Glu], so we pooled the data across trials for each animal and then determined whether the effect of GnRH differed for the four odorant stimuli. Within the experiments conducted with each odorant, EOG responses differed significantly throughout the course of the trial for all odorants [F(2,24) = 12.9, P < 0.001 for Lys, F(2,27) = 122.9, P < 0.001 for Met, F(2,18) = 6.66, P = 0.007 for CysH, and F(2,18) = 15.4, P < 0.001 for Glu]. Specifically, during GnRH application, EOG responses were reduced relative to baseline for all odorant stimuli (Fig. 3, all Ps <0.05). During the subsequent wash period, EOG responses evoked by Glu were significantly enhanced relative to baseline (Fig. 3, P = 0.03), but responses to Lys, Met, and CysH were not significantly different from baseline (Fig. 3, P > 0.05). The overall effect of GnRH on EOG responses did not differ significantly among the different types of amino acids tested [Fig. 3, F(6,99) = 1.04, P = 0.41].
Although the overall effect of GnRH application was a reduction in EOG
responses, in some cases, EOG responses appeared to be enhanced in the
presence of GnRH. During GnRH application, the magnitude of the mean EOG
response was reduced in 62 of 78 trials (79.5%). In these trials, the
magnitude of the EOG responses averaged 79.4 ± 1.6% of baseline EOG
magnitude (range: 47.0–99.4%). In contrast, EOG responses during GnRH
application were enhanced in the remaining 16 trials with an average magnitude
of 112.3 ± 3.4% of the baseline (range: 100.0–140.7%). The
difference in effect of GnRH could not be attributed to individual
differences, differences across trials, or differences across odorant stimuli.
Fourteen of 33 animals (42.4%) showed reduced responses during GnRH
application in some trials and enhanced responses during other trials. The
percentage of trials in which reduction of EOG responses was observed did not
differ: reduction occurred in 81.5% of cases in the first GnRH trial (22 of 27
trials), 87.0% in the second (20 of 23 trials), and 71.4% in the third (20 of
28 trials; 2, P = 0.89). The effect of GnRH did not
depend on the stimulus used: reduction was observed in 85.0% of trials with
Lys (18 of 21 trials), 75.0% with Met (18 of 24 trials), 66.4% with CysH (10
of 15 trials), and 89.9% with Glu (16 of 18 trials; 2,
P = 0.94).
GnRH may affect temporal properties of odorant adaptation
Although we did not observe evidence of odorant adaptation during EOG
recordings on the first trial for any individual, we frequently observed a
successive decrement in EOG response magnitude over the first few recordings
during the second and third trials. We interpret this progressive decrement in
magnitude, illustrated in Fig.
4, as indicating that odorant adaptation is occurring.
Interpretable data consisting of at least three baseline EOG responses were
collected for 17 animals; of these, odorant adaptation was observed on the
second or third trial, or both, for 13 animals (76.5%). The frequency of
occurrence of adaptation was similar among odorants: adaptation occurred in
five of five animals (100%) for which Lys served as the stimulus, three of
five animals (60%) tested with Met, two of three animals (66.7%) tested with
CysH, and three of four animals (75%) tested with Glu. (Because we noticed
this unexpected phenomenon toward the middle of our experiments, the sample
sizes obtained using different odorants varies.) Differences among odorants
were not significant (2, P = 0.96).
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For further analysis of this phenomenon, we examined all 17 sets of EOG recordings for which data were available and analyzed the first three EOG responses at the beginning of the second trial before the second application of GnRH. For comparison, we also analyzed the first three EOG responses of the second trial for all control subjects for which such data were available (n = 6). EOG responses were normalized such that the magnitude of the very first EOG response was designated 100% and the magnitudes of the second and third EOG responses were expressed as a percent of this value.
Because the pattern of reduction in EOG response magnitude did not differ among the four stimuli used [F(6,51) = 0.21, P = 0.97], we pooled the data obtained using different odorants. As illustrated in Fig. 4, the overall pattern of EOG response magnitude during the first three recordings differed significantly between the control and experimental groups [F(2,69) = 8.42, P < 0.001]. In addition, we found that the magnitude of the second and third EOG responses in GnRH-exposed animals were significantly smaller than those obtained from animals in the control group (Fig. 4; t = 2.68, df = 21, P = 0.01 for the 2nd EOG response, t = 4.03, df = 21, P < 0.001 for the 3rd EOG response). Within-group analyses indicate that the magnitude of the first three EOG responses differed significantly in GnRH-exposed animals [Fig. 4; F(2,48) = 24.6, P < 0.001], but not within the control group [Fig. 4; F(2,15) = 0.73, P = 0.50]. Post hoc tests demonstrate that the magnitude of the second and third EOG responses in the experimental group were significantly smaller than that of the first, and that the magnitude of the third EOG response was smaller than that of the second (Fig. 4; both P < 0.04).
