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First published online September 19, 2006
Journal of Experimental Biology 209, 3898-3912 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02432
Perfusion with cGMP analogue adapts the action potential response of pheromone-sensitive sensilla trichoidea of the hawkmoth Manduca sexta in a daytime-dependent manner
Biologie, Tierphysiologie, Philipps-Universität Marburg, Karl-von-Frisch-Straße, D-35032 Marburg, Germany
* Author for correspondence (e-mail: stengl{at}staff.uni-marburg.de)
Accepted 6 July 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: insect olfaction, pheromone transduction, moth, cyclic nucleotide, antenna, circadian difference
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Keil, 1989;
Lee and Strausfeld, 1990).
Both ORNs are sensitive to different components of the pheromone blend. Only
one of the ORNs, which elicits the larger action potentials (APs), responds to
the main sex pheromone component bombykal (BAL)
(Starratt et al., 1979;
Tumlinson et al., 1989;
Kaissling et al., 1989;
Dolzer et al., 2001;
Kalinová et al.,
2001).
Much is already known about the peripheral signal transduction processes
underlying the detection of pheromone by the male moth. Pheromone-dependent
rises in inositol triphosphate (IP3)
(Breer et al., 1990) cause an
influx of Ca2+ into insect ORNs
(Stengl, 1994). This triggers
the opening of Ca2+-dependent ion channels involved in the
generation of pheromone-dependent receptor potentials
(Stengl, 1993;
Stengl, 1994;
Zufall and Hatt, 1991;
Zufall et al., 1991). In
contrast, little is known about the modulation of the pheromone transduction
cascade leading to adaptation and sensitization. Previous studies in
Manduca sexta distinguished desensitization from short-term and
long-term adaptation (Dolzer et al.,
2003). Desensitization is the decline in excitation, as seen
during a phasic-tonic response to a stimulus of long duration, while
short-term adaptation is the rapidly (within seconds to minutes) reversible
reduction of sensitivity due to prior stimulation. Long-term adaptation is the
more slowly occurring and longer persisting (for minutes to hours) reduction
of sensitivity due to previous strong stimulation
(Ziegelberger et al., 1990;
Marion-Poll and Tobin, 1992;
Boekhoff et al., 1993;
Stengl et al., 2001;
Dolzer et al., 2003).
Increasing evidence supports the involvement of cyclic guanosine monophosphate
(cGMP) in long-term olfactory adaptation and regulatory processes in moths.
After strong pheromone stimulation, cGMP levels rise slowly in antennae and
antennal homogenates of Antherea polyphemus and Bombyx mori,
remaining elevated for nearly 30 min and, thus, matching the time course of
long-term adaptation (Ziegelberger et al.,
1990; Boekhoff et al.,
1993). In the silkmoth Bombyx mori the perfusion of the
sensillar lymph with dibutyryl guanosine 3',5'-cyclic
monophosphate caused a suppression of the sensillar potential and a reduction
of the AP response in pheromone-dependent sensilla, mimicking a state of
adaptation (Redkozubov,
2000).
In tip recordings of trichoid sensilla of Manduca sexta, adapting
pheromone stimulation shifted the dose-response curves to higher stimulus
intensities (Dolzer et al.,
2003). In other moths, as for Manduca sexta, this shift
is also larger for the AP than for the sensillar potential response,
suggesting the presence of more than one adaptation mechanism
(Zack, 1979;
Kaissling et al., 1986;
Kaissling et al., 1987;
Dolzer et al., 2003). At least
one adaptation mechanism acts on the first step of the olfactory transduction
cascade, the generation of the receptor potential, which is assumed to occur
in the outer dendritic segment of the ORNs
(Kaissling and Thorson, 1980;
Stengl et al., 1998;
Dolzer et al., 2003). An
additional adaptation mechanism apparently acts on the second step of the
transduction cascade, the transformation of the amplitude-modulated receptor
potential into frequency-modulated APs. The APs are apparently elicited in the
soma- or axon-hillock region, as indicated by the polarity of APs and the
current necessary to elicit them, and not at a dendritic location, as
suggested by other experiments (Kaissling
and Thorson, 1980; de Kramer,
1985; de Kramer et al.,
1984; Dolzer et al.,
2001). The molecular basis and the behavioral consequences of
these different mechanisms of insect olfactory adaptation are still not fully
understood.
