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First published online July 20, 2006
Journal of Experimental Biology 209, 2911-2919 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02339
Neuromodulation of the locust frontal ganglion during the moult: a novel role for insect ecdysis peptides
1 Department of Zoology, Faculty of Life Sciences, Tel Aviv University, Tel
Aviv 69978, Israel
2 Entomology Department, Cornell University, Ithaca, NY 14853,
USA
* Author for correspondence (e-mail: ayali{at}post.tau.ac.il)
Accepted 8 May 2006
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Summary |
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Here, we focus on the locust frontal ganglion (FG), an important player in moulting behaviour, as a previously unexplored target for ecdysis peptides. We show that application of 10-7 mol l-1 ecdysis-triggering hormone (ETH) or 10-7 mol l-1 and 10-6 mol l-1 Pre-ecdysis-triggering hormone (PETH) to an isolated FG preparation caused an increase in bursting frequency in the FG, whereas application of 10-6 mol l-1 eclosion hormone (EH) caused an instantaneous, though temporary, total inhibition of all FG rhythmic activity. Crustacean cardioactive peptide (CCAP), an important peptide believed to turn on ecdysis behaviour, caused a dose-dependent increase of FG burst frequency. Our results imply a novel role for this peptide in generating air-swallowing behaviour during the early stages of ecdysis. Furthermore, we show that the modulatory effects of CCAP on the FG motor circuits are dependent on behavioural state and physiological context. Thus, we report that pre-treatment with ETH caused CCAP-induced effects similar to those induced by CCAP alone during pre-ecdysis. Thus, the action of CCAP seems to depend on pre-exposure to ETH, which is thought to be released before CCAP in vivo.
Key words: ecdysis, neuropeptides, neuromodulation, crustacean cardioactive peptide, frontal ganglion, Schistocerca gregaria
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), locusts (Hughes,
1980a), moths (Reynolds,
1980; Truman and Endo,
1974; Weeks and Truman,
1984; Miles and Weeks,
1991) and flies (Park et al.,
2002; Park et al.,
2003; Clark et al.,
2004): (i) the pre-ecdysis phase, which includes motor patterns
that are believed to loosen the old and new cuticles, and (ii) ecdysis, during
which the insect extricates itself from its old cuticle by means of anteriorly
directed peristaltic contractions.
At least four different peptide hormones are involved in the control of
these two phases (Hewes and Truman,
1994; Gammie and Truman,
1997; Truman et al.,
1997; Zitnan et al.,
1999): Pre-ecdysis triggering hormone (PETH) and ecdysis
triggering hormone (ETH), both of which are co-released from the Inka cells in
the peripheral epitracheal glands; eclosion hormone (EH), which, depending on
the species, is synthesised by one or two pairs of ventral medial (VM)
neurones located in the protocerebral region of the brain; and crustacean
cardioactive peptide (CCAP), which is expressed in 1-2 pairs of neurones in
each ganglion. Though still somewhat controversial
(Ewer et al., 1997;
Kingan et al., 1997;
Gammie and Truman, 1999;
Zitnan and Adams, 2000), the
generally accepted model of the hormonal interactions leading to ecdysis
behaviours is as follows: the decline of the moulting hormone,
20-hydroxyecdysone triggers the onset of a reciprocal interactions between EH
and ETH, with each peptide causing the release of the other. PETH and ETH act
on the central nervous system (CNS) to initiate pre-ecdysis behaviours.
Centrally released EH is believed to then trigger ecdysis behaviour, either
directly (Hesterlee and Morton,
1996) or by a subsequent release of CCAP within the central
nervous system, in a cGMP-dependent manner. CCAP then triggers the motor
activities necessary to complete ecdysial behaviour
(Truman et al., 1997;
Zitnan et al., 1999;
Gammie and Truman, 1999;
Park et al., 2002;
Park et al., 2003) (for a
review, see Ewer and Reynolds,
2002). This model is consistent with most of the available data.