Effects of GnRH on EOG responses may be concentration-dependent
As in the preceding text, all data obtained using varying concentrations of GnRH were normalized relative to the magnitude of the baseline EOG response for each trial. Because the pattern of EOG responses during a trial did not differ significantly across trials, we pooled the data across trials for each animal [F(4,63) = 0.79, P = 0.54 for 1 µM GnRH, F(4,72) = 0.56, P = 0.69 for 5 µM GnRH]. As illustrated in Fig. 5A, 5 and 10 µM GnRH produced similar effects on EOG responses, but the effect of 1 µM GnRH was not as pronounced. Overall, EOG responses before, during, and after GnRH application differed significantly with GnRH concentration [F(4,78) = 4.09, P = 0.005]. We further determined whether EOG responses during and after GnRH application differ across GnRH concentrations. EOG responses during GnRH application did not differ across groups [F(2,23) = 0.32, P = 0.73], but responses during the wash period differed significantly [F(2,23) = 7.62, P = 0.003]. The maximum EOG magnitude during the wash period was 96.5 ± 2.6% (n = 9) in 1 µM GnRH, 120.9 ± 6.9% (n = 10) in 5 µM GnRH, and 122.0 ± 4.3% (n = 7) in 10 µM GnRH. Post hoc tests revealed that EOG responses in experiments using 5 and 10 µM GnRH were significantly different from those in which 1 µM GnRH was used, and were significantly larger during the wash period than in trials in which 1 µM GnRH was used (Fig. 5, all Ps < 0.02).
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The results of within-group analyses indicate that EOG responses before, during, and after GnRH application differed significantly for all three concentrations of GnRH [F(2,24) = 14.77, P < 0.001 for 1 µM GnRH, F(2,27) = 16.55, P < 0.001 for 5 µM GnRH, and F(2,18) = 36.45, P < 0.001 for 10 µM GnRH]. Post hoc tests indicate that EOG responses during GnRH application were significantly reduced relative to baseline at all three concentrations (Fig. 5; all Ps < 0.05). EOG responses during the wash period were significantly enhanced in trials using 5 and 10 µM GnRH (Fig. 5; both Ps < 0.01) but not using in 1 µM GnRH (Fig. 5, P = 0.69).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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).
The magnitude of EOG responses to 1 mM L-amino acids is
relatively small in axolotls, ranging from 100 µV to 1–2 mV, and
is comparable to that of EOG responses recorded from a marine elasmobranch
(Dasyatis sabina) and marine ariidae catfish (Arius felis)
(Caprio 1980;
Silver 1979). In the marine
catfish, the magnitude of EOG responses to some amino acids, like
L-glutamic acid, is smaller than to others, such as
L-cysteine, L-methionine, and L-alanine
(Caprio 1980). The relatively
small EOG magnitude that we recorded in this study may be due to the use of
relatively small amounts of odorants or may be due to the use of Holtfreter's
solution, which contains NaCl and may cause electrical shunting of EOG signals
(Caprio 1980;
Silver 1979). Although the
magnitude of EOG responses we obtained was relatively small, the
signal-to-noise ratio was good, producing reliable, analyzable data throughout
the study.
In our preliminary studies, we delivered 10 µM GnRH to the main
olfactory epithelium of axolotls using the same method we later used to apply
odorants and found that GnRH alone did not induce any EOG response. This
result is consistent with another report that GnRH application alone does not
elicit odorant responses in single olfactory receptor neurons from rodents
(Wirsig-Wiechmann et al.
2000). Although application of GnRH to the olfactory epithelium of
rainbow trout (Oncorhynchus mykiss) has previously been reported to
elicit EOG responses (Andersen and
Døving 1991), more recent evidence suggests that Andersen
and Døving's result may have been caused by contamination (K. B.
Døving, personal communication). Thus we conclude that GnRH does not
serve as an odorant stimulus, and that any effects of GnRH application that we
observed were due to modulatory rather than sensory processes.
GnRH alters EOG responses to L-amino acid odorants. During GnRH
application, reduction of EOG responses was the most common effect, although
we observed both reduction and enhancement of EOG responses. The effect of
GnRH did not differ substantially among odorants nor among trials for each
individual. Similar inhibitory effects of GnRH application have been observed
in isolated rat uterine muscles, although the mechanism underlying this
inhibition is not known (Medeiros et al.
1988). In uterine muscles, GnRH does not directly affect
contractility of the muscles but decreases contractile responses to
acetylcholine and oxytocin (Medeiros et
al. 1988). This modulatory effect of GnRH is detectable 10 min
after initial GnRH exposure and reaches a plateau within 30–60 min,
reducing the response to 80–85% of the control response
(Medeiros et al. 1988).