Previous studies have revealed that the responsiveness of male moths to
pheromone is daytime-dependent. Males of the nocturnal moths Trichoplusia
ni and Agrotis segetum showed a distinct daily rhythm in their
response to pheromone, with a maximum response during the scotophase
(Linn et al., 1996;
Rosén et al., 2003).
Also, daytime-dependent changes in responsiveness to pheromone persist under
constant conditions so are controlled by an endogenous circadian clock
(Baker and Cardé, 1979;
Rosén et al., 2003).
Not only male responsiveness, but also calling behavior and pheromone release
of female moths, show a diurnal distribution with a maximum at the end of the
scotophase for Manduca sexta and the middle of the scotophase for
Agrotis segetum (Itagaki and
Conner, 1988; Rosén,
2002). For Manduca sexta it was shown that peaks in the
calling behaviour of female moths and male flight activity are correlated
during the scotophase (Sasaki and
Riddiford, 1984). In addition, Spodoptera littoralis
moths mated significantly less when males and females were raised in different
light:dark cycles out of phase (Silvegren
et al., 2005). This indicates that circadian clocks rule rhythmic
mating preference in male and female moths and that photoperiod and pheromones
synchronize the mating behavior of both sexes
(Silvegren et al., 2005).
To determine whether daytime-dependent changes in pheromone sensitivity already occur at the periphery in male Manduca sexta moths and to determine whether cGMP-dependent mechanisms of long-term adaptation might be employed, we applied the membrane-permeant cGMP analogue 8-bromo guanosine 3',5'-cyclic monophosphate (8bcGMP) in extracellular tip recordings from trichoid sensilla at two Zeitgebertimes (ZTs; ZT 1-4 and ZT 8-11). ZT 1 is at the beginning of the day (lights on at ZT 0) when the nocturnal moths switch from their active to their inactive phase, and ZT 8 is at the middle of the day when the moths are resting. Pheromone responses to the main pheromone component BAL and spontaneous APs of unstimulated ORNs of the hawkmoth Manduca sexta were investigated.
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)]. Male pupae were kept under a long-day photoperiod (17 h:7
h L:D) at 24-27°C and 40-60% relative humidity and were isolated 1 day
before emergence, gently cleaned with 70% ethanol, and allowed to hatch
without contact with pheromone. After their second dark phase, 30 min prior to
the start of each recording, the adults were taken out of the isolation boxes
and fixed in a TeflonTM holder. Thus, moths at both ZTs spent the same
time in the TeflonTM holder. The flagellum of the right antenna was
immobilized with dental wax (Boxing wax, Sybron/Kerr, Romulus, MI, USA), and
the 15-16 most apical annuli were clipped off. A glass electrode filled with
haemolymph Ringer (Kaissling,
1995), which served as the indifferent electrode, was inserted
into the flagellar lumen. To stop the first annuli from drying out the end of
the antenna was sealed with ECG electrode gel (PPG, Hellige, Freiburg,
Germany) afterwards. The tips of long trichoid sensilla from the apical row on
the second remaining annulus were clipped off using sharpened forceps. The
recording electrode, filled with sensillum lymph Ringer
(Kaissling, 1995), was slipped
over one sensillum. To minimize contributions of the electroantennogram, we
recorded from an annulus close to the tip of the haemolymph electrode. The
connection to the amplifier inputs was established with Ag/AgCl wires immersed
in the electrolytes. Signals were amplified about 200-fold in a custom-built
amplifier (0 Hz-2 kHz, input impedance 1012 
), and passed
through an anti-aliasing filter with a cut-off frequency of 2 kHz (900C/9L8L,
Frequency Devices, Haverhill, MA, USA). For data acquisition, a Digidata 1200
B digitizer and pCLAMP software (version 8) (Axon Instruments, Union City, CA,
USA) were used. The electrophysiological signal and a high-pass filtered
equivalent (cut-off frequency 5 Hz) were continuously recorded on a strip
chart recorder (EasyGraf, Gould, Valley View, OH, USA). Voltage polarity is
given with the sensillum lymph electrode in reference to the haemolymph
electrode. To be able to record in the photophase we compared the beginning of
the day with the middle of the day to search for ZT-dependent effects of
8bcGMP. All experiments were performed with room lights switched on, starting
either at the beginning of the day, 1 h after lights on at ZT1, or at the
middle of the day, at ZT8.
