Adaptation in pheromone-sensitive trichoid sensilla of the hawkmoth Manduca sexta
1 Biologie, Tierphysiologie, Philipps-Universität Marburg, D-35032
Marburg, Germany
2 Institut für Zoologie, Universität Regensburg, D-93040
Regensburg, Germany
* Author for correspondence (e-mail: stengl{at}mailer.uni-marburg.de)
Accepted 18 February 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Manduca sexta, insect olfaction, adaptation, pheromone sensillum, tip recording, bombykal, action potential, sensillar potential
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Moths distinguish pheromone mixtures according to the concentration ratios
of the different pheromone components. Thus, differentiation of pheromone
concentrations is very crucial for recognition of prospective mates.
Turbulences and wind velocities determine the structure of the pheromone
filaments that stimulate the antenna of a flying moth. Thus, adapting and
non-adapting pheromone stimuli of variable concentrations and stimulus
durations reach different parts of the antenna at various time intervals. It
is still unknown how the moth can recognize relevant pheromone ratios in
various states of adaptation. In our study of pheromone-sensitive ORNs, we
examine how adapting pheromone stimuli affect the encoding of different
pheromone concentrations (quantity coding) in the intact moth. We distinguish
the rapidly (within seconds to minutes) reversible reduction of sensitivity
due to prior stimulation (short-term adaptation) from the decline in
excitation, as seen during a phasictonic response to a stimulus of long
duration (desensitization; Zufall and
Leinders-Zufall, 2000). Thus, we compare quantity coding in
response to short (50 ms) and long (1000 ms) pheromone stimuli, as possibly
encountered during flight to the calling female. We do not examine the more
slowly occurring (within several minutes to hours) reduction of sensitivity
due to previous strong stimulation as seen during long-term (i.e.
long-lasting) adaptation (Ziegelberger et
al., 1990
; Marion-Poll and
Tobin, 1992
; Boekhoff et al.,
1993
; Stengl et al.,
2001
; Dolzer,
2002
).
Quantity coding in response to adapting pheromone stimuli has been studied
in extracellular tip recordings in saturniid moths, in which the different
terms `short-term' versus `long-term' adaptation were coined for
insect olfaction (Zack, 1979;
Zack-Strausfeld and Kaissling,
1986
; Kaissling et al.,
1986
,
1987
). Concerning ORNs of the
hawkmoth Manduca sexta, however, there is only one study about
olfactory adaptation in temporal coding of pheromone pulse trains
(Marion-Poll and Tobin, 1992
)
and a few studies on quality odour coding
(Kaissling et al., 1989
;
Kalinová et al.,
2001
).
Moths detect the pheromones using specialized ORNs, which innervate long
multiporous trichoid sensilla on the antenna in pairs
(Keil, 1989). It has been
shown that in each trichoid sensillum, one of the ORNs (the bombykal cell)
responds to bombykal, the main component in the conspecific pheromone blend
(Starratt et al., 1979
;
Tumlinson et al., 1989
). The
second cell (the non-bombykal cell) is tuned to other different pheromone
components in different sensilla. While the study by Kaissling et al.
(1989
) examined the coding of
different odour qualities, not much attention was paid to the coding of odour
concentrations. Quantification of pheromone responses in M. sexta was
not reliable, because at that time it could not be consistently distinguished
whether an action potential originates from the spontaneously active
non-bombykal cell or from the bombykal cell. A more recent study of trichoid
sensilla in M. sexta could distinguish two different nerve impulse
classes, termed the small and the large action potential classes, by amplitude
(Dolzer et al., 2001
).
However, it was still not completely resolved whether the bombykal cell always
generates the large or the small action potentials.
Thus, the present study first examines which action potential class the bombykal cell from a specific region of a distal flagellar annulus belongs to, since a clear distinction of both cells is a prerequisite for an unequivocal quantification of the bombykal responses. Then, we examine how a previous strong stimulus (a conditioning stimulus) can adapt and thus change the coding of bombykal concentrations at the level of the sensillar potential and the action potential response and whether these stimuli affect flight behaviour. In doseresponse curves to short (short-term adaptation) and long (desensitization) bombykal stimuli, we show that the bombykal cell can resolve four log10-units of pheromone concentrations but is apparently unable to encode stimulus duration with all parameters tested. Finally, we present evidence for the presence of several functionally distinct mechanisms of short-term adaptation, which affect the rising phase of the sensillar potential, its decline and the action potential response. In addition, we show at least one additional mechanism of desensitization. None of these bombykal stimuli affected flight behaviour.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cartridge stimulation
All recordings were performed at room temperature (1823°C).
Charcoal-filtered and moistened air was permanently blown over the preparation
through a glass cartridge (13 l min1). The airstream could
be redirected through cartridges containing a piece of filter paper
(approximately 1 cm2) loaded with synthetic bombykal
(E,Z-10,12-hexadecadienal) generously provided by T. Christensen (Tucson, AZ,
USA). Bombykal doses are always given in log10-unit intervals; for
example, in Fig. 9, 6
log10 bombykal dose (µg) means 106 µg
bombykal. The cartridges were placed with the outlet in a distance of
4.56 cm from the recording site. The airstream was switched between the
cartridges using solenoid valves (JFMH-5-PK and MFH-5-1/8; FESTO, Esslingen,
Germany) controlled by the computer. Airstream velocity was monitored with a
thermistor (BC32L1; Fenwal, Framingham, MA, USA) placed near the recording
site and connected to a custom-built anemometer. Limited by the specified
switching time of the solenoids (15 ms), and verified by the anemometer
recording, the shortest applicable stimulus duration was approximately 50 ms.
