OSCILLATIONS OF THE TRANSEPITHELIAL POTENTIAL OF MOTH OLFACTORY SENSILLA ARE INFLUENCED BY OCTOPAMINE AND SEROTONIN
1
Biologie, Tierphysiologie,
Philipps-Universität Marburg,
Karl-von-Frisch-Straße, D-35032 Marburg, Germany
2
Institut für Zoologie,
Universität Regensburg, D-93040 Regensburg,
Germany
*
Author for correspondence at address 1 (e-mail:
stengl{at}mailer.uni-marburg.de
)
Accepted June 8, 2001
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Summary |
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In extracellular tip recordings of pheromone-dependent trichoid sensilla on the antennae of male Manduca sexta moths, we investigated the effects of octopamine and serotonin on the transepithelial potential, which is generated by the activity of V-ATPases in sensillar accessory cells. In addition, the action potential activity of unstimulated olfactory receptor neurons was examined in the presence of biogenic amines. Under constant environmental conditions, the transepithelial potential oscillated regularly with periods of 2-8 min and with a 1-25 mV peak-to-peak amplitude over periods of several hours. These oscillatory intervals were interrupted by periods of relatively stable transepithelial potential, correlated with flight activity by the moth. Octopamine reduced the amplitude of the transepithelial potential oscillation and decreased the resistance of the sensillum preparation in a dose-dependent manner. Serotonin altered the waveform of the transepithelial potential, but did not change the resistance of the preparation. Thus, both amines affect the accessory cells, but have different targets in the regulation of the transepithelial potential. Neither amine significantly influenced the spontaneous action potential activity of the olfactory receptor neurons.
Key words: Manduca sexta, pheromone sensillum, tip recording, transepithelial potential, oscillation, action potential, octopamine, serotonin
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Introduction |
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In tip recordings of pheromone-dependent sensilla trichoidea, we tested
whether the biogenic amines octopamine and serotonin affect the
accessory-cell-dependent TEP in Manduca sexta antennae or the
spontaneous action potential activity of the two pheromone-dependent ORNs
found in each sensillum (Kaissling et al.,
1989; Keil,
1989
). Our experiments
revealed no significant effects of the biogenic amines on the spontaneous
activity of unstimulated ORNs, but showed that both amines, either directly or
indirectly, affect different targets in the accessory cells that generate the
TEP.
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Materials and methods |
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Environmental conditions
All recordings were performed at room temperature (18-23 °C). The
long-term recordings were performed in constant room light to exclude
circadian effects. No part of the electrophysiological apparatus had been in
contact with pheromone for at least 15 months. Charcoal-filtered and moistened
air was blown continuously over the preparation (131 min-1).
Drug application
Octopamine and serotonin (Sigma, Deisenhofen, Germany), dissolved in
haemolymph Ringer, were injected through the hole in the head capsule with a
glass capillary. We injected a minimum of 2 µl of a 0.5 mmol l-1
octopamine stock solution and a maximum of 5 µl of a 500 mmol
l-1 solution, resulting in doses of 1-2500 nmol. The highest dose
was of the same order of magnitude as in previous studies by Pophof (Pophof,
2000) and Grosmaitre et al.
(Grosmaitre et al., 2001
), who
injected a maximum dose of 1 µl of a 100 µg µl-1 solution
(527 mmol l-1). Because an adult moth contains approximately 1 ml
of haemolymph (J. Truman, unpublished observation), the final octopamine
concentration in the haemolymph was between 1 µmol l-1 and 2.5
mmol l-1.
In addition, we injected a minimum of 2 µl of a 5 mmol l-1 serotonin solution and a maximum of 15 µl of a 50 mmol l-1 solution, resulting in a dose of 10-750 nmol and a haemolymph concentration of 10-750 µmol l-1. In a set of pilot experiments, food dye injected at the same site was transported into the antenna with a delay of <1 to 3-5 min.
Polarity conventions
Voltage polarity is given for the sensillum lymph electrode with reference
to the haemolymph electrode. Current flow is defined in terms of the movement
of positive charge. The sign of current values was chosen to match the
polarity of the corresponding voltage response. Therefore, current is termed
positive if it flows out of the sensory hair into the sensillum lymph
electrode, and negative if it flows the opposite way (see Redkozubov,
2000).