In single neurons, inhibitory effects of GnRH on ion channels have been
reported in bullfrog sympathetic neurons
(Bley and Tsien 1990;
Boland and Bean 1993;
Elmslie et al. 1990;
Lewis and Ikeda 1997). Neurons
exposed to 300 nM GnRH show an 80% reduction in the magnitude of N-type
Ca2+ inward currents
(Boland and Bean 1993). The
inhibition begins almost immediately after GnRH application, develops over
several seconds, and reverses completely after peptide removal. In addition, a
decrease in the magnitude of Ca2+-dependent
K+ currents has been observed in sympathetic neurons, most likely
as a consequence of inhibition of Ca2+ influx by GnRH
exposure (Boland and Bean
1993). In this system, the decrease in the magnitude of the
Ca2+-dependent K+ current results in reduced
neurotransmitter release evoked by K+ depolarization and in a late
slow excitatory postsynaptic potential
(Lewis and Ikeda 1997).
Additional inhibitory actions of GnRH have been reported in other types of
neurons. For example, in GT1 neurons, GnRH transiently hyperpolarizes membrane
potentials by increasing the internal concentration of
Ca2+; during GnRH application, spike amplitude is
significantly decreased, although the frequency and duration of spikes are
increased (Van Goor et al.
1999). In a previous study using whole cell recordings from
mudpuppy olfactory neurons, we observed a few cases suggesting that 10 µM
GnRH alters an outward current 5–10 min after GnRH application
(Eisthen et al. 2000). These
results imply that GnRH may reduce odorant responses via inhibitory
effects.
We found that EOG responses are enhanced during the wash period in trials
in which Glu was used as the stimulus but not in trials with other odorants.
This enhancement was observed after application of both 5 and 10 µM GnRH
but not after application of 1 µM GnRH. These results suggest that GnRH
could increase the sensitivity of peripheral olfactory systems to selected
odorants. In rainbow trout (Salmo gairdneri) and catfish
(Ictalurus punctatus), the four L-amino acids used in our
study show relatively low cross-adaptation at the olfactory epithelium
(Caprio and Byrd 1984;
Rhein and Cagan 1983); thus
these amino acids probably bind to different receptors and may activate
different signal pathways. GnRH may therefore interact with specific receptors
or signaling pathways, altering responses to specific odorants.
Two possible mechanisms may underlie the enhanced response that we observed
during the wash period. First, enhanced EOG responses may simply represent
over-recovery of the responses. Previous studies of a variety of cell types
demonstrate over-recovery of the response during the wash period after GnRH
application in about half the individual cells
(Bley and Tsien 1990;
Boland and Bean 1993;
Bosma and Hille 1989).
Over-recovery is also observed after GnRH inhibition of both
Ca2+ (Bley and Tsien
1990) and K+ currents
(Boland and Bean 1993;
Bosma and Hille 1989), but the
mechanism underlying the over-recovery is not known. This explanation does not
completely explain our results, as enhanced EOG responses during the wash
period were only observed in experiments in which Glu served as the
stimulus.
Alternatively, GnRH may sensitize odorant responses to specific stimuli.
Enhancement of specific odorant responses was reported in a study of frog
(Rama esculenta/ridibunda) olfactory receptor neurons:
enhanced responses to odorants that generate adenosine 3:5-cyclic
monophosphate (cAMP) are observed 10 min after the application of compounds
such as carbachol and serotonin (Frings
1993). Protein kinase C (PKC) activated by intracellular
Ca2+ serves as a key factor in determining the
responsiveness of adenylyl cyclase (AC) in response to odorant stimulation
(Anholt and Rivers 1990;
Frings 1993). In
GnRH-releasing neurons in the terminal nerve, GnRH induces the release of
Ca2+ from intracellular stores
(Abe and Oka 2000). In
salamander olfactory receptor neurons, GnRH enhances voltage-activated
Na+ currents and also alters outward currents
(Eisthen et al. 2000). If GnRH
interacts with one of the second-messenger-mediated processes that sensitize
odorant responses in olfactory neurons, the result may be enhanced EOG
responses as observed in this study.
In our experiments, we did not detect any signs of odorant adaptation
before GnRH application. After the first trial, however, odorant adaptation to
consecutive odorant presentations was often observed, even though the
inter-stimulus interval and the volume and concentration of stimulus were kept
constant among trials. To date, there are no similar reports. Two different
mechanisms may account for the induction of odorant adaptation after GnRH
application. First, odorant adaptation may be a long-lasting effect of
enhanced EOG responses from GnRH exposure on the previous trial. EOG responses
may adapt during consecutive odorant presentations if some components that
were activated by GnRH and led to enhanced EOG responses are gradually
inactivated during the next trial. If this hypothesis is correct, we should
not find odorant adaptation occurring during trials in which the previous
exposure to GnRH did not enhance EOG responses. Nevertheless, we have detected
odorant adaptation after trials in which EOG responses were not enhanced,
suggesting that this hypothesis is incorrect. Alternatively, GnRH may directly
affect pathways involved in odorant adaptation. For example, in GnRH-releasing
neurons, GnRH releases Ca2+ from intracellular stores
(Abe and Oka 2002).