Application of 8bcGMP
Drug was applied to pheromone sensilla during tip recordings by perfusion
with water-soluble agents, as first suggested by Kaissling et al.
(Kaissling et al., 1991).
Long-term recordings from intact animals revealed no damage of the sensilla
when continued for up to several days
(Dolzer et al., 2001). So we
allowed the sensillum lymph ringer to passively perfuse the sensillum lymph
cavity and did not apply any pressure or suction. In recordings of stimulated
trichoid sensilla, 8bcGMP diluted in sensillum lymph ringer was applied over
the recording electrode. Therefore, we used an altered sensillum lymph ringer
with a concentration of 10 mmol l-1 8bcGMP. This application method
is further referred to as sensillum lymph perfusion. Biogenic amines injected
into the head capsule near the base of the antenna influenced oscillations of
the transepithelial potential (TEP) of trichoid sensilla
(Dolzer et al., 2001). Thus,
the agents were transported into the antenna after injection. In recordings of
the spontaneous activity of ORNs, 8bcGMP diluted in haemolymph ringer was
injected through a hole in the head capsule, which was pierced with a syringe
needle approximately 1 mm dorsocaudal to the right antennal base. We injected
a minimum of 3 µl of 10 mmol l-1 8bcGMP solution and a maximum
of 5 µl of 100 mmol l-1 solution, resulting in a concentration
of 30-500 nmol l-1 8bcGMP. Because an adult moth contains
approximately 1 ml of haemolymph the final 8bcGMP concentration in the
haemolymph was between 30-500 µmol l-1. This application mode is
further referred to as haemolymph injection. The ringers used for sensillum
lymph perfusion and the respective controls were prepared with
N-(2-hydroxyethyl)-piperazine-N'-(2-ethane sulfonic
acid) (Hepes; all chemicals from Sigma, Deisenhofen, Germany); the ringers
used for haemolymph injection were prepared with a phosphate buffer
(monopotassium phosphate). The pH was adjusted to 6.5, and osmolality was
adjusted to 475 mosmol l-1 with mannitol for sensillum lymph ringer
and to 450 mosmol l-1 for the haemolymph ringer.
Pheromone stimulation
All recordings were performed at room temperature (18-23°C).
Charcoal-filtered and moistened air was permanently blown over the preparation
through a glass cartridge (13 l min-1). The air stream could be
redirected through cartridges containing a piece of filter paper (about 1
cm2) loaded with synthetic bombykal (E,Z-10,12-hexadecadienal; BAL)
generously provided by T. Christensen (Tucson, AZ, USA). The air stream was
switched between the cartridges using solenoid valves (JFMH-5-PK and
MFH-5-1/8, Festo, Esslingen, Germany) controlled by the computer. Doses of 10
µg BAL dissolved in n-hexane (Merck, Frankfurt, Germany) were
applied to the filter papers (10 µl per paper), and the solvent was allowed
to evaporate. Stimulus intensity is always given in terms of the BAL dose
applied to the filter paper. The cartridges were placed in the outlet in a
distance of 4.5-6 cm from the recording site about 25 s prior to stimulation.