Data-acquisition protocols
At the beginning of each recording, a series of 5 mV calibration pulses was
applied to the haemolymph electrode, which was otherwise grounded. The
resistance of the preparation (Rprep) was then determined
by injecting current pulses of -100 pA through the recording electrode (de
Kramer, 1985). To improve the
signal-to-noise ratio, 10 current steps were subsequently applied, and the
voltage responses were averaged. For current injections, the input resistance
of the amplifier (Ri) was reduced to
109
. Since the amplification factor changes, if
Ri and Rprep are in the same range/of
the same order of magnitude, an additional calibration step was performed for
voltage measurements during current injection. The correct calibration of the
injected currents was regularly verified with a reference resistor connected
to the amplifier.
Current step protocols were used to determine the current amplitude required to elicit action potentials electrically. Positive current pulses of 50 ms duration, incrementing by 25 pA, were injected, and the capacitative transients originating from the passive properties of the sensillum in combination with the capacitance neutralization circuit of the amplifier were eliminated by adding the voltage responses to two pre-pulses of half the amplitude and of opposite polarity (P/N leak subtraction). The current that elicited action potentials was determined for large and small action potentials separately using the averaged results of five sequential protocols. To account for spontaneously occurring action potentials, action potentials were only considered to be elicited if they were also present in at least the succeeding two sweeps (i.e. at a higher current amplitude). For the same reason, protocols with more than 36 action potentials in the baseline region (50 ms pre-step and 50 ms post-step for each sweep) were discarded. In these cases, the mean of the remaining protocols was analyzed.
For long-term recordings, the signals were acquired in segments of 10 min, at a sampling rate of 19.6 kHz (Clampex, fixed-length events). Each action potential triggered a sweep of 12.75 ms duration, and the highpass-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. Thus, the time course of the TEP was monitored with variable sampling intervals depending on the occurrence of action potentials. The baseline of all action potential sweeps was then adjusted to 0 mV (see Fig. 5A) to identify sweeps that were triggered by artefacts. The peak-to-peak amplitude of each action potential was measured and plotted versus the time of its occurrence (see Fig. 5B). In addition, amplitude distribution histograms were created (see Fig. 5C). A combination of these plots was used to determine the threshold for action potential sorting.
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Action potential sorting
Action potentials separated by an interspike interval (ISI) of no more than
50 ms were defined as members of bursts. The decreasing amplitude of the
action potentials within a burst (see Fig.
4C, Fig. 5B) did
not allow a simple threshold-based procedure for action potential sorting.
Instead, an MS-Excel macro was used that performed the following steps: if the
interspike interval preceding the action potential under consideration was
larger than 50 ms, only the peak-to-peak amplitude was used for action
potential classification. Any action potential that occurred 50 ms or less
after an action potential classified as large was also considered to be large.
Since the amplitude only decreased, but never increased, during bursts, all
action potentials with an amplitude above threshold were classified as large,
even if they occurred within 50 ms of a small action potential. This could
cause misclassifications, however, if a single large action potential were to
occur within a burst of small ones, since the subsequent members of the burst
would incorrectly be classified as large. The action potential sorting
algorithm recorded such cases, and these action potentials were classified
manually.
|
Bursting behaviour
After separating the action potentials into two classes, we determined the
interspike intervals (ISIs) for each 10 min data segment. The variables
computed to describe the spontaneous action potential activity and the
bursting behaviour of the ORNs were (i) the mean action potential frequency,
(ii) the percentage of action potentials that were members of bursts, (iii)
the mean number of action potentials per burst and (iv) the coefficient of
variation (CV) of the ISIs, where CV=S.D./mean. In a sequence of events that
occur independently of preceding events, i.e. in a Poisson process, the CV of
the intervals between every two successive events is equal to 1 (Rospars et
al., 1994). Thus, a CV
significantly different from 1 indicates that the action potentials are not
randomly distributed.
Resistance and transepithelial potential
At the beginning of each recording, when typically no TEP oscillations were
present, the TEP and the resistance of the preparation were measured. The
measured TEP value was corrected for the electrode potential (-35.6±1.4
mV; median ± S.E.M.; N=6). To investigate the influence of
drug injections, the resistance was measured twice within 2-10 min before the
injection, twice within a period of 2-10 min after the injection, and once
more 10 min later. Since no significant difference in the group of
pre-injection measurements and in the group of post-injection measurements was
found, each group was averaged to yield the resistances before
(Rbefore) and after (Rafter) drug
injection. The normalized resistance
Rafter/Rbefore was then computed.
