Chem. Senses 28: 339-348,
2003
© Oxford University Press 2003
Olfactory Signal Modulation by Molluscan Cardioexcitatory Tetrapeptide (FMRFamide) in Axolotls (Ambystoma mexicanum)
Department of Zoology, Michigan State University, East Lansing, MI 48824, USA
Correspondence to be sent to: Heather L. Eisthen, Department of Zoology, Michigan State University, 203 Natural Sciences Building, East Lansing, MI 48824, USA. e-mail: eisthen{at}msu.edu
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Abstract |
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The terminal nerve, which innervates the nasal epithelia of most jawed vertebrates, is believed to release neuropeptides that modulate activity of sensory receptor neurons. The terminal nerve usually contains gonadotropin-releasing hormone as well as at least one other peptide that has not been characterized, but which bears some structural similarity to molluscan cardioexcitatory tetrapeptide (FMRFamide) and neuropeptide tyrosine (NPY). We investigated the effects of FMRFamide on both voltage-gated currents and odorant responses in the olfactory epithelium of axolotls (Ambystoma mexicanum), using whole-cell patch clamp and electro-olfactogram (EOG) recording techniques. In the presence of FMRFamide, the magnitude of a voltage-gated inward current was dramatically increased, reaching an average of 136% of the initial (pre-exposure) magnitude in neurons that showed a response to the peptide. This increase is detectable within
1–2 min
of exposure to FMRFamide and is sustained for at least 10 min. In EOG
experiments, odorant responses are not affected during FMRFamide application,
but are sometimes increased or decreased during the subsequent wash period. On
average, the largest single EOG response in each trial was detected 25
min after initial FMRFamide application, and ranged from 110 to 147% of
baseline. These results suggest that a compound similar to FMRFamide, if
released from the terminal nerve, may function in peripheral olfactory signal
modulation.
Key words: salamander, terminal nerve, whole-cell patch clamp, electro-olfactogram (EOG), neuropeptide, neuromodulation
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Introduction |
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Studies of the peripheral processing of odorant information generally focus on mechanisms involving single olfactory receptor neurons. The role of multicellular interactions, such as those involved in neuromodulation, receives relatively little attention. In the retina and cochlea of vertebrates, transduction of sensory stimuli into neural signals is generally modulated both locally and by centrifugal inputs (Akopian, 2000
;
Ashmore et al., 2000;
Weiler et al., 2000).
A complete understanding of peripheral processing of odorant stimuli must
include a thorough exploration of the role of neuromodulators.
The terminal nerve is an anterior cranial nerve present in most jawed
vertebrates. The fibers of these bipolar neurons extend rostrally to the nasal
cavity and caudally to the hypothalamic/preoptic area
(Wirsig-Wiechmann et al.,
2002). The cells and fibers of the terminal nerve contain several
potentially modulatory compounds, including gonadotropin-releasing hormone
(GnRH) (Oka, 1992), chemicals
that display immunoreactivity to neuropeptide tyrosine (NPY) or FMRFamide
(Phe-Met-Arg-Phe-NH2)
(Wirsig-Wiechmann, 1990;
Eisthen and Northcutt, 1996;
Chiba, 2000) and to tyrosine
hydroxylase (White and Meredith,
1995), and acetylcholinesterase
(Wirsig and Leonard, 1986;
Wirsig-Wiechmann, 1990). These
compounds appear to be released into the nasal epithelia or nasal cavity
(Wirsig-Wiechmann and Jennes,
1993).
The terminal nerve seems to play a role in reproductive behavior, although
the nature of this role has not been clearly established
(Wirsig and Leonard, 1987;
Yamamoto et al.,
1997). In dwarf gouramis (Colisa lalia), lesions of
terminal nerve cells inhibit initial nest-building behaviors
(Yamamoto et al.,
1997). In male hamsters, lesions of the terminal nerve decrease
mating frequency, and reduce behavioral responses to female vaginal odors
(Wirsig and Leonard, 1987).
The terminal nerve may play a modulatory role in peripheral sensory systems
(Walker and Stell, 1986;
Oka, 1992), and this
modulation may underlie its behavioral effects.