Ca2+ activates Ca2+-dependent
calmodulin kinase II (CaMKII) in olfactory receptor neurons
(Zufall and Leinders-Zufall
2000). In salamander olfactory neurons, CaMKII determines the
temporal properties of odorant adaptation by altering the sensitivity of AC
(Leinders-Zufall et al. 1999).
Thus it is possible that GnRH may affect one of the mechanisms of odorant
adaptation, for example by causing release of intracellular
Ca2+ which could stimulate CaMKII, altering the
sensitivity of AC.
The effect of GnRH on EOG responses appears to be concentration-dependent.
In our study, the pattern of EOG responses following the application of 5 and
10 µM GnRH was similar, whereas application of 1 µM GnRH produced only a
subset of these effects. The reduction of EOG responses during GnRH
application did not differ with GnRH concentration, but during the wash period
EOG responses were enhanced more by application of 5 and 10 µM GnRH than by
1 µM GnRH. A similar concentration effect has been observed in response to
GnRH application in other neurons. For example, in terminal nerve GnRH
neurons, the increased rate of firing is correlated with the concentration of
GnRH applied (Abe and Oka
2000). In pituitary gonadotrophs, low (picomolar) concentrations
of GnRH induce irregular and low-amplitude changes of intracellular
Ca2+ concentrations, whereas at higher concentrations,
GnRH induces high-amplitude changes
(Krsmanovic et al. 2000). In
intact animals, inputs from hormones and from other neurons may increase or
decrease the amount of GnRH released
(Wirsig-Wiechmann 1993;
Yamamoto and Ito 2000).
Given that we have only recorded from semi-intact preparations of olfactory
epithelium, it is possible that nonneural cells within the olfactory
epithelium may change the physiological environment of olfactory receptor
cells in response to stimulation by GnRH, altering the EOG response
indirectly. For example, supporting cells in the olfactory epithelium of frogs
and mice possess voltage-dependent K+ and Na+
conductances and are highly permeable to K+
(Ghiaroni et al. 2003;
Trotier 1998). In addition,
supporting cells in the main olfactory epithelium can be depolarized by
odorant stimuli or an increase in extracellular K+ concentrations
(Trotier 1998). These studies
suggest that supporting cells in the olfactory epithelium may play an active
role in signal transduction in the olfactory epithelium. If peptides that are
present in the nasal epithelium activate nonneural cells, such as
sustentacular cells or Bowman's cells
(Getchell et al. 1989), the
physiological milieu of the olfactory receptor neurons could be altered,
possibly resulting in altered EOG responses.
Odorant responses in the olfactory epithelium may be modulated by
endogenous chemicals released from the terminal nerve or from the other, as
yet unidentified, sources. The effects of GnRH on EOG responses that we
observed are comparable to those resulting from the application of adrenaline.
In our study, EOG responses during GnRH application were reduced to 80%
of the baseline, and some enhancement was observed during the wash period. In
experiments in which adrenaline has been used, EOG responses are enhanced
20–50% above baseline magnitude within 5–10 min after adrenaline
application, but no reduction is observed
(Arechiga and Alcocer-Cuaron
1969). In studies of single salamander olfactory neurons, GnRH
increases the voltage-activated Na+ current, and may reduce outward
currents (Eisthen et al.
2000). Adrenaline exposure increases the inward Na+
current to 18% above the baseline level in newt olfactory neurons, but no
information about outward currents is available
(Kawai et al. 1999). These
results demonstrate that both GnRH and adrenaline can modulate activity of
olfactory receptor neurons, but the mechanisms of modulation may differ.
Signal modulation in peripheral olfactory systems may be important for
odorant information encoding in the CNS, but few studies have examined the
modulation of odorant responses in the olfactory epithelium. Our results
indicate that GnRH reduces odorant responses in the olfactory epithelium in
the early phase of its application but in some trials enhances responses
during the wash period, possibly in an odorant-specific manner. Given that the
terminal nerve may also release other chemicals into the nasal cavity
(White and Meredith 1995;
Wirsig-Wiechmann 1990;
Wirsig-Wiechmann et al. 2002),
understanding peripheral signal modulation by multiple endogenous chemicals in
olfactory systems could greatly increase our understanding of signal
processing in the olfactory epithelium.
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DISCLOSURES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests: H. Eisthen, Dept. of Zoology, 203 Natural Sciences Bldg., Michigan State University, East Lansing, MI 48824 (E-mail: eisthen{at}msu.edu).
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