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Results |
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The recordings could be maintained for up to 100 h when no drugs were injected, but were usually terminated earlier. No steady trend in action potential frequency could be detected during the course of long-term recordings (Fig. 1), and the action potentials of both amplitude classes appeared to undergo random fluctuations. If a sensillum appeared to be damaged, as judged from rapid TEP breakdown or the disappearance of one action potential class, the recording was discarded. In total, 177 sensilla from 138 animals were recorded, but most measurements were only performed on a subset.
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Oscillations of the transepithelial potential
In 57 of 58 recordings that lasted more than 15 h
(Table 1), the TEP exhibited a
periodically oscillating time course with a peak-to-peak amplitude between 1-2
and >25 mV and a period of 2-8 min. The waveform was typically
asymmetrical, with a shoulder on the decay phase of the positive peak
(Fig. 1, see
Fig. 3B), but sinusoidal
signals were also observed. The oscillations were occasionally present from
the beginning of a recording, but typically started between 2 and 15 h after
the recording had been established. In most recordings, the oscillations
developed gradually, with slowly increasing amplitude, thus preventing a
quantitative analysis of the onset time. During the periods of oscillating
TEP, referred to here as oscillatory intervals, the animals typically
exhibited no flight activity (Table
1). The oscillatory intervals were interrupted by periods during
which the time course of the TEP fluctuated less periodically or remained
constant (Fig. 1). These
periods are termed non-oscillatory intervals. Flight activity typically was
observed only during non-oscillatory intervals, occurring in bursts of
approximately 5-15 s duration separated by periods without flight activity of
15 to >100 s.
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Oscillatory intervals typically lasted 1-5 h, but their duration was highly variable (up to >20 h). Non-oscillatory intervals, in contrast, had a shorter and more constant duration of approximately 20-50 min. Because the spontaneous occurrence and the duration of non-oscillatory intervals were very variable and appeared random, we did not attempt any further analysis. The sequence of oscillatory intervals without flight activity and non-oscillatory intervals associated with flight activity was observed in 47 of the 58 recordings of more than 15 h duration. Flight activity occasionally decreased over the course of long recordings and could be completely absent by the end. Flight activity during oscillatory intervals, however, was only observed in six experiments (Table 1).
The injection of 1-2500 nmol of octopamine into the head capsule suppressed the TEP oscillations after a delay of one to several minutes (Fig. 2; Table 2). The TEP stabilized at variable values between the maximum and minimum potential during the previous oscillations. Both the degreee of suppression and the duration of the effect were dose-dependent. At doses above 1000 nmol, the oscillations were completely absent after 89% of the injections during an oscillatory interval. At these doses, the amplitude of the oscillation did not fully recover for the rest of the recording (Fig. 2A). At lower doses, the suppression was usually incomplete and more transient (Fig. 2B-D). After some control injections with haemolymph Ringer, we observed a transient suppression of the TEP oscillations or a phase shift (Fig. 2B, see Fig. 7A; Table 2) reminiscent of the effect of a low octopamine dose. To account for these effects, for the quantitative analysis (Table 2), we scored the complete absence of oscillations for at least 20 min or a clear reduction in the oscillation amplitude (to two-thirds or less) for at least 30 min as suppression. More transient changes or the absence of any obvious influence were scored as no effect, which applied to 90% of the control injections during oscillatory intervals.
|
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Serotonin injections (N=15) led to a less pronounced reduction in the oscillation amplitude. The effect of serotonin typically resembled that of a 10-fold lower octopamine dose. However, the waveform of the oscillation was more regular after 13 of 14 serotonin injections that were associated with a suppression of the oscillation that was sufficiently transient to allow an analysis. In addition, after five of these injections, the shoulder after the positive peaks was less prominent than before. After five injections, the shoulder was completely absent, as shown in Fig. 3. A similar change in the oscillation waveform was never observed after octopamine injections. When injected during non-oscillatory intervals (N=10), none of the doses of octopamine or serotonin tested had any detectable effects. Because of their variable duration, it was not possible to analyze whether the non-oscillatory intervals were prolonged by drug injections. In none of the experiments did oscillations occur earlier than 20 min after the injection.