The only terminal nerve peptide that has been positively identified is
GnRH, and its effects on peripheral olfactory systems have recently been
examined. Recordings from slices of olfactory epithelium of salamanders
suggest that GnRH increases the excitability of olfactory receptor neurons
(Eisthen et al.,
2000). Specifically, GnRH applied to olfactory neurons of
mudpuppies (Necturus maculosus) increases the magnitude of a
tetrodotoxin (TTX)-sensitive sodium current, and also alters outward currents.
In another study, Park and Eisthen
(2003) found that
electro-olfactogram (EOG) responses to L-amino acids in axolotls
(Ambystoma mexicanum) were reduced during the application of GnRH,
but then recovered and were sometimes enhanced above the baseline magnitude
during the subsequent wash period. These studies demonstrate that GnRH
released from the terminal nerve into the nasal cavity can modulate peripheral
processing of olfactory signals.
In addition to GnRH, the terminal nerve of most jawed vertebrates contains
a second neuropeptide that has not been identified, but which can be labeled
using antisera directed against FMRFamide or NPY (Chiba,
1997,
2000). These peptides are
evolutionarily unrelated and differ in length, but share a C-terminus that
contains the motif -RFamide or -RYamide
(Greenberg and Price, 1992;
Larhammar, 1996). FMRFamide
was first isolated and characterized from the nervous system of molluscs
(Price and Greenberg, 1977);
since then, FMRFamide and other FMRFamide-like peptides (FLPs) have been
isolated in many invertebrates and vertebrates, including amphibians
(Koda et al., 2002).
FLPs have been shown to serve neuroendocrine functions in vertebrates and
neuromodulatory functions in invertebrates, but neuromodulatory effects of
FLPs have not been explored in detail in vertebrates
(Henry et al., 1999;
Lange and Cheung, 1999;
Loi and Tublitz, 2000;
Tsutsui et al., 2000;
Satake et al., 2001;
Koda et al., 2002).
In cultured spinal neurons from mice, FMRFamide alters ion conductances in
different ways in different neurons
(McCarthy and Cottrell, 1984).
FMRFamide depolarizes horizontal cells from the retinae of white perch,
Morone americana (Umino and
Dowling, 1991), and induces excitatory, inhibitory or biphasic
inhibitory–excitatory responses in retinal ganglion cells from goldfish,
Carassius auratus (Walker and
Stell, 1986). FMRFamide-like immunoreactivity has been described
in the olfactory epithelium of more than a dozen vertebrate species, including
frogs, salmon, shrews, and chickens (Muske
and Moore, 1988; Wright and
Demski, 1996; Rastogi et
al., 2001; Malz and Kuhn,
2002), but the role of FLPs in peripheral olfactory signal
modulation has not yet been investigated.
In the present study, we examined the effects of FMRFamide on both voltage-gated currents and odorant responses in the olfactory epithelium of axolotls, using whole-cell patch clamp and EOG recording techniques. We find that FMRFamide increases the magnitude of a voltage-gated inward current in some olfactory neurons, and modulates odorant responses in the olfactory epithelium of some individuals.
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Methods |
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Subjects
Adult axolotls obtained from the Indiana University Axolotl Colony were
kept in aquaria (80 x 40 x 50 cm) containing 100% Holtfreter's
solution, a standard medium for maintaining amphibians
(Mattison, 1982). All aquaria
were equipped with a recirculating filter system in which water 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 Inc., Buhl, ID) twice weekly. The temperature of the tanks ranged between 18 and 22°C, and the photoperiod was adjusted monthly to match that of the animals' native habitat in Mexico City.
The original research reported in this paper was conducted according to animal care and use guidelines established by the Society for Neuroscience and the US Public Health Service.
Whole-cell recordings
Olfactory epithelial slices were prepared using a protocol described
previously (Eisthen et al.,
2000). Axolotls were immersed in ice water for 20 min and then
decapitated. The nasal sac was dissected out, opened flat, and attached to a
support with cyanoacrylate glue. Slices 200–250 µm thick were cut
with a vibrating blade and stored in amphibian physiological saline containing
(in mM): 120 NaCl, 10 HEPES, 8 CaCl2, 5 glucose, 5 pyruvate, 2.5
KCl, and 1 MgCl2. The pH was adjusted to 7.6 using 1 N HCl or 1 M
NaOH. Slices of olfactory epithelium prepared in this manner remained viable
for recording at room temperature for at least 4 h, and when stored at 4°C
could be used for 48 h.