However, recent reports suggest that the endocrine control of ecdysis might be
even more intricate (McNabb et al.,
1997; Park et al.,
2003; Clark et al.,
2004). Thus, ecdysis offers an important point of interaction
between endocrine and neural control, allowing integration of environmental
cues, in order to ensure the proper timing and sequence of its behavioural
components.
We have previously described a novel central pattern generator (CPG)
network situated in the locust frontal ganglion (FG), and the motor patterns
it generates (Ayali et al.,
2002; Zilberstein and Ayali,
2002). In the desert locust, Schistocerca gregaria, FG
neurones innervate foregut dilator muscles and play a critical role in the
control of foregut motor patterns in different physiological and behavioural
states (Ayali, 2004). We have
recently presented the FG as an important target for chemical modulation
(Zilberstein et al., 2004).
During the moult, the foregut and FG are involved in air-swallowing behaviour.
By filling the gut with air, the larval locust can generate enough internal
pressure on the body wall to eventually split open the old cuticle, and to
then stretch and shape the new adult cuticle and wings after the old cuticle
has been shed at ecdysis (Bernays,
1972). Frontal ganglionectomy abolishes air-swallowing and results
in difficulty or failure in eclosion and wing expansion
(Bell, 1983; Zilberstein and
Ayali, unpublished). A role for the FG during moulting has been reported for a
number of insect species (Bounhiol,
1938; Clarke and Langley,
1963; Penzlin,
1971; Hughes,
1980a; Carlson and O'Gara,
1983; Bell, 1983;
Bestman et al., 1997;
Miles and Booker, 1998).
Here we focus on the FG CPG as a previously unexplored target for ecdysis peptides. We identified CCAP as a potent modulator of the locust FG motor patterns, and our results imply a novel role for this peptide in generating air swallowing behaviour during the early stages of ecdysis. We show that the modulatory effects of CCAP on the FG motor circuits are dependent on the behavioural state and physiological context.
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). The
cages were kept at 30°C under a 12 h:12 h L:D regime. Additional heat,
including direct radiant heat, was supplied during the daytime by incandescent
electric bulbs to bring the day temperature up to 36-38°C. Locusts were
fed daily with fresh Kikuyu grass and flaked oats. Adult and fifth instar
locusts of both sexes were used. Animals were staged relative to ecdysis as
described by Hughes (Hughes,
1980a).
Electrophysiology
Locusts were briefly anaesthetised with CO2, and their wings and
legs removed. The FG and the nerves leaving it are easily accessible by
cutting out a window in the head cuticle (frons). For the in vitro
preparation, the FG and nerves leaving it were dissected out, pinned in a
Petri dish lined with Sylgard (Dow Corning, Midland, MI, USA), and
continuously superfused with locust saline at 26-27°C. Extracellular
recordings were made with suction electrodes. Data were recorded in real time
using a four-channel differential AC amplifier (model 1700, A-M Systems Inc.,
Carlsborg, WA, USA) and stored using an A-D board (Digidata 1320A, Axon
instruments, Molecular Devices, Sunnyvale, CA, USA) and Axoscope software
(Axon instruments).
Cross-correlation analysis
Cross correlation analyses were carried out as described previously
(Zilberstein et al., 2004;
Segev et al., 2004). Briefly,
peak detection was performed on each burst of action potentials. A time window
was selected that included the entire burst duration. A smoothened
representation of the burst activity profile (the activity of all the neurones
firing throughout the burst) is then convoluted with a normalised Gaussian
filter. A matrix representing all recorded convoluted bursts is composed and
the cross-correlation between all pairs of bursts is computed using a standard
algorithm under a MatLab environment (The MathWorks Inc., Natick, MA, USA). A
colour code is then assigned to the values of the crosscorrelation. The
correlation values of sets of bursts (distinct physiological or experimental
conditions) can be averaged and the significance of the results can be
determined.