Doses between 106 µg and 100 µg bombykal dissolved in
n-hexane (Merck, Frankfurt, Germany) were applied to the filter papers (10
µl or 100 µl per paper), and the solvent was allowed to evaporate.
Stimulus intensity is always given in terms of the bombykal dose applied to
the filter paper. Stimulus durations between nominally 10 ms and 1000 ms were
employed. Due to the switching times of the solenoid valves, stimulus
durations below 50 ms are less clearly defined than above this duration and
are only included for completeness.
|
|
|
Local stimulation
While cartridge stimulation, as described above, was used in most of the
experiments, a small subset of the sensilla was stimulated locally. In these
experiments, the pheromone was applied to a piece of thread, which was then
inserted into a glass pipette with an opening of approximately 40 µm
(Kaissling, 1995). The
stimulation pipette was placed below the recorded sensillum, and the stimulus
airstream was redirected through the pipette for stimulation. The only dose
tested with local stimulation was 1 µg bombykal; stimulus durations were
between 20 ms and 2000 ms. Of the data presented here, only the recordings
shown in Fig. 10 were obtained
using local stimulation; all other data and results were obtained with
cartridge stimulation.
|
Acquisition protocols and data analysis
In the beginning of each recording, a series of 5 mV calibration pulses was
applied to the haemolymph electrode, which was otherwise grounded. The
pheromone responses were recorded in sweeps of 3 s duration at sampling
frequencies of 5 kHz and 1.67 kHz (Clampex, episodic stimulation mode with a
gear shift after 1000 ms). The solenoids were controlled by a digital output
signal switched to `high' 20 ms after the sweep start. The stimulus airstream,
as monitored by the anemometer, arrived at the recording site approximately 50
ms after the trigger signal.
The recordings were evaluated using macros in Clampfit 6, Microsoft Excel,
versions 7 and 8, and Automate 4 (Unisyn Software; Los Angeles, CA, USA)
(Dolzer, 2002). For the
analysis of the sensillar potential, the responses were lowpass-filtered at a
cut-off frequency of 50 Hz or 70 Hz (Clampfit, Gaussian filter). The evaluated
parameters of the sensillar potential, as illustrated in
Fig. 1A,B, were: (1) the
overall amplitude (SP amplitude), (2) the initial slope between the onset of
the sensillar potential and the half-maximal SP amplitude (slope), (3) the
half-time of the rising phase (t1/2 rise) and (4) the
half-time of the declining phase (t1/2 decline). The
half-times of the rising and declining phases were analyzed to compare our
results with studies on silkmoths (Antheraea sp.;
Zack, 1979
;
Kaissling et al., 1987
;
Kodadová, 1993
;
Kodadová and Kaissling,
1996
), and the initial slope was analyzed as an important
parameter describing the kinetics of the sensillar potential.
|
For the analysis of the action potentials
(Fig. 1C), the lowpass-filtered
trace was subtracted from the original response. This pseudo-highpass
filtering procedure, in contrast to actual highpass filters, does not distort
the shape of the action potentials and therefore allows the analysis of their
amplitude and waveform (Dolzer,
2002). The action potential response was characterized by: (1) the
peak frequency computed from the first five interspike intervals (AP
frequency; Fig. 1C) and (2) the
latency between the beginning of the sensillar potential and the first action
potential (AP latency; Fig.
1B). In the background activity, action potentials of two
amplitude classes were recorded (Dolzer et
al., 2001
). Therefore, when action potentials of two classes,
distinct by amplitude or by frequency and latency, were observed during the
response, they were analyzed separately. Because responses of trichoid
sensilla from different intact moths varied greatly in amplitude
(Table 1), it was impossible to
quantify these variable data sets without normalization. Thus, for the
quantitative analysis, the response parameters were normalized to the highest
response during the recording from the same sensillum. The highest response
was defined as the largest value of those parameters that were positively
correlated to the stimulus intensity and as the smallest value of parameters,
which were negatively correlated to the stimulus intensity. Thus, the
normalized responses were computed from: Responsenorm =
Response/Responsemax and Responsenorm =
Responsemin/Response, respectively. The sensillar potential
amplitude, the initial slope of the SP, t1/2 decline and
the action potential response were normalized to the largest value during each
recording from the same sensillum. Since t1/2 rise and AP
latency were negatively correlated with stimulus strength, both were
normalized to the smallest value during one recording. The inverse
normalization of the negatively correlated parameters (t1/2
rise and AP latency) allowed us to focus on responses in the
physiological range. Direct normalization of, for example, AP latency, either
to the largest or smallest value of the data set, exaggerates long latencies
in response to low stimulus intensity and obscures differences in the response
to physiological stimuli of a higher dose. For the investigation of
desensitization, stimuli of 1000 ms duration were applied with 60 s between
consecutive stimulations. The action potential response characteristics were
analyzed with peri-stimulus-time histograms to evaluate changes in the action
potential frequency over time. In time windows (bins) of 10 ms, action
potential responses were added up and plotted over time, with t=0
being the start of the DC response.
|
Stimulation protocols
In total, 1462 stimulations (all doses including controls) were applied to
70 sensilla of 42 animals. Since the experiments were done with a whole-animal
preparation, no rundown of the responses was observed, even during recordings
of more than 3 h with repetitive stimulation.