Epithelial slices were mounted in a recording chamber and viewed with a
40x water immersion objective on a Zeiss Axioskop FS microscope. Patch
electrodes of borosilicate glass were pulled on a Flaming-Brown programmable
micropipette puller (Sutter Instruments, Novato, CA). For whole-cell
voltage-clamp recordings, pipette resistance was generally 3–5 M
.
All recordings were conducted at room temperature using an Axopatch 200B
amplifier (Axon Instruments, Foster City, CA) with a low-pass Bessel filter
set at 5–10 kHz. Raw data were collected and leak current subtracted
before analysis using AxoGraph software (Axon Instruments, Foster City,
CA).
For some recordings, we did not use ionic substitutions in the
intracellular solution; in these experiments, the recording pipette was filled
with a solution containing (in mM): 105 K gluconate, 25 KCl, 10 HEPES, 5 ATP,
3 MgSO4, 1 EGTA, 0.5 GTP and 0.5 CaCl2. Our preliminary
experiments suggested that the effects of FMRFamide on outward currents were
variable but that effects on inward currents were more consistent and more
reliably obtained, so we chose to study the latter. In most experiments, then,
cesium was substituted for potassium to block large outward potassium currents
that might mask FMRFamide-sensitive inward currents; calcium-dependent outward
currents were blocked through the substitution of BaCl2 for
CaCl2 in the amphibian physiological saline described above. In
such experiments, the intracellular solution contained (in mM): 135 CsCl, 10
HEPES, 5 ATP, 3 MgSO4, 1 K4BAPTA, 0.5 GTP and 0.085
CaCl2. FMRFamide (Phe-Met-Arg-Phe-NH2; Bachem, Torrance,
CA) was dissolved in amphibian physiological solution at a concentration of 10
µM and bath-applied to slice preparations during recording; the dose was
selected based on a study using an endogenous amphibian FLP
(Koda et al., 2002).
We added a dye, fast green, to this solution to allow us to verify the timing
of FMRFamide application and wash. In previous studies, we have found that
fast green does not function as an odorant or alter activity of voltage-gated
ion channels in salamander olfactory epithelia
(Eisthen et al.,
2000; Park and Eisthen,
2003).
Solutions were introduced into the recording chamber using a gravity-feed
system. Our observations indicate that the amphibian physiological solution
covering a slice was completely replaced 60 s after solution sources were
changed. In some experiments, a bath solution containing fast green but not
FMRFamide was used as a control for the effects of changing solutions. In the
remainder of the paper, we refer to this solution as the `control solution';
note that the control solution is different from the `wash', which consisted
of plain physiological saline without fast green.
For each recording, we followed a standard protocol to ensure that
comparable data were collected from all cells. Once a seal of 1–5
G was attained, the membrane under the electrode was ruptured, and a
holding potential between –50 and –90 mV was applied. We recorded
responses to a series of 15 ms voltage pulses ranging from –100 mV to
100 mV in 10 mV steps. We then initiated the flow of amphibian physiological
solution over the slice and recorded responses to a similar set of pulses 1
min later. Recordings were made every 2 min in a flowing bath for 3–6
min until the seal and series resistance stabilized; usually two or three sets
of recordings were made in the flowing bath before FMRFamide was applied.
Recordings were made 1 min after the flow of FMRFamide was initiated, then
again at 2 min intervals for 10–12 min. While washing off the FMRFamide
with amphibian physiological saline, we recorded responses at 2 min intervals
for 6–16 min. Once a slice had been exposed to FMRFamide, the slice was
discarded.
In some experiments, the effect of FMRFamide appeared to depend on the holding potential applied. To test this possibility directly, we performed some recordings using a protocol similar to that described above, except that the holding potential was adjusted from –50 mV to –80 mV in a cycle of 10 mV steps once per minute. At least two sets of recordings at the four different holding potentials were made before FMRFamide was applied. Beginning 1 min after FMRFamide application, another four or five sets of recordings at each holding potential were made. We recorded responses to at least three sets of pulses at each holding potential during the washing off of the FMRFamide. In this experiment, recording pipettes were filled with cesium-substituted intracellular solution and the amphibian physiological saline contained BaCl2 instead of CaCl2. This experiment was completed in five cells.