Spontaneous action potentials
The distribution of the spontaneous action potentials was not random
(Table 3). On average,
45.7±2.1% of the small action potentials occurred in bursts made up of
3.31±0.12 action potentials and 47.4±2.7% of the large action
potentials occurred in bursts made up of 3.36±0.09 action potentials
(means ± S.E.M.; Fig. 4,
Fig. 5;
Table 3). The action potential
amplitude decreased during bursts, depending on the burst duration. Bursts
were also observed in each of five recordings made using tungsten electrodes
(Fig. 4D). Occasionally, a
single large action potential occurred within a burst of small ones or both
ORNs fired bursts simultaneously. Unless the frequency of both action
potential classes was rather high (>2 Hz), these cases were very infrequent
(<<1% of the total action potentials), indicating that bursts were
independent in the two ORNs. The coefficient of variation computed for the
interspike intervals (ISIs) of both action potential classes was clearly
higher than 1 (Table 3). The
action potential frequency of both ORNs was highly variable between
preparations and even between different sensilla of the same animal. In the
complete absence of pheromone, the highest measured frequencies were 6.7 Hz
for the small and 5.8 Hz for the large action potentials when averaged over 10
min. Both ORNs, however, also had transient periods when less than one action
potential occurred in 10 min. The mean action potential frequencies evaluated
for 438 data segments without drug injection from 66 recordings were
0.542±0.129 Hz (small action potential) and 0.390±0.057 Hz
(large action potentials) (means ± S.E.M.). The frequency of each
action potential class changed during the recordings, with periods of
increased activity and quiescent periods in apparently random sequence
(Fig. 6). However, when
examined over the duration of a recording, the more active ORNs were
consistently more active, while the more silent ones maintained a low average
activity.
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Neither with subjective judgement nor with cross-correlation tests on three sample recordings (not shown) did we find any significant correlation between the time course of the TEP oscillation and the action potential frequency. Furthermore, there was no detectable difference between oscillatory and non-oscillatory intervals with respect to the action potential frequency or any of the variables evaluated that describe the burst behaviour (Fig. 6). Consequently, injections of octopamine or serotonin did not significantly influence the action potential activity either (Fig. 7, Fig. 8).
|
Electrically elicited action potentials
During the application of current step pulses
(Fig. 9), large action
potentials were elicited by a significantly smaller step amplitude
(208.3±7.3 pA) than small ones (243.7±8.0 pA; means ±
S.E.M., N=123, P<0.01, Student's t-test). No
significant changes were found after the injection of any dose of the tested
drugs or of haemolymph Ringer (not shown).
|
Resistance and transepithelial potential
At the beginning of each recording, when TEP oscillations were not usually
present, the resistance of the preparation and the TEP were measured. The mean
resistance of 159 sensilla was 68.3±2.3 M (mean ±
S.E.M.). Octopamine injections reduced the resistance of the preparation by up
to 20 % in a dose-dependent manner (Fig.
10). Serotonin, in contrast, did not significantly alter the
resistance.
|
The TEP, as measured at the beginning of the recordings, was +33.8±0.8 mV (mean ± S.E.M.; N=177) irrespective of whether or not a drift or oscillation was present at the time of the measurement. During non-oscillatory intervals, the TEP fluctuated around variable values between the maximum and minimum potential in preceding and subsequent oscillatory intervals (Fig. 1). Similarly, after amine-dependent suppression of the oscillations, no consistent change in the absolute value of the TEP was found. Instead, the TEP stabilized around variable potentials between the upper and lower bounds of the previous oscillations, apparently dependent on the value of the TEP at the moment that drug action occurred (Fig. 2A-C, Fig. 3A, Fig. 7A).
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Discussion |
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Amine-dependent effects on the transepithelial potential
Interestingly, we found that octopamine suppressed the oscillations of the
TEP in a dose-dependent manner, while serotonin changed the waveform of the
oscillation. In accordance with our findings, octopamine did not alter the
mean value of the TEP in trichoid sensilla of Antheraea polyphemus
(Pophof, 2000). In contrast to
studies in moths, Küppers and Thurm
(Küppers and Thurm,
1975
) found an increase in the
amplitude of the TEP in whole antennae of the cockroach Blaptica
dubia after the application of serotonin. In both these studies, the
recordings lasted only a few minutes, so slow TEP oscillations were not
observed. Octopamine-dependent suppression of TEP oscillations in M.
sexta could stabilize the TEP at variable values, suggesting that an
additional mechanism determines the mean value of the TEP.