Non-adapted responses were obtained by applying either 50 ms stimuli or
1000 ms stimuli of increasing bombykal dose, separated by 60 s (dose ramp).
Control stimuli with only hexane on the filter paper were applied before each
dose ramp. In some recordings, additional control stimuli were applied.
Adapted responses were achieved by conditioning the sensilla with 10 µg
stimuli of 250 ms duration, applied 20 s before the test stimuli of 50 ms
duration (Fig. 2). For the 1000
ms stimuli, the sensilla were adapted by 10 µg stimuli of 1000 ms, applied
60 s before the test stimuli. To ensure identical conditions, each test
stimulus was preceded by its own conditioning stimulus. The sensilla were
allowed to recover for at least 10 min between every two stimulus pairs to
avoid accumulative adaptation. This interval between the stimulus pairs was
chosen because pilot experiments had suggested the complete recovery from
adaptation by a stimulus as used in the adaptation protocol after
approximately 5 min (Dolzer,
1996). To avoid contamination of our short-term adaptation
experiments by long-term adaptation (which lasts for hours), we did not employ
pheromone doses higher than 10 µg bombykal for the 50 ms stimuli and we
used a maximal dose of 100 µg for the stimuli of 1000 ms duration.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
General parameters of the pheromone response
Bombykal application causes depolarizing sensillar potentials in olfactory
receptor neurons (ORNs), which elicit action potential responses that are
carried along the axons of the ORNs to the brain to excite postsynaptic
antennal lobe neurons. In extracellular tip recordings, the sensillar
potential (SP) in response to pheromone stimulation is measured as a negative
deflection of the transepithelial potential. According to previous practice
and because the SP is assumed to reflect the depolarizing receptor potential
of the ORNs, its increase to maximal amplitude is described as the SP's rising
phase. Its repolarization to the steady-state potential is described as its
decline. Superimposed on the SP, action potentials can be recorded (Figs
3,
4,
5,
6). The amplitude of action
potentials decreased in response to strong stimulation (Figs
1C,
3,
4,
5). When action potentials of
two classes occurred during a response (Figs
3B,
4A,B), they are referred to as
small and large action potentials according to the amplitude of the first
action potential, i.e. before the stimulus-correlated amplitude reduction took
place. This terminology was introduced previously in recordings of
spontaneously active ORNs (Dolzer et al.,
2001). Action potentials of two amplitude classes were also
observed between the stimulations. All action potentials, whether spontaneous
or in a pheromone response, were recorded with their positive phase first.
There was a large variability in both the sensillar potential and action
potential responses among individual sensilla from different moths
(Table 1). Sixty-nine of the 70
sensilla tested responded to bombykal. The only non-responding sensillum had
no spontaneous action potential activity, indicating that it was damaged.
|
|
|
|
|
To determine whether stimulation with the different stimuli of the main
pheromone component bombykal alone elicits flight activity in the intact,
tethered moth, flight activity was recorded with a piezo-electric element
placed at the thorax. In addition, since a correlation between flight activity
and transepithelial potential oscillations was observed previously
(Dolzer et al., 2001), the
transepithelial potential was continuously monitored between the stimulations.
In 18 of 22 animals analyzed, bombykal stimulation did not elicit flight
activity. In three cases, the animals exhibited continuous flight activity. In
only one animal, flight activity occurred immediately after strong
stimulation, whereas the animal was silent before. Oscillations of the
transepithelial potential were observed in the recordings from 16 sensilla. In
no case did bombykal stimulation suppress or detectably influence the
oscillations.
Specificity of the ORNs
Because the two ORNs per trichoid sensillum can be clearly distinguished
according to the amplitude of their action potentials, we wanted to know
whether the bombykal cell always produces the large or the small action
potentials. Unless the stimulus dose exceeded 10 µg, only action potentials
of the large amplitude class were observed during the bombykal responses. When
small action potentials occurred at these dosages, they could not be
distinguished from spontaneously occurring action potentials by their
frequency or reduction of the amplitude. At doses of 10 µg bombykal,
however, the small action potentials occurred at a frequency that could not be
considered spontaneous in 22 of 68 stimuli of 50 ms duration
(Fig. 3;
Table 2). Apparently, the
non-bombykal cell was `cross-excited'. In the presence of small action
potentials, the SP amplitude was significantly larger (10.85±0.64 mV,
N=22) than in responses with only large action potentials
(8.75±0.50 mV; means ± S.E.M.; P<0.01,
N=46; Student's t-test). The action potential frequency of
the small action potentials was significantly lower and the action potential
latency was significantly longer than for the large action potentials of the
bombykal cell (Table 3).
|
When cross-excitation occurred during a response to 10 µg bombykal, the stimulus-correlated amplitude reduction of the small action potentials was less prominent than that of the bombykal cells (Fig. 3B). At lower doses, the amplitude and frequency of small action potentials occurring during the response was the same as during spontaneous activity (Fig. 4).
Dose dependence
After distinction of the action potential responses of the bombykal and the
non-bombykal cells, we measured doseresponse relationships of the
bombykal cell in response to 50 ms bombykal stimuli to determine the
threshold, range of resolution and saturation (Figs
5,
7). The bombykal responses
varied considerably among individual sensilla and among individuals
(Table 1). Therefore, for a
quantitative analysis it was necessary to normalize the response parameters
(see Materials and methods; Fig.