Tissue processing for CCAP immunoblotting
Brain, and frontal and abdominal ganglia were collected over dry ice and
stored at -70°C until use. Tissues were then homogenised on ice in 1.5 ml
Eppendorf tubes containing homogenisation buffer (40 mmol l-1 Tris
(pH 7.4), 1 mmol l-1 EDTA, 1 mmol l-1 EGTA 1 µg
ml-1 PMSF, 0.01% Triton X-100). Following microcentrifugation at
3360 g for 5 min at 4°C, the supernatant was removed and
analysed for CCAP by immuno dot blot, as described below.
Immuno dot blotting
Nitrocellulose membranes were spotted with 3 µl of ganglion extracts.
After air drying, the membrane was blocked for 1 h with Tris-buffered saline
(0.05 mol l-1 Tris 0.9% NaCl, pH 7.4) containing 0.1% Tween 20
(TBST) and 2.5% BSA. The membrane was then washed three times, for 10 min
each, in TBST and incubated overnight at 4°C in primary antiserum against
CCAP, diluted 1:5000 [code antiCCAP 1TB, kindly provided by Prof. Heinrich
Dircksen, Stockholm University, Sweden; Dircksen and Keller
(Dircksen and Keller, 1988)].
Following incubation, the membranes were washed three times with TBST.
Immunoreactivity was assessed using a secondary goat anti-rabbit antibody,
coupled to horseradish peroxidase, and visualised with a chemiluminescence
reaction (EZ-ECL chemiluminescence detection kit for HRP; Biological
Industries, Beit Haemek, Israel) on Kodak X-OMAT low sensitive film. Films
were scanned and relative CCAP content was estimated by densitometry of the
immunoreactive dots using the Scion Image analysis program (Scion Corporation,
Maryland, USA). The specificity of the primary antiserum has been established
previously in several immunocytochemical and radioimmunological studies on
crustacean and insect tissues (Dircksen
and Keller, 1988; Stangier et
al., 1989; Dircksen et al.,
1991; Ewer and Truman,
1996). An additional control was performed by omitting the
incubation in primary antiserum. No signal was detected after this
procedure.
Immunohistochemistry
CCAP and cGMP immunohistochemistry was carried out as described by Ewer and
Truman (Ewer and Truman,
1996). Animals were anaesthetised with CO2, ganglia
dissected and fixed in 4% buffered paraformaldehyde overnight at room
temperature. They were then rinsed three times for 10 min in
phosphate-buffered saline (PBS) + 0.3% Triton X-100 (PBSTX) at room
temperature and incubated in 0.5-1.0 mg ml-1 collagenase (Type IV;
Sigma Chemical Co., St Louis, MO, USA) in PBSTX for 30-60 min at room
temperature to aid in antibody penetration. They were then rinsed three times
for 10 min in PBSTX and incubated simultaneously in rabbit anti-CCAP and sheep
anti-cGMP. Anti-CCAP was kindly provided by H.-J. Agricola (Universität
Jena, Jena, Germany) and used at a dilution of 1:5000 (cf.
Park et al., 2003). Anti-cGMP
was a generous gift from J. de Vente (Limburg University, Maastrich, The
Netherlands) and was used at a dilution of 1:500 (cf.
Clark et al., 2004). Primary
antibodies were diluted in 2% normal donkey serum (NDS) (Jackson
ImmunoResearch Laboratories, West Grove, PA, USA) in PBSTX. Tissues were
incubated in primary antibody for 24-36 h at 4°C, rinsed four times for 10
min in PBSTX and incubated overnight in Texas-Red donkey anti-rabbit + FITC
donkey anti-sheep (Jackson ImmunoResearch, Inc.) in 2% NDS. Tissues were then
rinsed three times for 20 min in PBSTX, twice for 10 min in PBS, mounted onto
polylysinecoated slides, and dehydrated, cleared and mounted under DPX
(Fluka). The preparations were then examined using a ZEISS LSM 510 confocal
microscope (Carl Zeiss, Jena, Germany). Data from all ganglia were collected
during the same session using the same gain and settings. Horizontal optical
sections were taken at 3 µm steps through each ganglion, and the resultant
z-series was then projected as a flat image. The CCAP-immunoreactive
arborization boundaries in the ganglion neuropil and the innervated axons were
than analysed using LSM 5 Image Browser (Carl Zeiss, Jena, Germany).