We analyzed data for all cells for which we obtained responses to at least
four sets of pulses during the 10 min FMRFamide application, provided the seal
was >1 G and the series resistance did not increase more than
10% throughout the recording. Using these criteria, we report data
obtained from a total of 39 cells from 20 axolotls.
EOG recordings
Before surgery, axolotls were anesthetized with pH-corrected 0.1% MS 222 (tricaine methanesulfonate, pH 7.5, Sigma Chemical, St Louis, MO) in Holtfreter's solution, and immobilized with an intramuscular injection of gallamine triethiodide dissolved in amphibian Ringer's 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 was injected intramuscularly as necessary throughout the experiment.
The main olfactory epithelium was exposed by removing the tissue dorsal to
the nasal capsule. To record electrical field potentials, a glass capillary
electrode (100–200 µm tip diameter) was filled with 1% agar in
Ringer's solution bridged to a chloride-coated silver wire. An Ag–AgCl
reference electrode was placed under the skin of the head
(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 Co., Great Neck, NY), and then displayed, recorded, and analyzed
on a Macintosh computer using AxoGraph software (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.
During each trial, a continuous flow (3.5–4 ml/min) of Holtfreter's
solution bathed the olfactory mucosa. Holtfreter's solution contains (in mM):
60 NaCl, 2.4 NaHCO3, 0.67 KCl, 0.81 MgSO4 and 0.68
CaCl2 in distilled water (pH 7.5–7.6, adjusted by the
addition of 1 N HCl or 1 M Tris base). For each EOG recording, 50 µl
of 1 mM L-methionine dissolved in the Holtfreter's solution at room
temperature (23–25°C) was injected from a 1 ml syringe connected to
a pressure injector (Picospritzer II, General Valve, Fairchild, NJ) into the
flow of the Holtfreter's solution. We selected L-methionine (Sigma
Chemical) as an odorant because it is a potent stimulus for EOG responses in
axolotls (Park and Eisthen,
2003). To ensure that identical amounts of odorant were delivered
throughout EOG recordings, the settings on the Picospritzer were not adjusted
during a trial. The time of arrival of the injected stimulus at the olfactory
mucosa was measured by adding fast green to the odorant solution on some
trials; using this method, we found that the odorant solution arrived at the
epithelium 10 s after injection into the carrier stream and remained on
the epithelium for 2–3 s.
To determine whether FMRFamide affects EOG responses, we recorded EOG
responses to 50 µl of 1 mM L-methionine before, during and after
FMRFamide application. To establish a baseline response level, we recorded two
to four EOG responses to the stimulus odorant before FMRFamide application.
The interval between consecutive odorant presentations was 4 min, and did not
produce any sign of odorant adaptation during baseline recordings of EOG
responses. Once the EOG responses were relatively consistent (less than
10% difference in EOG magnitude), 5 µM FMRFamide prepared in
Holtfreter's solution was delivered to the olfactory epithelium continuously
for 12 min. The dose of FMRFamide was based on a previous study using
endogenous FLPs in bullfrogs (Koda et
al., 2002). Three EOG responses were recorded at 4 min
intervals during FMRFamide application. During the period after FMRFamide was
applied (`wash'), we recorded another nine EOG responses while bathing the
olfactory epithelium in running Holtfreter's solution.
To investigate the effects of consecutive exposures to FMRFamide, we repeated this procedure for another two trials for each subject, with a 60–80 min interval between consecutive trials. To optimize the signal, the recording electrode was sometimes relocated at the beginning of a trial. Fourteen animals were used in these experiments.
In control experiments with five animals, we repeated the procedures described above, except that we substituted Holtfreter's solution for FMRFamide solution. As in experiments with FMRFamide, three trials, separated by a 60 to 80 min interval, were conducted with each animal.
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Results |
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Whole-cell recordings
FMRFamide did not affect the magnitude of the inward current in all olfactory receptor neurons examined; thus, we categorized cells as `responders' and `non-responders' for further analysis. A cell was considered a `responder' if the magnitude of the inward current was elevated above baseline for at least four consecutive data points while exposed to FMRFamide. A cell for which the inward current was steady was categorized as a `non-responder'. Cells that displayed a variable inward current were categorized as uninterpretable and excluded from further analyses; we did not observe any cases in which the inward current was decreased relative to baseline for 4 or more consecutive readings. Using these criteria, 10 of 39 olfactory receptor neurons (25.6%) were classified as `responders'. An example of data from a `responder' is illustrated in Figure 1. Twenty-three additional cells (59.0%) were classified as `non-responders', and the responses of the remaining six cells were uninterpretable. Data from the 10 cells that responded to FMRFamide are described below.