7). The SP amplitude, the initial slope of the SP and the
half-time of the decay phase (t1/2 decline), as well as
the action potential frequency and latency, but not the half-time of the
rising phase (t1/2 rise), were dose-dependent (Figs
5,
6,
7). With higher pheromone
concentrations, the amplitude of the SP increased, its initial slope became
steeper and its decline to baseline became slower
(Fig. 7). The action potential
frequency increased, and the latency to the first action potential was
shortened. The threshold was between 103 µg and
102 µg bombykal for all these variables. The ORN was able
to resolve at least four log10-units of odour concentrations. The
t1/2 rise exhibited no dose dependence
(Fig. 7C).
Short-term adaptation
To evaluate changes in quantity coding after previous pheromone experience,
the sensilla were short-term adapted. Short-term adaptation is defined here as
a rapidly (within seconds to minutes) reversible reduction of sensitivity due
to prior stimulation with a conditioning stimulus. Short-term adaptation is
distinguished from long-term adaptation, which lasts for hours and involves
rises of intracellular cyclic GMP (cGMP) concentrations
(Stengl et al., 2001). When
the sensilla were adapted by a strong conditioning stimulus (10 µg
bombykal; 250 ms duration) 20 s prior to the test stimulus, the SP amplitude
and the action potential frequency were reduced (Figs
6,
7A,E). In addition, the decay
of the sensillar potential was accelerated (Figs
6B,
7D), as characterized by a
faster t1/2 decline. The doseresponse curves of the
SP amplitude and the initial slope were shifted to higher stimulus intensities
by approximately one log10-unit
(Fig. 7A,B), which means that
10 times more pheromone was needed to elicit the same response amplitudes. The
t1/2 rise was virtually dose-independent at a stimulus
duration of 50 ms and was not significantly altered by the adapting stimulus
(Figs 6B,
7C). The doseresponse
curve of the action potential frequency was shifted by two
log10-units, and the shift of the doseresponse curve of the
latency between the onset of the SP and the first action potential was even
larger (Fig. 7E,F). For none of
the variables was there an obvious difference in the slopes of the adapted and
the non-adapted doseresponse curves.
Desensitization
Desensitization is the decline in excitation as seen during a
phasictonic response to a stimulus of long duration and, thus, is
distinct from short-term adaptation (Zack,
1979; Zufall and
Leinders-Zufall, 2000
). To determine whether the stimulus duration
affects coding of pheromone quantity in the non-adapted and adapted state, we
employed pheromone stimuli of 20 times longer durations. With stimulations of
1000 ms duration in the absence of a preceding adapting stimulus
(N=129), the action potential response was phasictonic at all
doses between 106 µg and 100 µg bombykal
(Fig. 8). The phasic component
was more prominent with increasing pheromone dose. Thus, this stimulus
duration was not reliably encoded by the phasic part of the action potential
response. After the phasic peak, the action potential frequency declined to a
tonic plateau with a time constant of approximately 150 ms. Thresholds were
between 102 µg and 101 µg bombykal
for all variables, and approximately four log10-units of bombykal
concentrations were resolved (Fig.
9).
In contrast to short stimuli of 50 ms duration, in response to 1000 ms
stimuli, the t1/2 rise was dose-dependent at higher doses
of bombykal in the non-adapted state as well as in the adapted state
(Fig. 9C;
Dolzer, 1996). As for short
stimuli, after a preceding adapting stimulus of 10 µg bombykal, the
amplitude of the sensillar potential was decreased, the initial slope was less
steep and the frequency of the action potential response was reduced
(Fig. 9A,B,E). In contrast to
short stimuli, the t1/2 rise was further prolonged after
an adapting stimulus, the decline not further accelerated at all stimulus
concentrations and the action potential latency not further increased at all
high pheromone concentrations (Fig.
9C,D,F).
Encoding stimulus duration
After testing responses to different stimulus concentrations, we examined
how the bomykal cell can encode different stimulus durations. The stimulus
duration of 50 ms was neither encoded by the sensillar potential nor by the
duration of the phasic or tonic part of the action potential response (Figs
5,
10). When different stimulus
durations from 20 ms to several seconds were tested (at a dose of 10 µg
with an interstimulus interval of 60 s), none of the parameters of the
sensillar potential reliably encoded all stimulus durations (Figs
10,
11). Only stimulus durations
of >100 ms were distinguished by t1/2 decline and by
the total number of action potentials, but not by the SP amplitude,
t1/2 rise and action potential frequency (i.e. the first
five interspike intervals; Fig
11).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vertebrate olfaction, three stages of adaptation are distinguished in
current recordings in vitro by their effects and the underlying
mechanisms: short-term adaptation, desensitization and long-lasting adaptation
(Zufall and Leinders-Zufall,
2000). In vertebrates, short-term adaptation reduces the amplitude
of brief odour-dependent currents after previous stimuli, is
Ca2+-dependent and cGMP-independent, and declines within several
seconds. By contrast, desensitization, which reduces the amplitude and slows
down the kinetics of the odour response during maintained stimulation, is
Ca2+-dependent and calcium/calmodulin-dependent protein kinase (CaM
kinase)-dependent and lasts longer than short-term adaptation. Long-lasting
adaptation, however, is triggered by stronger stimulation, is
Ca2+-, CO2- and cGMP-dependent and lasts many minutes.
In the following, we will discuss the general characteristics of the bombykal
cell in M. sexta and the different mechanisms of adaptation in moths
in the context of known olfactory signal transduction pathways, to look for
common features between insects and vertebrates and to suggest a testable
hypothesis of olfactory adaptation in insects.