Saline and chemicals
Locust saline consisted of 147 mmol l-1 NaCl, 10 mmol
l-1 KCl, 4 mmol l-1 CaCl2, 3 mmol
l-1 NaOH (Frutarom, Haifa, Israel) 10 mmol l-1 Hepes
(Biological industries, Beit Haemek, Israel) pH 7.2
(Abrams and Pearson, 1982;
Penzlin, 1985).
Synthetic Manduca sexta PETH, ETH and EH were a generous gift from
Dr Dusan Zitnan (Slovak Academy of Sciences, Slovakia). CCAP was purchased
from Bachem (Bubendorf, Switzerland). Although the sequence of locust EH, ETH
and PETH is currently unknown, the synthetic CCAP used is identical to locust
CCAP (Stangier et al., 1989).
All chemicals were prepared at different final concentrations, as noted, in
normal saline just before bath application. The FG pattern was examined
before, during, and after 5-10 min of application, as well as after washing
for at least 30 min.
The significance of results was tested using one-way analysis of variance (ANOVA) test, followed by Tukey post test. Kruskal-Wallis test was used for nonparametric tests (Instat, GraphPad software Inc, San Diego, CA, USA).
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) and Gammie and Truman
(Gammie and Truman, 1999)]
caused an acceleration of the rhythm (Fig.
1A). Synthetic Manduca PETH also had an excitatory,
albeit more modest, effect on the FG rhythm.
Fig. 1B summarises the
excitatory effects of ETH and PETH on the in vitro FG burst
frequency. Data are presented as the average change in percentages from
control. 10-7 mol l-1 and 10-6 mol
l-1 PETH significantly increased the burst frequency by
28.5±12.5% and 31.8±16.4%, respectively (N=6). The
effect of the two doses did not differ significantly from each other.
Application of 10-7 mol l-1 ETH significantly increased
the frequency by 54.1±16.6% (N=9; P<0.05). Thus,
the responsiveness of the FG to ETH is much higher than that to PETH. In
contrast to the previous effects of PETH and ETH, application of
10-6 mol l-1 EH generated an instantaneous, though
transient, total inhibition of all FG rhythmic activity
(Fig. 1C; N=5).
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80% compared to control; see also
Fig. 3). Furthermore,
application of 10-5 mol l-1 CCAP directly on the FG of
an intact adult animal through a small opening in the head capsule resulted in
clear swallowing movements and strong activation of gut motor patterns [as
seen during the pre-ecdysis behaviour
(Zilberstein and Ayali,
2002)]. When the experiment was performed on animals in which the
foregut was exposed we also observed subsequent appearance of air bubbles in
the crop (Fig. 2B). These
results indicate that CCAP is a potent modulator of the FG rhythm and since
air-swallowing behaviour starts before the onset of ecdysis behaviour
(Hughes, 1980c), they may
indicate an early role for CCAP in generating air-swallowing behaviour during
the moult.
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State-dependency of the excitatory dose-dependent effect of CCAP
We further examined the dose-dependent effects of CCAP on the rhythmic
activity of FG dissected out from feeding versus (vs)
moulting (i.e. in the stage where the gut was already filled with air and the
old cuticle started to split) animals. We found that CCAP caused a
significantly greater acceleration of the FG rhythm in moulting vs
feeding animals (Fig. 3A). In
Fig. 3B we used a recently
introduced advanced cross-correlation analysis technique
(Zilberstein et al., 2004) to
determine the effects of CCAP on the temporal characteristics of the FG rhythm
(spike timing within the burst; data marked by dashed line in
Fig. 3A were used).
