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First published online October 5, 2006
Journal of Experimental Biology 209, 4000-4010 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02479
The control of anterior foregut motility during a larval molt of the moth Manduca sexta involves the modulation of presynaptic activity
Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA
* Author for correspondence (e-mail: bestman{at}cshl.org)
Accepted 1 August 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Key words: molting, foregut, Manduca sexta, insect, ecdysis, CCAP, modulation
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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;
Truman et al., 1998).
Recently, we found that the proper modulation of anterior foregut motility
is essential for the successful completion of a larval–larval molt of
M. sexta (Bestman and Booker,
2003). As described by Miles and Booker
(Miles and Booker, 1994) the
anterior foregut of M. sexta larvae is composed of a muscular section
comprising the buccal cavity, the pharynx, esophagus, and the crop. The
rhythmic contractions of the anterior foregut musculature are controlled by
neurons originating from the frontal ganglion
(Miles and Booker, 1994).
During the intermolt larval stages, the alternating constrictions and
dilations of the muscles of the foregut (exclusive of the crop) power
ingestion and together constitute the peristaltic contractions of the anterior
foregut that we describe here (Miles and
Booker, 1994). Early in the molt between the 4th and 5th larval
instars, the old, outer cuticle separates from the newly forming cuticle that
lies beneath it. An enzymatic cocktail, referred to as molting fluid (MF), is
secreted into the space created as the two cuticle layers separate. The first
16–18 h of the molt after the old and new cuticles begin to separate,
the MF digests and weakens the old cuticle to aid in its removal. As the end
of the molt approaches MF is ingested before the old cuticle is shed a few
hours later. The movement of the MF during a larval–larval molt is
correlated with the level of anterior foregut motility. Just prior to the
first appearance of MF in the space between the old and newly developing
cuticle, the robust contractions of the anterior foregut are suspended, only
to return some 16 h later to power the ingestion of MF prior to ecdysis.
During a larval–larval molt, the presynaptic terminals on the
anterior foregut musculature are targeted for modulation
(Bestman and Booker, 2003).
With the beginning of the molt, the decline in anterior foregut activity is
accompanied by a sharp decline in the amplitude of the excitatory junctional
potentials (EJPs) recorded from the esophageal constrictor muscles of the
anterior foregut. Using the styryl dye, FM1-43 it was demonstrated that the
decline in EJP amplitude could be accounted for by a reduction in the efficacy
of the synaptic endings on the anterior foregut musculature. The return of
anterior foregut peristalsis near the end of the molt is correlated with a
dramatic rebound in EJP amplitude and synaptic efficacy. Here we examine the
potential roles of two signals in modulating anterior foregut motility during
a larval–larval molt. We present evidence that an element present in the
hemolymph of early-molt stage larvae may act to suppress the contractions of
the anterior foregut during the molt. The return of the robust peristaltic
contractions of the anterior foregut near the end of molt can be triggered by
the release of the nonapeptide, CCAP. In both instances the presynaptic
endings on the anterior foregut musculature appear to be the targets of these
potential modulators.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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). A
27°C, 16 h:8 h L:D photoperiod regimen was provided for the M.
sexta colony. Under these conditions, the durations of the 4th and 5th
instar stages were 3–4 days and 4–5 days, respectively. Because
head capsule slippage (HCS), when the old head cuticle separates from the
developing new cuticle and the resulting space fills with MF, is an easily
detected and externally recognizable feature of the molt cycle it was used as
the starting reference point for the molt cycle. Under the colony conditions
used, the duration of the molt after HCS between the 4th and 5th instars was
25 h. We used a number of developmental markers to track the progress of the
molt between the 4th and 5th larval instars. We define the early-molt stage as
the first 16 h of the molt after HCS, during which time the newly developing
mouthparts lack pigmentation. The late-molt stage, some 16–18 h after
HCS, is marked by the new mouthparts becoming yellow-brown as they begin to
tan. Approximately 21 h after HCS, air bubbles are first observed in the head
capsule, marking the first sign of the ingestion of the MF. The molt typically
ends some 4 h later with the shedding of the old cuticle during ecdysis.