Specificity of the olfactory receptor neurons and behavioural
aspects
As described previously (Dolzer et al.,
2001; Kalinová et al.,
2001
), but in contrast to other studies in M. sexta
(Kaissling et al., 1989
;
Marion-Poll and Tobin, 1992
),
two classes of action potentials, which can probably be assigned to the two
ORNs in each sensillum (Keil,
1989
), could be distinguished by their amplitude in most of the
recordings. Our experiments showed that in M. sexta, as in other moth
species investigated, the ORN that fires the action potentials with the larger
amplitude responds to the main component of the conspecific pheromone blend
(Bombyx mori, Kaissling et al.,
1978
; Antheraea polyphemus and A. pernyi,
Zack, 1979
; Mamestra
suasa, Lucas and Renou,
1989
; Mamestra brassicae,
Renou and Lucas, 1994
). Only
at very high pheromone doses was the cell with the smaller action potential
amplitude excited as well. Such cross-excitation was also described in A.
polyphemus (Kodadová and
Kaissling, 1996
;
Kodadová, 1993
).
Interestingly, none of the sensillar potential parameters and neither the
phasic nor the tonic part of the action potential response encoded all
stimulus durations tested. Only longer stimulus durations were distinguished
by the overall action potential frequency but were not reflected by the
duration of the action potential response. From analysis of the odour plume
and from antennal lobe recordings, it is known that odour intensity and
duration vary rapidly in nature and can be resolved by M. sexta
antennal lobe neurons on a millisecond time scale (Christensen et al.,
1996,
1998
;
Vickers et al., 2001
). Thus,
in their natural environment, moths have to resolve rapid changes in stimulus
strength and duration but might not need to respond reliably to ongoing odour
stimulation over a period of seconds to minutes.
In wind-tunnel assays, Tumlinson et al.
(1989) found that both
bombykal and (E,E,Z)-10,12,14-hexadecatrienal are required to elicit a
sequence of anemotaxis and mating behaviour. They did not test for activation,
however. Our results suggest that bombykal alone is not sufficient to activate
the moths. Neither did bombykal stimulation elicit flight activity nor
suppress oscillations of the transepithelial potential, as would have been
expected if activation is assumed to be correlated with octopamine release
(Roeder, 1999
;
Dolzer et al., 2001
). Although
the experiments were performed during the active phase of the nocturnal moths
(reared in an inverse culture), we cannot exclude influences of the
preparation procedure or the unnatural situation during the recordings. Thus,
this finding needs to be confirmed in experiments with freely behaving moths.
Finally, it remains to be tested in behavioural experiments how the different
forms of adaptation affect recognition of the species-specific pheromone
blend.
Adaptation of the rising phase and the amplitude of the sensillar
potential
In our study, the rising phase of the sensillar potentials (which in the
extracellular recording appears as a negative deflection from baseline to the
maximal amplitude) was characterized using two different parameters, the
initial slope and t1/2 rise. The initial slope, whether
determined by a straight line fit (not shown) or computed from
t1/2 rise and half the SP amplitude, exhibited a clear
dose dependence in correlation with the SP amplitude (Figs
7B,
9B). In contrast to studies
with pheromone sensilla of A. polyphemus and Antheraea
pernyi (Zack, 1979;
Kaissling et al., 1987
;
Kodadová, 1993
;
Kodadová and Kaissling,
1996
; Pophof,
1998
) and benzoic acid-sensitive sensilla of Bombyx mori
(Kodadová, 1993
), in
our experiments with short pheromone stimuli, t1/2 rise
showed virtually no dose dependence and was not influenced by adaptation
(Fig. 7C). The major difference
in the regimens of stimulation between our study and the studies mentioned
above is the stimulus duration (50 ms versus 2 s or 5 s). Thus, we
suspect that the longer stimulus duration, which resulted in a larger total
pheromone amount applied, produced desensitization and possibly also long-term
adaptation, which might be reflected by a change in the kinetics of the rising
sensillar potential phase. This assumption is supported by our recordings in
M. sexta at stimulus durations of 1000 ms (Figs
8,
9). These recordings showed a
dose dependence and prolongation of t1/2 rise after
adapting pheromone stimuli (Fig.
9C), as was also found in A. polyphemus
(Zack, 1979
;
Kaissling et al., 1987
). This
might indicate that a stimulus-duration-dependent mechanism of adaptation
exists that occurs during desensitization and possibly also during long-term
adaptation, which affects the amplitude and the kinetics of the rising phase
of the sensillar potential in different ways.
Based on the assumption that the sensillar potential predominantly reflects
the receptor potential of the adequately stimulated ORN, the waveform of the
sensillar potential is probably governed by the complex processes of the
chemo-electrical transduction cascade (reviewed by
Stengl et al., 1998). From
patch-clamp studies on cultured ORNs of M. sexta we know that after
application of a very low dose of bombykal, inositol (1,4,5)-trisphosphate
(IP3)-dependent Ca2+ channels open, causing a very
transient rise in the intracellular Ca2+ concentration
(Stengl, 1994
). This rapid
increase of intracellular Ca2+ then opens Ca2+-dependent
cation channels, and possibly also Ca2+-dependent
Cl and K+ channels, which, as counteracting
currents, may determine the kinetics of the rising phase and the amplitude of
the sensillar potential (Stengl,
1993
,
1994
; Stengl et al.,
1992
,
1998
). Because two of these
channel types (which share properties with trp- and
trpl-like channels) are permeable to Ca2+ and because both
are closed in a Ca2+-dependent manner, it is very likely that
intracellular Ca2+ rises caused by these channels are involved in
short-term adaptation, as has been shown in Drosophila
(Störtkuhl et al., 1999
;
Montell, 2001
). Biochemical
and physiological evidence suggests the involvement of cGMP
(Ziegelberger et al., 1990
;
Boekhoff et al., 1993
;
Stengl et al., 2001
;
Dolzer, 2002
) together with
Ca2+ concentration rises (Stengl,
1993
,
1994
;
Dolzer et al., 1999
) in
long-term adaptation in moths. Thus, it is likely that Ca2+- and
cGMP-dependent mechanisms affect the rising phase of the sensillar potential
in response to long and very strong pheromone stimuli.