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As illustrated in Figure 2,
the inward current was increased relative to baseline (the average magnitude
of the inward current on the last two recordings before FMRFamide
application), beginning 1–2 min after FMRFamide application began.
This increase was sustained throughout the FMRFamide exposure period and
recovered to the baseline level within 5–10 min after washing FMRFamide
off.
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To calculate the latency to and magnitude of the largest inward current, we
used four cells for which we were able to complete our recording protocol,
including the full wash period. In these cells, the largest inward currents
were detected 10 min (9.71 ± 0.34 min) after initial FMRFamide
application, and ranged from 119.5 to 146.2% (136.1 ± 6.02%) of the
baseline magnitude.
We investigated the possibility that the holding potential interacts with FMRFamide effects. In three of the five cells examined, the inward current was increased after FMRFamide application at all holding potentials (–50, –60, –70 and –80 mV). In two cells the inward current was only increased at a specific holding potential: in one case, –50 mV, and in the other, –80 mV. In these two cells, the inward current was unchanged at other holding potentials during FMRFamide application.
EOG recordings
As illustrated in Figure 3A,
EOG responses in control experiments varied within 5% of the baseline
magnitude throughout the experimental protocol. Overall, EOG responses did not
differ significantly among the three trials in control experiments
(Kruskal–Wallis test, P = 0.99), nor did they vary
significantly within any of the three trials (Kruskal–Wallis test,
P = 0.39 for the first trial, 0.73 for the second trial, and 0.57 for
the third trial). Thus, we pooled data within and across the three control
trials for presentation in Figure
4.
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As in our control experiment, 4 of 14 experimental animals displayed less
than 5% change in EOG magnitude during FMRFamide application in any of the
three trials. These animals were categorized as `non-responders' and excluded
from further analysis. We also excluded from analysis 3 of 30 trials from the
remaining 10 animals in which technical problems occurred, such as changes in
the subject's level of anesthesia during the trials. The percent of analyzable
data obtained did not differ among trials (
2, P =
0.94). Data from these 27 trials in the 10 animals that responded to FMRFamide
are described below.
Figure 3B illustrates EOG responses during an experiment in which 5 µM FMRFamide was applied to the olfactory epithelium. In such experiments, EOG responses were generally unaffected during the period of FMRFamide application, ranging from 93.6% to 103.8% of the baseline magnitude. Nevertheless, the magnitude of the EOG response was often increased or decreased relative to baseline during the subsequent wash period.
For statistical analysis and data display, we designated the mean magnitude of the two to four EOG responses recorded before the FMRFamide application as 100%, and normalized all other data collected in each trial relative to this mean. EOG responses from each trial were grouped into three categories, as follows: baseline EOG responses, the average magnitude of two to four EOG responses recorded before FMRFamide exposure; FMRFamide EOG responses, the average magnitude of three consecutive EOG responses recorded during FMRFamide exposure; and wash EOG responses, the average magnitude of nine consecutive EOG responses recorded during the wash period. The same analysis was performed for each trial, using the baseline magnitude from that trial as the standard for comparison. The values obtained for each treatment within a trial were averaged across animals, but we did not average the data across trials.
Statistical tests indicated that some of the data were not distributed
normally; thus, we used nonparametric statistics for most analyses. To
determine whether odorant responses differ before, during and after FMRFamide
application within a trial, we used the Kruskal–Wallis test, a
non-parametric analog of the one-way ANOVA test. A significant result in the
Kruskal–Wallis test indicates that the EOG responses are significantly
different between treatments, but does not indicate which particular
treatments differ. Therefore, when the Kruskal–Wallis test indicated
significant differences among treatments, we performed additional
nonparametric post hoc tests with an 
level of 0.05, as
described in Siegel and Castellan (Siegel
and Castellan, 1988) for two-point comparisons. In these cases, we
performed pairwise comparisons among all data points.