Fig. 3B shows a
cross-correlation matrix of a sequence of bursts recorded under three
experimental conditions: 10-6 mol l-1 CCAP and
10-5 mol l-1 CCAP in feeding animals, and
10-6 mol l-1 CCAP in moulting animals (separated by
dashed line). Three separate correlated clusters can be seen: as expected,
each group of bursts (experimental conditions) was most strongly correlated to
itself (shades of red; 
>0.95).
Interestingly, the next strongest correlation was between feeding animals
treated with 10-5 mol l-1 CCAP and moulting animals
exposed to 10-6 mol l-1 (cross-correlation value of
0.89±0.04). The lowest correlation was obtained in animals from the two
different stages treated with 10-6 mol l-1 CCAP
(=0.79±0.05). Thus, there
was a developmental stage as well as dose dependency in the effect of CCAP.
This dependency was manifest not only at the level of cycle period
(Fig. 3A), but also in other
burst characteristics (Fig.
3B).
In order to explore the bases of this state dependency, we pre-exposed
ganglia dissected from feeding locusts to the ETH peptide prior to CCAP
application. ETH is situated upstream of CCAP in the ecdysis hormonal cascade
(see Introduction). In addition, ETH receptors have recently been located in
FG neurones (M. Adams, UC Riverside, USA, personal communication), suggesting
that the FG may be a target of this peptide. The FG was first treated with
ETH, and 10 min later (once the rhythm had stabilised) with CCAP. We found
that in feeding animals pre-exposure of the FG to ETH increased the CCAP
response of the FG to levels similar to those induced by CCAP alone on the FGs
of moulting animals (53.2±13.3 and 59.6±8.7, respectively,
compared with 31.8±9.8 for 10-6 mol l-1 CCAP
treated FGs from feeding animals, Fig.
4). Even though the increased responsiveness induced by CCAP on
the ETH-pretreated group was similar to that induced on the FGs of the
moulting groups, cross-correlation analysis revealed a low correlation between
these two responses
(=0.77±0.05). This low
correlation suggests that ETH may not be sufficient to fully `prepare' the
frontal ganglion for its ecdysis-related task.
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CCAP levels in the FG and other ganglia during the moult
We used dot-blot analysis to measure CCAP levels in the FG, abdominal
ganglia and brain from the following five stages: I, mid fifth larval instar;
II, fifth larval instar, pre-eclosion (less than 12 h); III, early stages of
eclosion (from air-swallowing to cuticle rupture); IV, advanced stages of
eclosion; V, adult. We found that CCAP levels in abdominal ganglia showed a
tendency to increase as the animal approached the time of eclosion, but this
increase was not statistically significant. CCAP levels then showed a
statistically significant drop after eclosion (1.3±0.2; N=5
vs 0.6±0.1; N=9, in stages IV and V, respectively;
P<0.01; Fig. 5A).
This is consistent with findings from Manduca, which show that CCAP
activates the ecdysis motor program and its immunoreactivity drops
post-ecdysis in the abdominal ganglia neuropil
(Gammie and Truman, 1997).
Fig. 5B demonstrates that the
significant decrease in CCAP levels occurred earlier (prior to ecdysis) in FG
than in the abdominal ganglia (1.2±0.07, N=8 vs
0.9±0.09, N=5 and 0.77±0.07, N=9; stages
III-V, respectively; stage III vs stage IV, P<0.05; stage
III vs stage V, P<0.001;
Fig. 5B). In the brain the time
course of the changes in CCAP levels was similar to that seen in abdominal
ganglia (data not shown).
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), we found one to three CCAP-immunoreactive neurones
in the tritocerebrum that project through the frontal connective nerve and
give rise to extensive arborization within the neuropil of the FG.