Zack (Zack, 1979) concluded
from her studies that accessory cells generate the TEP in insect sensilla. It
is generally assumed that the TEP is generated by active K+
transport through the apical membranes of the tormogen/trichogen cells into
the sensillum lymph cavity (Thurm,
1972
; Thurm,
1974
). The high K+
concentration of approximately 200 mmoll-1 in the sensillum lymph
causes a potential difference of up to 50 mV between the sensillum lymph and
the haemolymph. This potential difference is assumed to add to the
transmembrane potential of the outer dendrites. The localization of the M.
sexta midgut V-ATPase in accessory cells of antennal moth sensilla
(Klein, 1992
; Klein and
Zimmermann, 1991
) suggests the
presence of the same two-step mechanism for generation of the TEP in the
antennal epithelium, as has been described for the midgut and for a number of
other epithelia in insects (for reviews, see Wieczorek,
1992
; Wieczorek et al.,
2000
). Therefore, it is likely
that the slow TEP oscillations reflect feedback-coupled regulatory mechanisms
both in H+ transport by the V-ATPase and in the subsequent
K+/H+ antiport. Because serotonin rather selectively
suppressed the shoulder of the TEP waveform, it is assumed that two distinct,
but coupled, mechanisms are involved in the generation of the TEP. Octopamine
injection, in contrast, reduced the amplitude of the oscillation, suggesting
that the octopamine-sensitive mechanism is the initial process, acting on the
V-ATPase. The serotonin-sensitive process, however, is delayed, and might
therefore reflect a mechanism modulating K+/H+ antiport.
We will test this hypothesis in future experiments by targeting the V-ATPase
and K+/H+ antiport. TEP oscillations also occur in
pheromone sensilla of the moth A. polyphemus (Zack,
1979
) and in Malpighian
tubules of mosquitoes (Beyenbach et al.,
2000
), which also contain
V-ATPases (Wieczorek et al.,
2000
). The question of whether
the oscillations have any biological significance, however, or are just a
by-product of feedback-regulated processes, remains to be determined in
pheromone-stimulated sensilla. Interestingly, Zack (Zack,
1979
) mentioned that TEP
oscillations measured in different sensilla are not necessarily in phase with
each other. Possibly, a stable phase relationship between TEP oscillations in
different sensilla on the antenna could affect the temporal resolution of
pheromone responses. This hypothesis needs to be examined in pheromone
stimulation experiments.
It is not clear how octopamine reaches the accessory cells and what the
effective drug concentration at the sensilla is. The maximal dose used in
these experiments was within the range reported to influence pheromone
responses in other moths (Pophof,
2000; Grosmaitre et al.,
2001
). We then reduced the dose
by three orders of magnitude until we reached a concentration that ceased to
be effective. An octopamine concentration of between 2 and 17
nmoll-1 has been reported in the haemolymph of M. sexta
(Lehman, 1990
). However, it is
not known whether active transport can cause octopamine to accumulate in
specific tissues, such as the antenna. In addition, it is not known how much
octopamine is released by the 1-2 octopaminergic neurons that project into the
antenna (U. Homberg, personal communication). Thus, effective doses are
difficult to determine. But the findings of amine-dependent effects on the TEP
in M. sexta reported here and on pheromone responses in other moths
(Pophof, 2000
; Grosmaitre et
al., 2001
) indicate that the
doses of haemolymph-carried octopamine employed have a functional significance
in the antenna. This assumption is further supported by the localization of a
neuronal type 3 octopamine receptor, which inhibits cyclic AMP synthesis, at
the base of olfactory sensilla on the antennae of the moths Bombyx
mori and Heliothis virescens (von Nickisch-Rosenegk et al.,
1996
). In addition, the weak
and transient octopamine-like effects observed after several of the control
injections (Fig. 2B, Fig. 7A) suggest that the TEP
oscillations are equally affected by endogenous octopamine. The manipulations
during an injection aroused the animals and occasionally caused flight
activity (Fig. 3). Flight
activity is known to be elicited and maintained by octopamine (Kinnamon et
al., 1984
; Orchard et al.,
1993
). Thus, we assume that,
during some control injections, a stress-dependent increase in endogenous
octopamine levels caused the observed transient suppression of the TEP
oscillations together with the initiation of flight activity.