Adaptation of the declining phase of the sensillar potential
The faster decline of the sensillar potentials in the short-term-adapted
state in response to short stimuli, as well as to long stimuli (Figs
6B,
7D,
9D), suggests the presence of
an additional adaptation mechanism, acting via stabilization of the
resting potential. Thus, we would expect that either K+ efflux or
Cl influx is increased via short-term adaptation,
possibly together with a faster closure of depolarizing ion channels. Since
preliminary measurements with Ca2+-sensitive dyes (M. Stengl and B.
Lindemann, unpublished results) suggest that short-term adaptation is caused
by prolonged rises in the intracellular Ca2+ concentration while
long-lasting adaptation is also due to cGMP-dependent mechanisms, it is likely
that opening of Ca2+-dependent K+ channels
(Zufall et al., 1991), and
possibly also Ca2+-dependent Cl channels
(Dolzer, 2002
), is responsible
for the faster decline of the sensillar potential. We are currently testing
whether large Cl channels or non-specific cation channels,
which are probably Ca2+- and/or cGMP-dependent
(Dolzer and Stengl, 1998
;
Dolzer, 2002
), might be
responsible for this stabilization of the resting potential.
Adaptation of the action potential response
In accordance with findings in A. polyphemus
(Kaissling et al., 1987), in
M. sexta the doseresponse relationship of the action potential
response to short and long bombykal stimuli after an adapting stimulus
underwent a larger shift to higher stimulus intensities than did the
relationships of the SP amplitude and the initial slope (Figs
7,
9). This suggests the presence
of at least one additional mechanism of adaptation, acting on the
transformation of receptor potentials into action potentials. This
transformation process probably takes place in the soma or axon hillock
region, morphologically and electrically remote from the origin of the
receptor potentials (de Kramer et al.,
1984
; Kodadová,
1993
), which favours the assumption of a functionally distinct
adaptation mechanism. We will test whether cGMP-
(Zufall et al., 1991
;
Zufall and Hatt, 1991
; Stengl
et al., 1992
,
2001
;
Dolzer, 2002
) or a
Ca2+-dependent phosphorylation of ion channels
(Zufall and Hatt, 1991
;
Stengl, 1993
) are involved in
the adaptation of the action potential response.
In A. polyphemus, a change in the slope of the doseresponse
curves of the action potential frequency, as well as the SP amplitude and
t1/2 rise, was found after adapting stimulation
(Kaissling et al., 1987).
Responses to weak stimuli were further reduced compared with responses to
strong stimuli, leading to a steeper slope of the doseresponse
relationship in the adapted state. In M. sexta, however, the
doseresponse curves were only shifted to higher stimulus intensities
but were not obviously altered in their slope. Our study aimed to investigate
stimuli in a physiological range. Thus, we did not routinely exceed a stimulus
dose of 10 µg and did not statistically analyze whether there is a change
in the slope. In the study by Kaissling et al.
(1987
), applied pheromone
doses were corrected for a nonlinearity in the ratio of the pheromone dose
loaded onto the filter paper and its release, as found by studies on
radioactively labeled pheromone
(Kaissling, 1995
). Because no
radioactively labeled bombykal is available to date, we do not know whether
the same nonlinearity exists for bombykal. Nevertheless, we assume that the
differences in the results are rather due to differences in the stimulus
protocols used to adapt the sensilla. Kaissling et al.
(1987
) used the strongest
stimulus of a protocol that resembles our dose ramp as the only adapting
stimulus for a sequence of test stimuli that were applied in increasing order
successively. Thus, the time interval between the adapting stimulus and the
test stimulus was longer for the test stimuli of higher doses, giving the
sensillum more time to recover. In our current study, however, the intervals
between conditioning and test stimuli were kept constant
(Fig. 2).
Desensitization
The phasictonic action potential response observed with long
stimulations (Fig. 8) indicates
the presence of desensitization (Zufall
and Leinders-Zufall, 2000). Desensitization quickly stops the
response to a stimulus and thus allows for resolving temporally patterned
odour stimuli. The phasictonic pattern of the action potential response
cannot be completely due to a nonlinearity in the stimulus concentration,
because the plateau of the sensillar potential lasts longer than the phasic
part of the action potential response
(Fig. 10;
Kaissling and Priesner, 1970
).