Fig. 6A demonstrates the
variation in the number of CCAP immunoreactive axons in the FG neuropil at the
different stages examined. In mid-larvae only one to two (mostly one) fibres
were stained, whereas at the air-swallowing stage at least three afferent
fibres were visible. As ecdysis progressed the number of stained axons then
declined. A quantitative analysis of size of the CCAP-immunoreactive neuropil
was consistent with the results obtained using dot blot. As exemplified in
Fig. 6B and quantified in
Fig. 6C, we found that the area
of CCAP-immunoreactive neuropil increased during air-swallowing and decreased
at late ecdysis.
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One to three CCAP-immunoreactive cell bodies were observed in the locust FG. The axons of these cells did not branch within the ganglion. No difference was found in the staining of these cell bodies at the different stages of the moult (not shown).
cGMP immunoreactivity was not observed in the tritocerebrum and FG CCAP
cells at different stages. However, one of the two pairs of
CCAP-immunoreactive cells in the abdominal ganglia showed elevation of cGMP
during ecdysis (data not shown), as observed during the ecdysis to the first
instar (Truman et al.,
1996).
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; Harris-Warrick,
1994; Grillner et al.,
1994; Marder et al.,
1994; Marder and Thirumalai,
2002). Insect ecdysis offers a model for the intricate
interactions between endocrine and neural control. It is composed of a series
of motor patterns whose timing must be precisely coordinated to ensure the
proper execution of its vital outcome.
The imaginal ecdysis of the desert locust has been described in details
(Hughes, 1980a). As the moult
approaches, the last larval instar locust ceases to feed (approximately 24 h
prior to shedding the old cuticle). The abdomen acts as a ventilatory pump,
performing characteristic movements that may help to loosen the old cuticle,
which are described as the preemergence behaviour. During this time the FG
acts in full synchrony with the ventilation rhythm
(Zilberstein and Ayali, 2002).
This synchronisation is momentarily switched off at the specific time of air
swallowing activity, during which the endogenous FG activity emerges
(Zilberstein and Ayali, 2002).
Gut inflation proceeds intensively until the old cuticle splits. As the gut
fully inflates, the FG pattern again becomes synchronous with the ventilation
rhythm. After eclosion, the gut remains inflated throughout the `expansional
motor program' (Hughes, 1980b)
that serves to expand the new cuticle into its final form and to expand the
wings of the emerged locust.
As mentioned previously, the FG plays a critical role in the moult
(Bounhiol, 1938;
Clarke and Langley, 1963;
Penzlin, 1971;
Hughes, 1980a;
Carlson and O'Gara, 1983;
Bell, 1983;
Bestman et al., 1997;
Miles and Booker, 1998;
Zilberstein and Ayali, 2002).
In this study we focused on the FG as a previously unexplored target for
ecdysis peptides. We first screened the known ecdysis peptides to look for a
potent FG modulator and a candidate for activating the air swallowing
behaviour. Using an in vitro preparation, we found that both PETH and
ETH accelerated the FG rhythm, whereas EH transiently inhibited it. We have
previously reported that haemolymph collected from non-feeding pre-moult
larvae inhibited the FG rhythmic activity
(Ayali et al., 2002). In
Manduca there is also evidence for foregut and FG modulation during
the moult, and haemolymph collected from moulting larvae and applied to a
larval FG-foregut preparation alters the ongoing feeding motor pattern to
resemble that observed in moulting larvae
(Bestman et al., 1997).
Interestingly, a rhythmic motor pattern that resembles air swallowing could be
generated in isolated Manduca heads 24-30 h before eclosion by
application of eclosion hormone (EH)
(Miles and Booker, 1998).
Carlson and O'Gara (Carlson and O'Gara,
1983) reported that the cricket FG could generate activity in
vitro only if isolated from near-moult, ecdysing, or expanding insects.
This state washed out within 1 h, during which the ganglion generated an
air-swallowing pattern.
These diverse, sometimes contradictory, observations could be explained in
light of our current results by considering the different and complex effects
of the various insect ecdysis-related peptides
(Truman et al., 1997;
Zitnan et al., 1996;
Kingan et al., 1997), and by
suggesting that the timing of peptide application can cause significantly
different responses.