Action potentials
The absence of effects of octopamine and serotonin on action potential
activity in the absence of adequate stimuli indicates that these amines alone
do not directly influence the action potential generator. It appears that a
synergistic factor is required to increase the background activity after
octopamine injections in the presence of low pheromone doses (Pophof,
2000; Grosmaitre et al.,
2001
). It has yet to be tested
whether rises in intracellular Ca2+ concentration or increases in
cyclic GMP or cyclic AMP levels mimic the presence of a low pheromone dose for
these octopamine effects. In addition, no correlation between the time course
of the TEP and any aspect of the spontaneous action potential activity of the
ORNs was found, despite the fact that the TEP adds to the driving force for
the generator potential (Thurm,
1972
). Thus, the ionic
conductivity of the plasma membrane in the outer dendritic segment, the
location of the chemo-electrical transduction machinery, is obviously very low
in the absence of adequate stimuli. If this were not the case, changes in the
driving force up to 20 mV, such as were present during oscillatory intervals,
would very likely be reflected in the action potential frequency. This
assumption is further supported by the fact that no ion channel openings
occurred in unstimulated pheromone-dependent ORNs either in situ or
in vitro (Zufall and Hatt,
1991
; Stengl et al.,
1992
).
Action potentials were elicited by the injection of positive, but not
negative, current (see de Kramer,
1985; de Kramer et al.,
1984
). Positive currents
hyperpolarize the outer dendritic segments of the ORNs, but depolarize
membranes that are situated basal to the zones of septate junctions located at
the transitional region between the inner and outer dendritic segment (Keil,
1989
). Together with the
polarity of the recorded action potentials (positive phase first), this
further confirms the hypothesis that the action potential generator is located
in the soma or axon hillock region. Action potentials with the opposite
polarity, as would be expected for dendritic action potentials, were never
observed. Unexpectedly large currents of more than 200 pA were necessary to
elicit action potentials. In current-clamp recordings of vertebrate (Lynch and
Barry, 1989
; Iida and
Kashiwayanagi, 1999
; Ma et al.,
1999
), lobster
(Schmiedel-Jakob et al., 1989
)
and cultured insect (M. Stengl, unpublished observations; I. Jakob, personal
communication) ORNs, currents as small as 1-10pA were sufficient to elicit
action potentials. Thus, it appears likely that most of the injected current
does not flow through the ORNs, but through a shunt pathway, i.e. through the
accessory cells and the surrounding epithelium. Since the measurement of
preparation resistance is also based on current injections, the measured
resistance is presumably governed by the resistance of the shunt pathway. This
was also assumed by Zack (Zack,
1979
) in A.
polyphemus. The reduction in the resistance of the preparation found
after octopamine injection therefore further supports the hypothesis that the
accessory cells are targets for the biogenic amines. It has yet to be resolved
whether and how this decrease in the resistance of accessory cells can
influence TEP oscillations. Zack (Zack,
1979
) also showed that the
preparation resistance oscillates. These resistance oscillations, however,
cannot be directly responsible for the TEP oscillations because the two types
of oscillation differed in their kinetics and showed a stable phase
difference. If resistance oscillations are also present in M. sexta,
the actual changes in the preparation resistance after octopamine injection
might be even larger than observed.
Physiological implications
What is the physiological relevance for a biogenic-amine-controlled
olfactory sensillum? Because biogenic amines can act as hormones, they could
adjust the general sensitivity (or state of adaptation) of various targets, at
the periphery as well as centrally, at the same time. Such a sensitivity
adjustment could possibly be triggered by a biologically relevant signal, such
as sex pheromones. The octopamine-dependent increase in the action potential
activity of ORNs only in the presence of a low pheromone dose (Antheraea
polyphemus, Pophof, 2000;
Mamestra brassicae, Grosmaitre et al.,
2001
) is consistent with this
hypothesis of a sensitivity adjustment in response to a relevant stimulus. In
addition, an aminedependent sensitivity adjustment could be governed by the
circadian clock of a nocturnal moth because increased sensitivity to pheromone
is strongly dependent on photoperiod cues (Linn,
1997
; Linn and Roelofs,
1986
; Linn and Roelofs,
1992
; Linn et al.,
1992
; Linn et al.,
1996
). It is known that
octopamine levels in the haemolymph show a circadian rhythm and are high at
night, during the activity phase of the moths (Lehman,
1990
), when the females
release pheromone (Itagaki and Conner,
1988
). In our experiments, we
tried to keep endogenous octopamine levels constant by disturbing the
circadian rhythm of endogenous octopamine release using constant light
application (which stops the circadian clock). Thus, we do not know whether
the TEP oscillations show a circadian rhythm in undisturbed moths. In future
experiments, we will test whether there is a circadian rhythm in the pheromone
response of M. sexta, whether it is dependent on the presence of
octopamine and whether it correlates with circadian changes in the kinetics or
the amplitude of the TEP.
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Acknowledgments |
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