Anterior foregut preparation and recording techniques
To gain access to M. sexta anterior foregut musculature and
nervous system, larvae were dissected along the dorsal midline and the
anterior foregut, brain and frontal ganglion were surgically isolated
according to methods described (Bestman and
Booker, 2003). The `semi-intact' anterior foregut preparations
prepared in this manner were transferred and pinned to a 35 mm plastic tissue
culture dish lined with silicone elastomer (Sylgard; Dow Corning, Midland, MI,
USA). This preparation preserves synaptic input to the constrictor and dilator
muscle groups of the foregut, and is capable of producing both the
alternating, posterior-directed muscle contractions associated with the
peristaltic swallowing motor pattern of the foregut as well as the `squeezing'
pattern described (Miles and Booker,
1994). Instances of the squeezing motor pattern were very
infrequent; this is probably because of the absence of sensory input from an
intact and full crop, which has been suggested to provide sensory feedback to
the foregut central pattern generator
(Miles and Booker, 1994). The
preparations were bathed in physiological saline [in mmol
l–1: NaCl, 140; KCl, 5; CaCl2, 4; dextrose, 28;
Hepes, 5; MgCl26H2O, 2; trehalose, 5 (modified from
Trimmer and Weeks, 1989)]. For
Ca2+-free saline, the CaCl2 was replaced by 20 mmol
l–1 MgCl2, NaCl was lowered to 124 mmol
l–1, and 0.5 mmol l–1 EGTA was added. For
high K+-free saline, the KCl was increased to 90 mmol
l–1 and NaCl lowered to 55 mmol l–1. The
CCAP (Peninsula Labs, San Carlos, CA, USA) was diluted in physiological saline
and applied to the anterior foregut preparations by exchanging the regular
saline with the CCAP saline solution of appropriate dilution. Extracellular
recordings from the muscles were obtained using custom-made
glass-pipette-tipped suction electrodes. In some experiments, a movement
transducer, constructed from a piezoelectric phonograph cartridge, was
attached to the anterior esophageal constrictor muscles in order to monitor
and record anterior foregut contractions. The brain and frontal ganglion were
removed in the `isolated anterior foregut' preparation and contractions were
elicited by delivering 500 ms trains of 5 ms pulses delivered at 40 Hz to the
severed recurrent nerve using a Grass S48 stimulator with a suction electrode.
The outputs of the movement transducer and the extracellular suction
electrodes were amplified using a differential amplifier (A-M systems,
Carlsborg, WA, USA). To elicit EJPs from the anterior foregut musculature, 10
ms pulses were delivered through a suction electrode to the recurrent nerve.
To record intracellularly from the anterior foregut constrictor muscles, a
piece of Sylgard was inserted into the lumen of the gut in order to stabilize
the foregut. Muscle recordings were made with loosely suspended 20–25
M
electrodes amplified with a Neuroprobe 1600 (A-M Systems) amplifier.
All signals were stored to a VHS cassette tape (A. R. Vetter Instruments,
Rebersburg, PA, USA) and real-time playback was conducted using a high-speed
chart recorder (Astro-Med MT95000, West Warwick, RI, USA).
Preparation of the active hemolymph fraction
To collect hemolymph, larvae were anesthetized on ice and their hemolymph
was extracted through a small incision cut on the proleg with scissors. The
hemolymph was collected in microcentrifuge tubes containing
phenylthiocarbamide (Sigma Chemicals, St Louis, MO, USA) to prevent oxidation.
The hemolymph was then spun in a bench top centrifuge at 20 000
g for 5 min. The supernatant was collected, boiled for
10–15 min, cooled and filtered under vacuum through Watmann no. 1 paper.
The sample was fractionated by serial liquid/liquid extraction first with
ethyl acetate followed by n-butanol. The peristalsis of the intact
anterior foregut preparation was used as a bioassay; intact anterior foregut
preparations were exposed to a sample of each blood fraction and the effects
on the ongoing contractions of the anterior foregut were noted.