Stimuli of 10 µg BAL and 50 ms duration were applied every 5 min during a
recording session of 3 h. A suction tube of 10 cm diameter was placed below
the animal for rapid removal of the pheromone component after stimulation, and
to avoid uncontrolled stimulation due to BAL leaking out of the stimulus
cartridges. Between the recording sessions, the cartridges were stored at
-20°C in individual glass scintillation vials. Control cartridges loaded
with hexane alone were prepared and treated the same way. A set of stimulus
cartridges was used for 5-10 recording sessions; the control cartridges were
used for up to 30 recordings.
Acquisition protocols and data analysis
Each sensillum trichodeum contains two ORNs, both generating spontaneous
APs and responses to different compounds of the pheromone blend. The APs of
both ORNs can be distinguished by their amplitude. The spontaneous activity of
the ORNs was acquired in segments of about 10 min with a sampling frequency of
19.6 kHz (Clampex 8, fixed-length events). Each AP triggered a sweep of
duration 12.75 ms, and the high-pass filtered signal served as a trigger
channel only. All analyses were performed using the direct-current-coupled
signal. The mean voltage during the initial 2.5 ms was defined as the baseline
and used to measure the TEP. The baseline of all AP sweeps was then adjusted
to 0 mV to identify sweeps that were triggered by artifacts. To evaluate the
shape of APs, the waveforms of both classes were averaged for intervals of 10
min. The pheromone responses were recorded in sweeps of 5161 ms duration at a
continuous sampling rate of 20 kHz (Clampex 8, Episodic Stimulation Mode) with
a pre-trigger portion of 180.6 ms and a post-trigger portion of 4930.2 ms. The
spontaneous activity between the stimuli was recorded in segments of
approximately 5 min in fixed length events, as described above. The recordings
of the pheromone responses were evaluated using the Microsoft Excel Add-in
XtraCell (Dolzer, 2002) and
Clampfit 8. For analysis of the sensillar potential, the responses were
low-pass filtered at a cut-off frequency of 50 Hz (Clampfit, Gaussian filter).
The evaluated parameters of the sensillar potential (SP), as illustrated in
Fig. 1A,B, were: (1) the
overall amplitude (SP amplitude), (2) the initial slope between the onset of
the DC response and the half-maximal SP amplitude (initial slope) and (3) the
half-time of the rising phase (t
rise). The second
portion of the rising phase of the sensillar potential was described by an
exponentional fit of first order, using only the time constant (
). The
fitting was performed with a non-iterative Chebyshev algorithm
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ISI [N=number of interspike
intervals (ISI)]. Due to the high variability of the evaluated response
parameters of each recording, the parameters were normalized to the first
response of a recording. Subsequently the data for each parameter was binned
to intervals of 5 min and the mean was plotted against the time of
recording.
The data of the evaluated parameters of 8bcGMP and associated control recordings in each time slot were compared using the Student's t-test. For statistical analysis of changes in several parameters over the recording duration, its time course was divided into three intervals and then analyzed using a one-way ANOVA followed by the Tukey HSD post-hoc test. For analysis of the AP amplitude reduction, action potentials of three consecutive responses within an interval (from 1 min before to 2 min after the stimulus) were binned to 10 ms intervals and plotted against time. To measure changes in the amplitude reduction, the ratio of the minimal and maximal AP amplitudes was calculated and the normalized parameter for each recording plotted against the time of recording. To evaluate changes in the AP frequency or distribution in time, the AP response characteristics were analyzed using post-stimulus-time histograms; the AP responses were added up, binned to 10 ms intervals and plotted over time, with t=0 being the start of the DC response.