Using this method to compare results within trials, we found that EOG responses before, during, and after FMRFamide application varied significantly in the second (H30/2 = 19.99, P < 0.001) and third trials (H24/2 = 6.86, P = 0.032), but not in the first trial (P = 0.86). Post hoc tests indicate that the magnitude of EOG responses obtained during FMRFamide application did not differ from baseline (both P > 0.05). Curiously, EOG responses during the wash period were enhanced relative to baseline in the second trial, but were depressed in the third trial (both P < 0.05). The results of these analyses are illustrated in Figure 4.
We determined whether the effects of FMRFamide differed among the
three trials using the Kruskal–Wallis test followed by nonparametric
post hoc tests for two-point comparisons
(Siegel and Castellan, 1988),
as described above. The magnitude of the baseline EOG response, calculated
from raw data, did not differ among trials (H28/2 = 0.124,
P = 0.94). We found that the effect of FMRFamide on EOG responses
differed significantly across the three trials (H81/2 =
12.70, P < 0.002). Specifically, the magnitude of the EOG
responses during the wash period differed significantly across trials
(Figure 4:
H27/2 = 11.64, P = 0.003), but those during
FMRFamide application did not (Figure
4: P = 0.62). Post hoc tests indicate that the
EOG responses during the wash period in the second trial were larger than
those in the third trial (P < 0.05), but other comparisons did not
indicate significant differences (P > 0.05, for all cases).
Because EOG responses during the wash period were significantly different
among three consecutive trials, we also measured and analyzed (i) the
magnitude of the largest single EOG response elicited during the wash period
for each trial, expressed as a percent of the mean baseline EOG magnitude, and
(ii) the duration of the time interval between the initial FMRFamide
application and the largest EOG response, or the `latency' to the largest EOG
response within each trial. The data concerning the magnitude of and latency
to the largest EOG responses were normally distributed (Shapiro–Wilk
test, P = 0.27 and 0.54, respectively), so we analyzed the data using
one-way ANOVA, followed by Tukey's post hoc test
(Sokal and Rohlf, 1981).
In accord with the results of across-trial analyses, we found that the magnitude of the largest EOG response elicited during the wash period differed significantly among the three trials (Figure 5A: F24/2 = 4.10, P = 0.029). The magnitude of the largest EOG response in the second trial (mean ± SEM, 146.54 ± 7.67%; n = 10) was larger than that in the third trial (mean ± SEM, 110.14 ± 6.30%; n = 8), and this difference was significant (Tukey's test, P = 0.028). The magnitude of the largest EOG response obtained during the first trial did not differ significantly from those obtained during other trials. In addition, the latency to the largest EOG response did not differ among trials, ranging from 21.3 to 27.5 min across trials (Figure 5B: one-way ANOVA, P = 0.50).
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Discussion |
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To investigate whether FMRFamide affects voltage-dependent currents in single olfactory neurons and odorant responses from the olfactory epithelium of axolotls, we used whole-cell patch clamp and EOG recording techniques. We found that FMRFamide increased voltage-dependent inward currents and altered EOG responses to L-methionine.
Given the magnitude, rate of onset and inactivation, and reversal potential
of the current (60 mV), the inward current that was increased by the
application of FMRFamide appears to be carried by sodium. In separate
experiments, we have found that a current with the same characteristics can be
reversibly blocked by application of 1 µM TTX, indicating that the current
that is enhanced in the presence of FMRFamide is a voltage-dependent sodium
current (data not shown). The voltage-gated inward current that was affected
by FMRFamide was robust after replacing CaCl2 with BaCl2
in the extracellular solution, indicating that the current is not dependent on
external calcium, but we cannot rule out the possibility that the altered
current is partially carried by calcium (or, in some experiments, barium).
It is possible that FMRFamide is directly gating amiloride-sensitive
(Cottrell, 1997;
Cottrell et al.,
1984) or acid-evoked ion channels that are members of the
degenerin/epithelial Na+ channel (DEG/ENaC) family
(Askwith et al.,
2000), although our data suggest that this is not the case. The
inward current that we found to be altered by FMRFamide is voltage-activated,
whereas DEG/ENaC channels are not
(Cottrell, 1997;
Askwith et al., 2000).
In addition, our extracellular solutions contained relatively high
concentrations of divalent cations (8 mM Ca2+ or Ba2+),
which may be sufficient to block amiloride-sensitive channels
(Green and Cottrell, 2002).