We found that CCAP was the most potent modulator of the FG rhythm. CCAP was
originally isolated and sequenced from the pericardial organs of the shorecrab
Carcinus maenas. It has been shown to increase the amplitude and
frequency of the crayfish heart constrictions
(Stangier et al., 1987).
Further studies revealed an extensive network of CCAP-IR neurones in the
central nervous system of Carcinus maenas, suggesting that the
peptide may also function as a neurotransmitter (Dircksen and Keller, 1998) or
neurohormone (Dircksen et al.,
1991; Donini et al.,
2001). CCAP has since been discovered in insects, including the
tobacco hawkmoth, Manduca sexta
(Cheung et al., 1992), and the
migratory locust, Locusta migratoria
(Stangier et al., 1989;
Dircksen and Homberg, 1995).
The pattern of CCAP-IR cells and fibres was described in the brain and the
nervous system of L. migratoria
(Dircksen et al., 1991;
Dircksen and Homberg, 1995).
Most importantly, CCAP has been implicated in the onset of insect ecdysis
behaviour (Gammie and Truman,
1999). Furthermore, the sequence of the CCAP peptide is identical
in all arthropods sequenced to date (including the locust)
(Stangier et al., 1989;
Veelaert et al., 1997) Thus,
the synthetic CCAP we applied in vitro corresponds to locust
peptide.
In order to investigate the exact role of CCAP in the execution and
regulation of ecdysis, Park et al. (Park
et al., 2003) used transgenic Drosophila bearing targeted
ablations of CCAP neurones. These insects expressed specific defects at
ecdysis, and at adult eclosion showed a failure in abdominal inflation. These
results are consistent with our current data on the role of CCAP early in
ecdysis, during air-swallowing (e.g. gut inflation) behaviour. This is a novel
effect of CCAP, which takes place earlier than the traditional role attributed
to this neuropeptide in the final stages of the moult.
We were not able to fully mimic, in our in vitro preparation, the
motor patterns observed during the ecdysis in vivo
(Zilberstein and Ayali, 2002).
In addition to being due to complex effects of the various insect
ecdysis-related peptides, this could also be due to another important finding
of the current study, i.e. that the modulatory effect of CCAP is strongly
dependent on physiological context. We found the effects of CCAP on FG burst
frequency to be greater in moulting than in feeding animals. The doses we used
for moulting animals were consistent with those reported in the literature
(Zitnan and Adams, 2000),
whereas higher doses were needed to affect the FG activity of feeding animals.
Interestingly, pre-treatment of FGs from feeding animals with ETH increased
the effect of CCAP to levels similar to those obtained in moulting animals.
Thus, the response to CCAP seems to depend on pre-exposure to ETH, which is
thought to be released before CCAP in vivo (for a review, see
Ewer and Reynolds, 2002;
Mesce and Fahrbach, 2002).
Practically nothing is known about the ETH in locusts; however, evidence in
the literature suggests that, although Inka cells of different insects are
very variable in number and morphology, they have a conserved function in
activation of the pre-ecdysis and ecdysis motor programs within the CNS (e.g.
Zitnan et al., 2003).
An animal's physiological and behavioural state is known to be an important
factor affecting the specific actions of neuromodulators in the case of
modulation of sensory pathways (Kay and
Laurent, 1999; Christensen et
al., 2000; Cardin and Schmidt,
2004). Whether the same applies to modulation of motor circuits
has been unclear, as fewer examples are known of context-dependent modulation
of motor output (Nusbaum and Marder,
1989). Thus, our results strengthen the importance of insect
ecdysis as a model of neuromodulation for behaviour.
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Acknowledgments |
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References |
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Ayali, A., Zilberstein, Y. and Cohen, N.
(2002). The locust frontal ganglion: a central pattern generator
network controlling foregut rhythmic motor patterns. J. Exp.
Biol. 205,2825
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