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describing the
second half of the rising phase of the sensillar potential
(Fig. 1A,B). Parameters
describing the phasic part of the AP response are the AP frequency computed
over the first five interspike intervals, the AP latency in relation to the
onset of the sensillar potential, and the stimulus-dependent amplitude
reduction (Fig. 1B-D). The
beginning and end of the recordings were compared
(Fig. 2). In both time slots
from ZT 1-4 and ZT 8-11, tip recordings with perfusion of the sensillar lymph
with 10 mmol l-1 8bcGMP applied over the recording electrode were
compared to control recordings without cyclic nucleotides. In control
recordings at both ZTs, no changes in the evaluated parameters could be
observed throughout the 3 h of recordings (Figs
3,
5). In the presence of 8bcGMP
the AP frequency was continuously decreased, and in a few recordings the
latency between the onset of the sensillar potential and the occurrence of the
first AP increased (Figs 2,
4). All other parameters that
describe the AP and the sensillar potential response remained constant over
the time of recording (Fig. 4).
For recordings from ZT 8-11 (Fig.
5), significant differences were found in both the comparison of
the AP frequency of 8bcGMP and control recordings (Student's t-test
for independent samples, P<0.01) and also within the time course
of the AP frequency (ANOVA and Tukey HSD post-hoc test; 
=0.01,
P<0.01). No significant difference was found between intervals 2
and 3 of the time course. In contrast no significant differences were found
for the sensillar potential amplitude. Due to the high variability for the AP
latencies no significant difference for recordings with or without 8bcGMP was
found (data not shown).
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As in recordings from ZT 8-11, the AP frequency of recordings from ZT 1-4
(Fig. 5) showed a significant
decrease when 10 mmol l-1 8bcGMP was included in the recording
electrode (Students t-test for independent samples,
P<0.01), but no significant difference was found between groups 2
and 3 of the time course (ANOVA and Tukey HSD post-hoc test;
=0.01, P<0.01). The sensillar potential amplitude and the
evaluated parameters of control recordings
(Fig. 5) did not change over
time (N=9). Comparison of the AP frequency depletion between both
time slots revealed a 12% stronger decrease for recordings at ZT 8-11.
It was next determined whether there are any daytime-dependent differences in the distribution of APs in response to stimulation with BAL in controls or in the presence of 8bcGMP. To analyse the distribution of APs occurring during the first 1000 ms, for responses from the beginning (0-20 min), middle (80-100 min) and end (160-180 min) of the recordings, the counted APs were binned to 10 ms intervals and plotted as post-stimulus-time histograms (Figs 6, 7). In control recordings from ZT 1-4 during the first 100 ms of the responses the response peak occurred later and the number of APs in the first 100 ms decreased significantly over the 3 h recording, leading to a slightly less phasic characteristic of the responses (Fig. 6, insert in C). Under the influence of 8bcGMP a weak decline in the number of APs over the course of the 3 h recording was also recognizable (Fig. 6A-C); owing to high variability, however, the decrease over the first 100 ms of the responses was not significant (Fig. 6C, insert).
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Effects on the amplitude reduction
Characteristic for responses to BAL stimuli of higher dose is the reduction
of the AP amplitude (Fig.