FM1-43 labeling
The fluorescent dye, FM1-43 (Molecular Probes, Eugene, OR, USA) was used to
monitor the effects of potential modulators of the presynaptic endings on the
anterior foregut musculature. In all experiments, the anterior foregut
preparations were viewed under a 40x, 0.8 water-immersion lens using a
Nikon Eclipse 600-FN epifluorescent microscope equipped with a 100 W mercury
lamp. The images were captured with a CCD SPOT2 camera (Diagnostic
Instruments, Sterling Heights, MI, USA) with identical acquisition settings
throughout each experiment. Using PhotoShop 7 (Adobe Systems, San Jose, CA,
USA), the amount of FM1-43 taken up by the nerve endings was calculated by
measuring the average luminosity of individual FM1-43 fluorescent puncta from
which average background levels for each image were subtracted. We estimated
the density of FM1-43 labeled puncta by counting the number of fluorescent
puncta per 20 µm2.
To monitor the effects of the inhibitory agent found in the hemolymph of
early-molt stage larvae, the anterior foreguts of intermolt 5th instar larvae
were first `loaded' with the FM1-43 dye by incubating anterior foregut
preparations in normal saline containing 10 µm FM1-43 for 15 min
(Bestman and Booker, 2003). The
preparations were quickly rinsed in Ca2+-free saline, and then
incubated for 20 min in Ca2+-free saline. The presynaptic endings
on the esophageal dilator muscles terminals were then imaged to determine the
amount of dye loaded. The dye was then allowed to unload for 20 min by placing
the anterior foregut preparations in either normal saline or in saline to
which the fraction from the hemolymph of early-molt larvae had been added. The
anterior foregut preparations were then rinsed in Ca2+-free saline,
and imaged. Terminals were selected individually and the `percentage of FM1-43
unloading' was calculated by taking the ratio of the average luminosity of
each selected terminal after and before the period of unloading (unloaded
luminosity/loaded luminosity). Although synapses all over the anterior foregut
became loaded with FM1-43, the signal of the constrictor muscles was difficult
to resolve because of the autofluorescence of the cuticular lining of the
foregut. In addition, synapses tended to lie between constrictor muscles and
even beneath the constrictors, making them more difficult to image. By
contrast, the esophageal dilator muscles are flat and in many places are just
a single fiber thick. In the generation of foregut peristalsis, the activities
of the dilator and constrictor groups are temporally and spatially
interdependent, and therefore because the esophageal dilator muscles provide a
better FM1-43 signal, they were used for these imaging experiments.
To assess the potential role of CCAP in triggering the increase in synaptic activity observed near the end of the molt, the intact anterior foregut preparations of early-molt 4th instar larvae (<16 h after HCS) were loaded in normal saline containing FM1-43 and imaged as above. Next, the preparations were incubated for 20 min in normal saline containing 10–8 mol l–1 CCAP. The preparations were then returned to normal saline solution containing FM1-43 for 20 min then quickly rinsed in Ca2+-free saline and imaged. After each treatment the density of the FM1-43 labeled endings was determined.
CCAP immunocytochemistry
Cold-anesthetized larvae that were in the process of molting between the
second and third instar were cut along the dorsal midline, pinned out and
fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 1–2
h at room temperature. The fixed preparations were then rinsed in PBS for 2 h
at room temperature before being switched to PBS containing 0.3% Triton X-100
(PBST) overnight at 4°C. Tissues were then blocked for 4 h in 10% normal
horse serum in PBS. A rabbit anti-CCAP antiserum (prepared by Dr H. Agricola,
University of Jena, Germany) was diluted 1:5000 in PBST and the tissues were
incubated for 24 to 36 h at 4°C. The specificity of this antibody has been
previously characterized (Ewer and Truman,
1996). After repeated washings in PBST, the preparations were
incubated in Alexa Fluor 488-conjugated goat anti-rabbit (Molecular Probes)
diluted 1:200 in PBST for 2 h at room temperature or overnight at 4°C.