The pH of our extracellular solution was 7.6, too high for FMRFamide to
potentiate acid-evoked channels (Askwith
et al., 2000). Thus, although we cannot rule out the
possibility that the effects we observed were due to activation of one or both
of these types of channels in axolotl olfactory receptor neurons, this
scenario seems unlikely.
In invertebrates, FMRFamide has been shown to alter the conductance of
sodium currents, resulting in modulation of neuronal activity. For example, in
peptidergic neurons of the central nervous system that are involved in egg
laying in the mollusc Lymnaea stagnalis, FMRFamide reduces the
magnitude of a TTX-sensitive, voltage-gated Na+ current
(Brussaard et al.,
1990). Such a change results in an increased threshold for action
potential generation, and arrests ongoing firing activity of the cells. In
addition, FMRFamide applied to the abdominal ganglion cells of Aplysia
californica induces a slow inward current by opening voltage-dependent
Na+ channels, resulting in depolarized cells
(Chiba et al., 1992).
Furthermore, in LFS neurons in the neural circuit underlying the siphon
withdrawal reflex of Aplysia, FMRFamide application typically
produces a biphasic response, involving a fast excitatory response followed by
a prolonged inhibitory response (Belkin and
Abrams, 1998). Activation of a TTX-insensitive Na+
conductance by FMRFamide plays a key role in the early transient
depolarization of the neurons (Belkin and
Abrams, 1998). These results lend support to our suggestion that
FMRFamide may modulate the excitability of olfactory receptor cells by
altering voltage-activated sodium currents.
FMRFamide alone does not appear to elicit an odorant response. In our
patch-clamping experiments, we found that FMRFamide altered the magnitude of
an inward (probably sodium) current, but observed no responses similar to
those elicited by odorants. Similar results have been obtained in response to
GnRH application in mudpuppy olfactory receptor neurons
(Eisthen et al.,
2000). The results of our EOG experiments also support our
contention that FMRFamide does not evoke odorant responses. We conducted a
preliminary experiment with one animal, in which FMRFamide solution alone was
applied to the olfactory epithelium, and we were not able to record an EOG
response to this stimulus (data not shown). In addition, in the data presented
here, we found that EOG responses to an odorant solution were not altered
during FMRFamide application, but only during the wash period. Taken together,
these observations indicate that FMRFamide does not directly induce odorant
responses, but instead is functioning to modulate activity of olfactory
receptor neurons.
FMRFamide appears to affect odorant responses in the olfactory epithelium.
In our EOG study, we found that odorant responses were not affected during the
period of FMRFamide application, but were enhanced during the wash period in
the second trial. Although the mechanism underlying this phenomenon is
unknown, similar effects have been described in other animals, using different
compounds. For example, in frog (Rana esculenta/ridibunda)
olfactory receptor neurons, enhanced responses to odorants that generate
adenosine 3:5-cyclic monophosphate (cAMP) were observed 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 studies of invertebrates, FMRFamide has been shown to
alter cAMP and AC concentrations: for example, FMRFamide applied to molluscan
heart cells increases both cAMP and AC concentrations
(Higgins et al.,
1978; Willoughby et
al., 1999), and markedly increases AC concentrations in
salivary glands of the freshwater snail, Planorbarius corneus
(Ferretti et al.,
1996). These results suggest that FMRFamide might modulate odorant
responses in peripheral olfactory systems by interacting with one or more
biochemical pathways involved in signal processing.
In our EOG study, we found that the effect of consecutive FMRFamide
exposures was significantly different across three trials: the effect of
FMRFamide on odorant responses was enhanced in the second trial, and was
significantly decreased in the third trial. This difference cannot be
explained by deterioration of the EOG responses or changes in the subjects'
state, for we found in control recordings that EOG responses were similar in
all three trials, and in FMRFamide experiments we found no cross-trial
difference in the magnitude of the baseline EOG response. A similar
differential response induced by consecutive FMRFamide applications has been
reported in a study of retinal ganglion cells in goldfish (Carassius
auratus). In several such cells, the excitatory response caused by the
first application of FMRFamide was much larger than those evoked by subsequent
applications (Walker and Stell,
1986). Thus, although we do not understand the mechanisms
involved, our data suggest that FMRFamide produces relatively long-term
changes in the functioning of the olfactory epithelium.