1C,D), which lasts for seconds to minutes. Especially in responses
to strong stimuli with BAL concentrations higher than 1 µg per filter
paper, the reduction of the peak-to-peak amplitude in the phasic portion is
very strong and, in many cases, after about 150 ms the APs cannot be
distinguished from noise. When comparing the beginning and end of a recording
(Fig. 2), or the positive
amplitude of APs in responses during an interval of 1 min before to 2 min
after stimulation, several effects of 8bcGMP on the amplitude reduction can be
observed (Fig. 8A). At the
beginning of the recordings the amplitude reduction is very strong, but it
gradually attenuates over the time of the recording until the reduction is
only very weak and transient in the presence of 8bcGMP
(Fig. 8A). In control
recordings no strong changes in the amplitude reduction are recognizable
(Fig. 8A). In addition to the
cGMP-dependent attenuation of the decline of the AP amplitude, the kinetics of
the amplitude attenuation also changed (data not shown). At the beginning of
the recording the APs did not return to their prestimulus amplitude until 2
min after the stimuli had been applied. Under the influence of 8bcGMP the
recovery phase was gradually attenuated until it disappeared. Also the minimal
positive AP amplitude linearly increased over the course of the recording
(data not shown). Furthermore, the normalized ratio of the minimal and maximal
positive AP amplitude (Fig. 8B)
(as a mean of the strength of the amplitude reduction) shows that 8bcGMP
perfusion attenuates the AP amplitude reduction more strongly than in the
controls at both ZTs. In addition, there are differences between both ZTs in
the control recordings and in those with 8bcGMP perfusion. At ZT 8-11 with
application of 8bcGMP, an attenuation of the amplitude reduction was
recognizable in all of the recordings, whereas in the associated controls the
strength of the amplitude reduction was reduced in only one recording over the
entire time course of the recordings. At ZT 1-4 the recordings were more
unequally distributed. In the control recordings there was higher variance
compared to recordings at ZT 8-11, which also increased with the duration of
the recording. Under the influence of 8bcGMP 2 populations of recordings were
recognizable. In one population that describes the majority of the recordings,
the amplitude reduction increased only slightly and this increase did not
start until 90 min after the beginning of the recording. In the second
population a very strong attenuation of the amplitude reduction was present
from the beginning. Changes in AP amplitudes of spontaneous APs between BAL
responses were not found.
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Spontaneous APs
In addition to the effects of 8bcGMP on pheromone responses, 8bcGMP also
had an effect on the waveform of spontaneous APs of unstimulated sensilla
trichoidea of isolated male moths that had never experienced pheromone.
Changes in the waveform of both AP classes were observed after 6 of 8
haemolymph injections of 30-500 nmol 8bcGMP for several hours. In one
recording (Fig. 9) the time
course of the AP waveform could be monitored almost continuously for 6 h after
the injection at the base of the antenna. The waveforms of both AP classes
were influenced independently, suggesting that the 8bcGMP effect took place
within the individual ORNs (N=6). After injection of 8bcGMP, two
aspects of the waveform were altered: the peak-to-peak amplitude increased and
the negative phase of the APs was prolonged. These effects then reverted and
reappeared later, suggesting the presence of feedback-coupled mechanisms that
were triggered by 8bcGMP injection. A consistent change in the frequency or
burst behaviour of the APs was not observed. In recordings of stimulated
olfactory neurons no effects of 8bcGMP on the waveform of spontaneous APs
between the stimuli were found (data not shown).
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The role of cGMP in insect olfactory adaptation
It was known from extracellular tip recordings from different moth species
that strong, long or high frequency pheromone stimuli can cause
desensitization, short- or long-term adaptation to pheromone responses
depending on stimulus strength and time course
(Zack, 1979;
Dolzer et al., 2003;
Marion-Poll and Tobin, 1992).
In Manduca sexta it was shown that, depending on BAL stimulus, length
and strength parameters of the sensillar potential and AP response adapt
differentially (Dolzer et al.,
2003). After adapting to pheromone stimuli, the time of rise of
the sensillar potential did not increase after short-, only after
long-adapting stimuli, whereas both stimulation schemes shifted the
dose-response curves of the sensillar potential amplitude, as well as the
initial slope of its rising phase, to higher stimulus concentrations. The
shift in the dose-response curve of the AP response was larger compared to the
shift in dose-response curve of the sensillar potential response, indicating
that depending on the properties of the stimulus unknown mechanisms of
adaptation occur at different levels of the transduction process
(Dolzer et al., 2003).