After repeated washings, the preparations were dehydrated through an ethanol
series, cleared in methyl salicylate, mounted in DPX (Fluka, Sigma-Aldrich
Corp., St Louis, MO, USA) and examined using a Bio-Rad 600 confocal
microscope. As discussed above, because the dilator muscles have an improved
signal to noise ratio, the photomicrographs show CCAP labeling of the anterior
foregut dilator muscle group.
Surgical manipulations
Late 4th instar larvae that were committed to entering the molt (mass >1
g) were anesthetized under CO2 gas. A small incision was made in
the cuticle near the target tissue and the CNS lesions made using iris
scissors. The procedures for the sham surgeries were similar except that the
neural tissue was touched with forceps through the incision. The incisions
were sealed with dermatological adhesive (New-Skin, Medtech Laboratories,
Jackson, WY, USA) and the larvae were placed back into the colony to
recover.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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). We
decided to test the possibility that a blood-borne factor might play a role in
triggering the decrease in anterior foregut motility observed during the molt
cycle. Hemolymph was extracted from larvae during the first 2–4 h after
larvae showed signs of apolysis during the molt between the 4th and 5th larval
instars. As a control we used hemolymph obtained from nonmolting, feeding 5th
instar (intermolt) larvae. We then assayed the effect of the hemolymph
extracts on the contraction patterns of semi-intact anterior foregut/frontal
ganglion preparations isolated from intermolt larvae (see Materials and
methods). In saline, anterior foreguts isolated from intermolt larvae show
regular alternating rhythmic contractions of the buccal and esophageal regions
of the anterior foregut, resulting in robust posteriorly directed peristaltic
contractions associated with swallowing
(Bestman and Booker, 2003).
Upon exposure to hemolymph collected from early-molt stage larvae, we failed
to detect peristaltic contractions in 77% (69/90) of the treated semi-intact
anterior foreguts preparations. This percentage was significantly greater than
when the semi-intact anterior foreguts were exposed to hemolymph collected
from intermolt larvae, where only 15% (2/13) of the preparations showed
disruption of anterior foregut activity (P<0.001,
2 test). The hemolymph from early-molt stage larvae was
subjected to fractionation (see Materials and methods) resulting in the
peristalsis-blocking activity of the hemolymph of early-molt stage larvae
remaining in the aqueous fraction. The aqueous extract of early-molt larval
hemolymph suppressed peristalsis by 84% (24/29) compared with that of the
active semi-intact anterior foregut preparations assayed. In comparison, the
ongoing peristalses of significantly fewer, just 15% (3/17) of the anterior
foregut preparations assayed, were disrupted after application of aqueous
extract of intermolt 5th instar hemolymph (P<0.001,
2 test).
Our previous results suggested that the presynaptic terminals on the
anterior foregut musculature are modulated during a larval–larval molt
(Bestman and Booker, 2003). The
decline in anterior foregut peristalsis during the early stages of the molt is
accompanied by a sharp reduction in synaptic efficacy, resulting in attenuated
EJP amplitude recorded from the esophageal constrictor muscles of the anterior
foregut (Bestman and Booker,
2003). If the inhibitory agent found in the hemolymph of
early-molt stage larvae plays a key role in modulating anterior foregut
motility during a larval–larval molt then it should do so by targeting
the presynaptic terminals on the anterior foregut musculature. In an effort to
identify potential targets of the inhibitory hemolymph factor, we used an
isolated anterior foregut preparation (without the brain and frontal ganglion)
and elicited foregut contractions through exogenous stimulation of the
recurrent nerve (Bestman and Booker,
2003). We quantified the strength of the elicited anterior foregut
contractions by attaching a movement transducer to the anterior esophageal
constrictors. When isolated anterior foregut preparations obtained from
intermolt larvae were bathed in saline, all of the foreguts were responsive to
the stimulation and the average amplitude of the elicited contractions was
51.4±5.0 µm (Fig. 1A;
N=6). However, following the application of the hemolymph fraction
from early-molt stage larvae, the average amplitude of the contractions
dropped to less than 10% of the control values, and we failed to elicit a
measurable contraction in 66% of the anterior foreguts tested (N=6;
P<0.001, paired t-test;
Fig. 1B). In all preparations,
there was a complete recovery of activity once the anterior foregut
preparations were washed with fresh saline. Treating the hemolymph isolated
from early-molt stage larvae with a number of proteases destroyed all
activity, consistent with the active factor being a peptide (J.E.B. and M. del
Campo, unpublished observations).