Since rises in the cGMP concentration after adapting pheromone doses are very
slow (Boekhoff et al., 1993
),
it is likely that mostly Ca2+- and/or phosphorylation-dependent
mechanisms and, to a lesser extent, also cGMP-dependent mechanisms are
involved in desensitization. Because after long pheromone stimuli
intracellular Ca2+ rises closed IP3-dependent
Ca2+ channels and Ca2+-dependent cation channels but
opened protein kinase C-dependent cation channels (Stengl
1993
,
1994
), it is likely that these
Ca2+-dependent mechanisms also underlie desensitization. We assume
that after long, strong pheromone stimuli the population of the more slowly
activating protein kinase C-dependent cation channels, which are less
Ca2+-permeable and which are not blocked by Ca2+,
dominates the late phase of the sensillar potential and underlies the tonic
depolarization of ORNs. Future studies will examine which ion channels and
second messenger cascades are involved in the different states of adaptation
to challenge our hypotheses.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bell, R. A. and Joachim, F. A. (1976). Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Ann. Entomol. Soc. Am. 96,365 -373.
Boekhoff, I., Raming, K. and Breer, H. (1990). Pheromone-induced stimulation of inositol-trisphosphate formation in insect antennae is mediated by G-proteins. J. Comp. Physiol. B 160,99 -103.
Boekhoff, I., Seifert, E., Göggerle, S., Lindemann, M., Krüger, B. W. and Breer, H. (1993). Pheromone-induced second-messenger signaling in insect antennae. Insect Biochem. Mol. Biol. 23,757 -762.[CrossRef]
Breer, H., Raming, K. and Boekhoff, I. (1988). G-proteins in the antennae of insects. Naturwissenschaften 75,627 .
Breer, H., Boekhoff, I. and Tareilus, E. (1990). Rapid kinetics of second messenger formation in olfactory transduction. Nature 344, 65-68.[CrossRef][Medline]
Burkhardt, D. (1961). Allgemeine Sinnesphysiologie und Elektrophysiologie der Rezeptoren. Fortschr. Zool. 13,146 -189.
Christensen, T. A., Heinbockel, T. and Hildebrand, J. G. (1996). Olfactory information processing in the brain: encoding chemical and temporal features of odors. J. Neurobiol. 30, 82-91.[CrossRef][Medline]
Christensen, T. A., Waldrop, B. R. and Hildebrand, J. G.
(1998). Multitasking in the olfactory system: context-dependent
responses to odors reveal dual GABA-regulated coding mechanisms in single
olfactory projection neurons. J. Neurosci.
18,5999
-6008.
de Kramer, J. J., Kaissling, K.-E. and Keil, T. A. (1984). Passive electrical properties of insect olfactory sensilla may produce the biphasic shape of spikes. Chem. Senses 8,289 -295.
Dolzer, J. (1996). Mechanismen der Adaptation bei olfaktorischen Sensillen des Schwärmers Manduca sexta. Diploma Thesis. Universität Regensburg, Germany.
Dolzer, J. (2002). Mechanisms of modulation and adaptation in pheromone-sensitive trichoid sensilla of the hawkmoth Manduca sexta. Ph.D. Thesis. Philipps-Universität, Marburg., Germany. http://archiv.ub.unimarburg.de/diss/z2002/0185.
Dolzer, J. and Stengl, M. (1998). Pharmacological investigation of ion channels in cultured olfactory receptor neurons of the hawkmoth Manduca sexta. Göttingen Neurobiol. Report 1998 II,380 .
Dolzer, J., Bittmann, K. and Stengl, M. (1999). Olfactory adaptation in Manduca sexta. ESITO VI, 54.
Dolzer, J., Krannich, S., Fischer, K. and Stengl, M. (2001). Oscillations of the transepithelial potential of moth olfactory sensilla are influenced by octopamine and serotonin. J. Exp. Biol. 204,2781 -2794.[Medline]
Kaissling, K.-E. (1995). Single unit and electroantennogram recordings in insect olfactory organs. In Experimental Cell Biology of Taste and Olfaction (ed. A. I. Spielman), pp. 361-377. Boca Raton, New York, London, Tokyo: CRC Press.
Kaissling, K.-E. and Priesner, E. (1970). Die Riechschwelle des Seidenspinners. Naturwissenschaften 57, 23-28.[Medline]
Kaissling, K.-E., Kasang, G., Bestmann, H. J., Stransky, W. and Vostrowsky, O. (1978). A new pheromone of the silkworm moth Bombyx mori. Sensory pathway and behavioral effect. Naturwissenschaften 65,382 -384.
Kaissling, K.-E., Zack-Strausfeld, C. and Rumbo, E. R. (1986). Adaptation processes in insect olfactory receptors: their relation to transduction and orientation. Chem. Senses 11, 574.
Kaissling, K.-E., Zack-Strausfeld, C. and Rumbo, E. R. (1987). Adaptation processes in insect olfactory receptors. Mechanisms and behavioral significance. Ann. N. Y. Acad. Sci. 510,104 -112.[Abstract]
Kaissling, K.-E., Hildebrand, J. G. and Tumlinson, J. H. (1989). Pheromone receptor cells in the male moth Manduca sexta. Arch. Insect Biochem. Physiol. 10,273 -279.
Kalinová, B., Hoskovec, M., Liblikas, I., Unelius, C. R.
and Hansson, B. S. (2001). Detection of sex pheromone
components in Manduca sexta (L.). Chem.
Senses 26,1175
-1186.
Keil, T. A. (1989). Fine structure of the pheromone-sensitive sensilla on the antenna of the hawkmoth, Manduca sexta. Tissue Cell 21,139 -151.[CrossRef]
Kodadová, B. (1993). Effects of temperature on the electrophysiological response of moth olfactory sensilla. Ph.D. Thesis. Max-Planck-Institut für Verhaltensphysiologie, Seewiesen, Germany.