Biochemical experiments implicated that rises in cGMP concentration are
involved in at least one mechanism of long-term adaptation, because strong and
long pheromone stimuli caused delayed and sustained rises in intracellular
cGMP concentrations in antennal homogenates of Antherea polyphemus
and Bombyx mori. These increased levels of cGMP concentration matched
the time courses of long-term adaptations
(Ziegelberger et al., 1990;
Boekhoff et al., 1993). In
immunocytochemical experiments and in situ hybridizations it could be
shown that at least subpopulations of pheromone-sensitive ORNs of male
Manduca sexta upregulate cGMP concentrations after minute-long (but
not seconds-long) exposure to female pheromones
(Stengl et al., 2001). These
cGMP rises were augmented via exposure to NO donors in the presence
of a pheromone-inducible NO-synthase-like enzyme in ORNs, but not via
stimulation of a soluble guanylyl cyclase
(Stengl and Zintl, 1996;
Stengl et al., 2001). Thus,
adapting, long and strong pheromone stimuli, as occur in close vicinity to the
calling female, cause long-lasting rises in cGMP concentrations. But it
remained unknown which antennal targets are affected via cGMP and
whether rises in cGMP concentration underlie at least one mechanism of
long-term olfactory adaptation.
In the present study we have shown in tip recordings of trichoid sensilla
of the hawkmoth Manduca sexta that brief but strong pheromone stimuli
applied in a non-adapting protocol with interstimulus intervals of 5 min cause
no changes in the pheromone response, either in the sensillar potential or in
the AP response. Only during long-term exposure to the cGMP analogue 8bcGMP
was the AP frequency decreased over the 3 h of the recordings; the sensillar
potential response remained unchanged. This selective decrease in the AP
frequency distinguishes one of the different forms of adaptation in
Manduca sexta for the first time. Interestingly, injection of
dibutyryl guanosine 3',5'-cyclic monophosphate into the antennae
of Bombyx mori decreased the AP frequency as well as the sensillar
potential amplitude (Redkozubov,
2000). The recording methods and the cGMP analogues employed
differed between both moth species, as well as the stimulation scheme. While
only very brief strong pheromone stimuli were applied in our recordings,
Redkozubov employed either 10 times longer and stronger pheromone stimuli, or
presented continuous pheromone stimuli of a lower dose. Most likely, these
differing pheromone stimulation protocols were responsible for the differences
observed.
The decrease in the AP frequency, the decrease in the number of APs in the
first 100 ms of the response, and the observed cGMP-dependent slowdown of the
negative phase of spontaneous APs, could all be explained by a closure of
K+-channels. Also, the increase in the peak-to-peak amplitude of
spontaneous APs could be explained by the closure of K+-channels
resulting in an increased resistance. This increase in the resistance of the
preparation could also be responsible for the 8bcGMP-dependent attenuation of
the AP amplitude reduction. In Manduca sexta, a pheromone-activated,
cGMP-blockable K+-channel was described without its function being
understood (Zufall et al.,
1991; Stengl et al.,
1992). Based on our findings and on FURA-measurements with
cultured ORNs from Manduca sexta (M.S., unpublished observations), we
assume that strong or long pheromone stimulation resulted in long-term rises
of intracellular Ca2+ levels. These Ca2+ elevations then
appeared to cause rises of intracellular cGMP levels, which then close the
fast BAL-dependent cGMP-dependent K+-channel. Whether other
cGMP-gated ion channels that were described in patch clamp recordings in
Manduca sexta (Dolzer,
2002) are also involved in the observed adaptations of the action
potential response remains to be shown.