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The decline in anterior foregut motility following exposure to the active fractionated early-molt stage hemolymph is also accompanied by a decrease in the amplitude of the EJP recorded from the anterior foregut esophageal constrictor musculature. In saline, the average EJP amplitude elicited by stimulation of the recurrent nerve was 12.5±1.1 mV (N=10; Fig. 2A). Following exposure to the aqueous hemolymph fraction from the early-molt larvae, the average EJP amplitude fell to 5.6% of the control value (Fig. 2B; N=10; paired t-test, P<0.001). Rinsing the preparations with fresh saline resulted in the full recovery of the EJP amplitude compared with initial values (N=5; P>0.05, paired t-test). The decline in the contraction amplitude of the anterior foregut musculature following exposure to the hemolymph fraction was not due to a drop in the resting potential of the muscle fibers. The resting membrane potential of the muscles recorded in control saline and following exposure to early-molt stage hemolymph aqueous fraction was –40.5±1.5 mV (N=12) and –36.8±0.8 mV potential (N=12) respectively; they were not significantly different (P>0.05, paired t-test).
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Next we used the styryl dye, FM1-43, to determine whether the hemolymph
fraction from early-molt stage larvae targeted the presynaptic endings on the
esophageal dilator muscle group of the anterior foregut. FM1-43 is taken up
exclusively into the presynaptic terminals that have undergone a vesicle
exocytosis/endocytosis event while exposed to the dye
(Betz and Bewick, 1992;
Cochilla et al., 1999) and
therefore the amount of dye present in the terminal serves as an indicator of
the level of synaptic activity. To examine the effect of the early-molt larval
hemolymph fraction on the presynaptic endings we used active semi-intact
anterior foregut preparations from intermolt larvae. We first allowed
endogenous frontal ganglion-driven activity to preload the FM1-43 dye into
synaptic terminals on the anterior foreguts. The dilator muscles of the
foreguts were imaged and the amount of dye loaded was determined by measuring
the luminosity of the synaptic terminals. We next tested whether exposure to
the active hemolymph fraction influenced the rate that FM1-43 was unloaded
from the terminals. For these experiments the preloaded anterior foreguts
(Fig. 3A,C) were allowed to
unload for 20 min in either control saline
(Fig. 3B) or saline containing
the hemolymph fraction (Fig.
3D). At the end of this period the previously identified terminals
were located and their level of luminosity was measured. As expected, when the
anterior foreguts with preloaded terminals were bathed in control saline,
there was a dramatic drop in the amount of dye remaining in the presynaptic
terminals (Fig. 3E). Within 20
min the luminosity of the FM1-43-loaded terminals bathed in control saline
declined by 67.6±9.4% (N=14). In comparison, a significantly
greater FM1-43 signal remained in the anterior foregut terminals exposed to
the active hemolymph, dropping just 20.9±9.1% (N=19;
P<0.01, unpaired t-test). Thus exposure to the blood
fraction resulted in a significant decrease in the efficacy of the presynaptic
terminals on the foregut dilator musculature.
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Crustacean cardioactive peptide triggers the precocious increase in anterior foregut motility
As the end of the molt approaches, there is a sudden rebound in the
amplitude of the anterior foregut contractions, followed a short time later by
the first signs of the MF ingestion
(Bestman and Booker, 2003). We
were curious to determine what factor(s) might play a role in the return of
anterior foregut peristalsis. For a number of reasons we focused on CCAP as a
potential modulator of anterior foregut motility around the time of MF
ingestion. CCAP is a nonapeptide and acts as a neuro- and myomodulator in a
number of different arthropods (Breidbach
et al., 1995; Groome and
Lehman, 1995; McNeil et al.,
1998; Mulloney et al.,
1997). In the central nervous system (CNS) of M. sexta
there are over 90 neurons that express the CCAP gene
(Loi et al., 2001). There is
also evidence for a role for CCAP in triggering the ecdysial motor patterns
responsible for removing the old cuticle at the end of the molt
(Ewer et al., 1997;
Truman et al., 1998).