Kodadová, B. and Kaissling, K.-E. (1996). Effects of temperature on silkmoth olfactory responses to pheromone can be simulated by modulation of resting cell membrane resistances. J. Comp. Physiol. A 179,15 -27.
Lucas, P. and Renou, M. (1989). Responses to pheromone compounds in Mamestra suasa (Lepidoptera: Noctuidae) olfactory neurones. J. Insect Physiol. 35,837 -845.
Marion-Poll, F. and Tobin, T. R. (1992). Temporal coding of pheromone pulses and trains in Manduca sexta. J. Comp. Physiol. A 171,505 -512.[Medline]
Montell, C. (2001). Physiology, phylogeny, and functions of the TRP superfamily of cation channels. Sci. STKE 2001,RE1 .
Pophof, B. (1998). Inhibitors of sensillar esterase reversibly block the responses of moth pheromone receptor cells. J. Comp. Physiol. A 183,153 -164.[CrossRef]
Renou, M. and Lucas, P. (1994). Sex pheromone reception in Mamestra brassicae L. (Lepidoptera): responses of olfactory receptor neurones to minor components of the pheromone blend. J. Insect Physiol. 40,75 -85.[CrossRef]
Roeder, T. (1999). Octopamine in invertebrates. Prog. Neurobiol. 59,533 -561.[CrossRef][Medline]
Starratt, A. N., Dahm, K. H., Allen, N., Hildebrand, J. G., Payne, T. L. and Röller, H. (1979). Bombykal, a sex pheromone of the sphinx moth Manduca sexta. Z. Naturforsch. 34,9 -12.
Stengl, M. (1993).
Intracellular-messenger-mediated cation channels in cultured olfactory
receptor neurons. J. Exp. Biol.
178,125
-147.
Stengl, M. (1994). Inositol-trisphosphate-dependent calcium currents precede cation currents in insect olfactory receptor neurons in vitro. J. Comp. Physiol. A 174,187 -194.[Medline]
Stengl, M., Zufall, F., Hatt, H. and Hildebrand, J. G. (1992). Olfactory receptor neurons from antennae of developing male Manduca sexta respond to components of the species-specific sex pheromone in vitro. J. Neurosci. 12,2523 -2531.[Abstract]
Stengl, M., Ziegelberger, G., Boekhoff, I. and Krieger, J. (1998). Perireceptor events and transduction mechanisms in insect olfaction. In Insect Olfaction (ed. B. S. Hansson), pp. 49-66. Berlin, New York, Heidelberg: Springer.
Stengl, M., Zintl, R., de Vente, J. and Nighorn, A. (2001). Localization of cGMP immunoreactivity and of soluble guanylyl cyclase in antennal sensilla of the hawkmoth Manduca sexta.Cell Tissue Res. 304,409 -421.[CrossRef][Medline]
Störtkuhl, K. F., Hovemann, B. T. and Carlson, J. R.
(1999). Olfactory adaptation depends on the Trp Ca2+
channel in Drosophila. J. Neurosci.
19,4839
-4846.
Tumlinson, J. H., Brennan, M. M., Doolittle, R. E., Mitchell, E. R., Brabham, A., Mazomemos, B. E., Baumhover, A. H. and Jackson, D. M. (1989). Identification of a pheromone blend attractive to Manduca sexta (L.) males in a wind tunnel. Arch. Insect Biochem. Physiol. 10,255 -271.
Vickers, N. J., Christensen, T. A., Baker, T. C. and Hildebrand, J. G. (2001). Odour-plume dynamics influence the brain's olfactory code. Nature 410,466 -470.[CrossRef][Medline]
Wegener, J. W., Boekhoff, I., Tareilus, E. and Breer, H. (1993). Olfactory signalling in antennal receptor neurones of the locust (Locusta migratoria). J. Insect Physiol. 39,153 -163.[CrossRef]
Wegener, J. W., Hanke, W. and Breer, H. (1997). Second messenger-controlled membrane conductance in locust (Locusta migratoria) olfactory neurons. J. Insect Physiol. 43,595 -605.[CrossRef][Medline]
Zack, C. (1979). Sensory adaptation in the sex pheromone receptor cells of saturniid moths. Ph.D. Thesis. Ludwig-Maximilians-Universität, München, Germany.
Zack-Strausfeld, C. and Kaissling, K.-E. (1986). Localized adaptation processes in olfactory sensilla of Saturniid moths. Chem. Senses 11,499 -512.
Ziegelberger, G., van den Berg, M. J., Kaissling, K.-E., Klumpp, S. and Schultz, J. E. (1990). Cyclic GMP levels and guanylate cyclase activity in pheromone-sensitive antennae of the silkmoths Antheraea polyphemus and Bombyx mori. J. Neurosci. 10,1217 -1225.[Abstract]
Zufall, F. and Hatt, H. (1991). Dual activation of a sex pheromone-dependent ion channel from insect olfactory dendrites by protein kinase C activators and cyclic GMP. Proc. Natl. Acad. Sci. USA 88,8520 -8524.[Abstract]
Zufall, F. and Leinders-Zufall, T. (2000). The
cellular and molecular basis of odor adaptation. Chem.
Senses 25,473
-481.
Zufall, F., Stengl, M., Franke, C., Hildebrand, J. G. and Hatt, H. (1991). Ionic currents of cultured olfactory receptor neurons from antennae of male Manduca sexta. J. Neurosci. 11,956 -965.[Abstract]