Daytime-dependent differences in the cGMP effects
In addition to daytime-dependent differences in the control recordings,
several daytime-dependent effects of cGMP were found. 8bcGMP-dependent
distribution of APs became more tonic and the number of APs during the first
100 ms of the BAL response only decreased at ZT 8-11. Furthermore, the
8bcGMP-dependent decrease of the AP frequency at ZT 8-11 was 12% stronger than
in recordings at ZT 1-4. Also, the 8bcGMP-dependent attenuation of the
reduction of the positive AP amplitude was more prominent at ZT 8-11. In
addition to daytime-dependent differences in the effects of 8bcGMP, a
different distribution in the timing of APs in control recordings between both
ZTs were observed. In recordings from ZT 8-11 the pheromone-dependent AP
response became less phasic and more tonic, and also fewer numbers of APs were
elicited during the first 100 ms of the BAL response, than at ZT 1-4. Thus,
the main differences between the responses of the recordings at ZT 1-4 and ZT
8-11 are the distribution and numbers of APs elicited in the phasic portion of
the response. An ORN with more tonic responses would very likely be less able
to resolve pheromone filaments of high frequencies.
The effects of the plume structure on behavioural and flight responses of
male Cadra cautella to pheromone were investigated in wind tunnel
experiments (Mafra-Neto and Cardé,
1995a; Mafra-Neto and
Cardé, 1995b). Faster frequencies of pulses were followed
by a higher percentage of males responding by shorter latencies and less time
spent in the search behaviour. Because the variation of the fine structure of
the plume had more influence on the flight pattern of males than a 1000-fold
increase in the pheromone dosage, the temporal resolution of ORNs appears to
be very crucial. In addition, it was shown that the antennal lobe network is
strongly modulated by the temporal pattern of the stimulus
(Christensen et al., 1998a;
Christensen et al., 1998b). The
antennal lobe is tuned to fast temporal discrimination of pheromone pulses,
which appears to be necessary for odor blend discrimination
(Christensen and Hildebrand,
1997; Stopfer et al.,
1997). Whether the decrease of the numbers of APs within the first
100 ms represents a further adaptation mechanism that decreases the ability of
the ORNs to encode pulsed pheromone signals and therefore blend discrimination
remains to be shown in future investigations.
Our results suggest for the first time that there might be
daytime-dependent differences in the sensitivity of moth ORNs to pheromone.
Whether the observed differences are controlled by an endogenous circadian
clock that regulates the sensitivity of ORNs remains to be studied. So far, it
has been assumed that daytime-dependent rhythms in male responsiveness to
pheromone occur at the level of the antennal lobe and midbrain
(Payne et al., 1969;
Worster and Seabrook, 1988;
Rosén et al., 2003). In
addition, investigations of circadian sensitivity changes to food odours in
Leucophaea maderae indicated that these rhythms were driven by a
circadian pacemaker in the optic lobes but not in the ORNs
(Page and Koelling, 2003),
while circadian changes in the electroantennogram (EAG) in Drosophila
melanogaster suggested that they depend on PER-dependent endogenous
circadian pacemakers within the antenna
(Krishnan et al., 1999;
Tanoue et al., 2004;
Zhou et al., 2005). The main
difference between our experiments and the studies mentioned is that we
performed tip-instead of EAG-recordings, which allowed us to analyse the AP
response of a single ORN with high temporal and spatial resolution. The shift
from a phasic-tonic to a tonic response pattern as an effect of 8bcGMP
application and circadian differences has never been shown before because it
cannot be resolved by the EAG-recording technique.
It is still unknown whether Manduca sexta males are maximally sensitive to their intermittently pulsed pheromone signals during the late night when the females are calling. Also, it is unresolved whether there is a circadian rhythm that might adapt the ORNs in the photophase when no pheromone is released by the females and when the moths are inactive. To investigate rhythms in the sensitivity of ORNs to pheromone and to challenge the hypothesis that the blend discrimination is affected via a cGMP-dependent mechanism, long-term recordings starting at the late scotophase, which comprises the shift from scoto- to photophase, will be performed. In addition, it will be investigated in behavioral studies whether male Manduca sexta show rhythmic changes in their responsiveness to pheromone. Furthermore, current biochemical experiments examine whether there are differences in cyclic nucleotide concentrations in the moth antenna at different ZTs depending on differing stimulation schemes (K. Riedinger and M.S., unpublished).
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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