We found it intriguing that one of the four pairs of CCAP-immunoreactive
(CCAP-ir) neurons, which have been repeatedly identified in the subesophageal
ganglion (SEG), has also been reported to send out processes terminating in
the immediate region of the anterior foregut
(Davis et al., 2001;
Ewer et al., 1994;
Klukas et al., 1996;
Loi et al., 2001). Using CCAP
antiserum, we labeled possible release sites on the muscles of the anterior
foregut (shown in Fig. 4, the
anterior foregut esophageal dilators). This labeling was reminiscent of
CCAP-ir neurohemal structures found on other M. sexta tissues and in
other species (Davis et al.,
2001; Donini et al.,
2002). With these data in mind, we wanted to test whether CCAP
also plays a role in triggering the return of the peristaltic contractions of
the anterior foregut observed at the end of the molt.
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A dose–response curve was constructed by measuring the amplitude of the anterior foregut contractions in the initial saline condition and then following a single exposure to saline containing CCAP (Fig. 6). For these experiments we used isolated anterior foregut preparations obtained from early-molt stage larvae and elicited anterior foregut contractions through stimulation of the attached recurrent nerve (see Materials and methods). In all instances the magnitude of the response was determined within 3 min of the single application of various CCAP doses diluted in physiological saline, and identical stimulation settings were used before and after CCAP application. Under these conditions the threshold concentration of CCAP to produce a detectable response was 10–12 mol l–1 (N=10; P<0.05, paired t-test). At a concentration of 10–7 mol l–1 CCAP the maximal response recorded was over fivefold that of the saline only values (578±217%, N=8), with the response reaching a plateau or slightly declining at higher concentrations. The anterior foreguts prepared from intermolt larvae also responded to the application of CCAP with an increase in contraction amplitude. However, although significant, these increases were smaller relative to the results obtained for the early-molt stage anterior foregut preparations. For example, following the application of 10–8 mol l–1 CCAP to the anterior foreguts isolated from intermolt larvae, the amplitude of the contractions increased less than twofold compared to controls (Fig. 6B; 170±42%, P>0.05, paired t-test).
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CCAP also triggered a rapid increase in the EJP amplitude recorded from the esophageal constrictor muscle fibers of an isolated anterior foregut preparation obtained from early-molt stage larvae (Fig. 7). In control saline the average muscle EJP recorded was 8.2±1.3 mV (N=20), significantly lower than the 14.6±5.9 mV (N=6) potential recorded from the esophageal constrictor muscle of intermolt larvae (Fig. 7B; unpaired t-test, P<0.01). Within 10 min of exposure to 10–8 mol l–1 CCAP, the average EJPs recorded from anterior foreguts isolated from early-molt stage larvae increased to 16.3±2.1 mV (N=18; paired t-test, P<0.001). By contrast, the application of CCAP had no significant effect on the amplitude of the EJPs recorded from muscles of anterior foreguts isolated from intermolt larvae.
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The subesophageal ganglia and brain are necessary for molting fluid ingestion
The data presented above reveal that the application of exogenous CCAP was
sufficient to trigger a return of the robust peristaltic contractions of the
anterior foregut late in the molt cycle. Although the published
immunocytochemical studies cited above indicate that the SEG is the source of
the CCAP-ir endings found on the anterior foregut musculature, at this point
there is no direct evidence supporting a role for these CCAP inputs
contributing to the modulation of anterior foregut activity during a
larval–larval molt. In an effort to establish that the SEG is necessary
for MF ingestion, we carried out a series of surgical manipulations on fourth
instar larvae that had already committed to the molt cycle. We then determined
whether the surgically manipulated animals ingest their MF and initiated
ecdysis within 35 h of the onset of the molt
(Table 1). Larvae that had
undergone sham surgery served as controls. All the sham-operated control
larvae ingested their MF and initiated ecdysis within 35 h of the onset of the
molt. By contrast, only 23% (3/13) of the SEG-lesioned larvae ingested their
MF and 8% (1/13) initiated ecdysis. We next tested whether the connections
between the SEG and the rest of the CNS were necessary for MF ingestion and
the initiation of ecdysis. All of the larvae in which the connectives between
the SEG and T1 were severed swallowed their MF, however, they all failed to
initiate ecdysis. Similarly, 78% of the larvae in which the connectives
between the brain and SEG were severed ingested their MF, while only 20%
initiated ecdysis. To determine whether the brain also plays a role in
regulating MF ingestion, the brain itself was also ablated but the frontal
ganglion and its projections to the anterior foregut were left intact.