Given that we have recorded only from semi-intact preparations of olfactory
epithelium, we do not know whether FMRFamide induces responses directly by
binding to receptors on olfactory receptor neurons, or indirectly by affecting
other types of cells in the olfactory epithelium. In addition, we cannot
determine whether the effects we observed are due to binding of FMRFamide to
FLP receptors, or whether we are activating receptors for other peptides, such
as NPY. Finally, we do not yet know whether the terminal nerve of axolotls
produces an endogenous FMRFamide-like peptide that is released into the nasal
epithelia. Nevertheless, FMRFamide-like peptides have been isolated from
several vertebrate species, including an amphibian
(Tsutsui et al.,
2000; Satake et al.,
2001; Koda et al.,
2002), and the brain, terminal nerve and olfactory epithelium of
amphibians show FMRFamide-like immunoreactivity
(Muske and Moore, 1988;
Rastogi et al.,
2001).
In both our patch-clamping and EOG experiments, we found that not all cells
and subjects respond to FMRFamide. Similar results have been obtained in
previous studies using GnRH, in which we found that some cells and subjects
were affected by GnRH and others were not
(Eisthen et al.,
2000; Park and Eisthen,
2003). We have demonstrated that the proportion of cells
responding to GnRH varies across the breeding season, suggesting that
endogenous factors regulate GnRH responsivity
(Eisthen et al.,
2000). Given that the terminal nerve seems to play a role in
reproductive behavior, perhaps the activity of the terminal nerve or
responsivity to its peptides generally varies with reproductive condition. If
so, we would expect to find that any terminal nerve-derived peptide only
affects a subset of cells and subjects.
Several different compounds have now been demonstrated to modulate activity
of olfactory receptor neurons. The effects of GnRH and adrenaline have been
examined using both whole-cell patch clamp and EOG recording techniques in
amphibians. The results from these studies are broadly comparable to the data
we present here, although the species examined, odorant stimuli, and
concentrations of modulatory compounds vary across studies. We find that only
some cells and animals respond to FMRFamide; similar results have been
obtained in studies of the modulatory effects of GnRH
(Eisthen et al.,
2000; Park and Eisthen,
2003), but information concerning the proportion of cells and
animals that respond to adrenaline is not available. In the present study, we
found that FMRFamide increased the magnitude of the inward current by as much
as 136% relative to baseline. GnRH has been shown to increase the magnitude of
the Na+ current 120%
(Eisthen et al.,
2000), and adrenaline increases the magnitude of the
Na+ current 118% (Kawai
et al., 1999). The time interval between application of
the compound and the maximal effect varies considerably, ranging from 4
min for adrenaline (Kawai et al.,
1999) to 10 min for FMRFamide and 15 min for GnRH
(Eisthen et al.,
2000). In EOG studies, the magnitude of the enhanced responses is
similar: 120–150% of the baseline for adrenaline
(Arechiga and Alcocer-Cuaron,
1969), 116–157% for GnRH
(Park and Eisthen, 2003), and
110–147% for FMRFamide. The time interval between application of the
compound and the maximal EOG response is similar for FMRFamide and GnRH:
21–27 min for FMRFamide and 25–40 min for GnRH
(Park and Eisthen, 2003). In
contrast, Arechiga and Alcocer-Cuaron
(Arechiga and Alcocer-Cuaron,
1969) report that adrenaline increases EOG responses to a maximal
level within 5–10 min. These results suggest that different compounds
may modulate activity of the olfactory epithelium over different time scales.
With all three compounds, studies using patch-clamping and EOG recordings
consistently reveal discrepant results, but it is not clear whether these
discrepancies reflect real differences in the processes and mechanisms
measured using the two recording techniques or are due in part to disruptions
of physiological processes caused by the different recording preparations.
In conclusion, our data demonstrate that terminal-nerve-derived compounds may be involved in peripheral olfactory signal modulation. Given that the terminal nerve may contain additional compounds, such as NPY, substance P and acetylcholine, a complete characterization of these compounds and their effects on the olfactory epithelium could greatly enhance our understanding of odorant processing in the periphery.
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Acknowledgments |
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We thank Jessica Fawley for assistance with some of the patch-clamping experiments reported here, and Jeanette McGuire, Jennifer Schlegel and Julie Ziobro for their diligence in taking care of our research animals. This work was supported by grants from the National Science Foundation (IBN 9982934) and the National Institutes of Health (DC05366).
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Accepted April 6, 2003
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