Following the removal of the brain none of the molting larvae swallowed their
MF or initiated ecdysis. These data indicate that both the brain and the SEG
were necessary for MF ingestion, but that they did not need to be physically
connected to the rest of the CNS in order for MF ingestion to occur.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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).
Near the beginning of the molt, the robust contractions of the anterior
foregut are suspended to prevent the premature swallowing of the MF. About
4–5 h prior to the initiation of ecdysis, as the end of the molt
approaches, the anterior foregut is reactivated and the MF is swallowed.
Results using the dye FM1-43 reveal that during a larval–larval molt it
is the presynaptic terminals on the anterior foregut musculature that are the
primary target for modulation (Bestman and
Booker, 2003).
The results of this work reveal that there are at least two factors
involved in modulating anterior foregut motility during a larval–larval
molt – an inhibitory factor found in the hemolymph of larvae having
recently started the molt cycle, and later in the molt cycle, CCAP. The yet
unidentified inhibitory factor is present only in the hemolymph of the
early-molt stage larvae and is sufficient to suspend anterior foregut
peristalsis when applied to the fully active anterior foreguts of intermolt
larvae (Fig. 2). The decline in
anterior foregut motility triggered by the inhibitory factor is accompanied by
a 90% decline in the average amplitude of the EJPs recorded from the
esophageal constrictor muscles of the anterior foregut. This inhibitory factor
appears to target the presynaptic terminals located on the anterior foregut
musculature. Results using FM1-43 reveal a sharp decline in the efficacy of
the presynaptic terminals of the esophageal dilators within a few minutes of
the application of the active factor. Because the changes in FM1-43 loading
were found to coincide with changes in anterior foregut contractions, it also
suggests that the inhibitory blood borne factor acts directly on the
presynaptic neurons as opposed to acting through a polysynaptic mechanism.
Given that the activity of the hemolymph fraction is lost following exposure
to several proteolytic enzymes, it is consistent that the active factor is a
peptide (J.E.B. and M. del Campo, unpublished observations). There are,
however, still many questions that need to be addressed. For example, we have
not examined and cannot comment on the many other myomodulatory agents
including biogenic amines such as octopamine
(Zilberstein et al., 2004) or
the numerous peptide hormones (Predel et
al., 2001a; Predel et al.,
2001b) that have been found to affect insect gut contractions
outside of the context of the molt. The isolation and a detailed chemical
characterization of the active factor, mechanisms governing its release, as
well as the identification of its source would provide valuable clues in the
effort to understand the mechanism responsible for coordinating anterior
foregut activity relative to the other motor patterns during a molt.
Reactivation of anterior foregut peristalsis by CCAP
As we have described, about two-thirds of the way through the molt cycle
there is a sharp increase in anterior foregut motility followed shortly by the
first signs of MF ingestion (Bestman and
Booker, 2003). We tested whether CCAP application was effective in
triggering the increase in anterior foregut peristalsis and found that after
CCAP application the foregut began peristaltic contractions similar to those
occurring normally as the end of the molt approaches. Within minutes of
exposure to CCAP, there was a dramatic increase in the contraction amplitude
of all of the early-molt stage anterior foreguts to levels similar to that
observed near the time of the onset of MF ingestion (Figs
5,
6). The application of CCAP
also triggered a doubling of the amplitude of the EJPs recorded from the
esophageal constrictor muscles of the anterior foregut of early-molt stage
larvae to a range similar to that observed around the time before the onset of
MF ingestion (Fig. 7). Our
results using the dye FM1-43 reveal that CCAP targets the presynaptic
terminals of the anterior foregut. The larval–larval molt is accompanied
by a 75% decrease in the density of active terminals as determined by FM1-43
labeling (Bestman and Booker,
2003). Shortly after the application of CCAP, the density of
FM1-43-labeled terminals on the dilator muscles of the anterior foreguts of
early-molt larvae increased to levels typically observed for anterior foreguts
isolated from late-molt stage and intermolt larvae
(Fig. 8).
Additional evidence in support of a role for CCAP in the modulation of
anterior foregut activity comes from previous neuron-labeling and
immunocytochemical studies. Using cobalt backfills, two cells that project
from the SEG to the esophageal dilator muscles through nerves of the corpora
cardiaca secretory system were uncovered
(Copenhaver and Truman, 1986).
Later Ewer and coworkers described how these SEG neurons are homologous to
CCAP and cGMP immunopositive neurons in the more posterior ganglia
(Ewer et al., 1994;
Ewer and Truman, 1996). Klukas
et al. confirmed that these SEG neurons were CCAP-IR
(Klukas et al., 1996), which
was also consistent with neurons identified in the CCAP gene expression study
(Loi et al., 2001). Lastly,
Davis et al. provided evidence for neurohemal release sites from the SEG of
CCAP in the vicinity of the aorta and anterior foregut
(Davis et al., 2001). Our
finding of CCAP-IR neurohemal-like structures on the muscles of the anterior
foregut (Fig. 4) is consistent
with these previous studies.
The SEG-CCAP neurons that project to the anterior foregut are two of
approximately 90 neurons in the CNS of larval M. sexta that express
the CCAP transcript (Loi et al.,
2001). Many of the CCAP-ir neurons play a role in coordinating the
motor patterns that characterize insect molting, and a tight correlation
exists between transient increases in cGMP expression in these neurons, the
release of CCAP and the triggering of the ecdysis motor pattern
(Ewer et al., 1994;
Ewer et al., 1997;
Ewer and Truman, 1997;
Fuse and Truman, 2002;
Zitnan and Adams, 2000). In
the SEG all but one pair of CCAP-ir neurons show an increase in cGMP
expression within the final hour of the start of the ecdysis motor patterns.
The lone exception is the pair of SEG CCAP-ir neurons that send projections
that terminate on the anterior foregut musculature
(Davis et al., 2001;
Ewer et al., 1994). This pair
of CCAP-ir anterior foregut-projecting neurons exhibits an increase an in cGMP
levels at the time of MF ingestion, approximately 6–8 h prior to ecdysis
(Ewer and Truman, 1996;
Ewer and Truman, 1997;
Zitnan and Adams, 2000). The
results of our lesion experiments reveal that the SEG is necessary for MF
ingestion (Table 1) and
together this collection of data suggests a further role of CCAP in the
regulation of larval–larval molts of M. sexta to include
triggering an increase in anterior foregut peristaltic activity to power the
ingestion of MF.
There is precedent for CCAP's role as a modulator of anterior foregut
motility in a number of crustaceans. In crabs, immunocytochemistry reveals
that CCAP is delivered to both central and peripheral targets of the
stomatogastric (foregut) motor system
(Christie et al., 1995;
Dircksen and Keller, 1998;
Stangier et al., 1987;
Stangier et al., 1988). The
effects of CCAP on the stomatogastric system of crabs were examined
(Weimann et al., 1997) and it
was found that concentrations in the 10–10 mol
l–1 range produced effects on the pyloric rhythm and the
concentrations in the 10–7 mol l–1 range
increased nerve-evoked contractions of a subset of pyloric muscles. Although
many modulators were tested, only the additions of CCAP to the silent lobster
stomatogastric system can routinely produce a recovery in its tri-phasic motor
pattern (Marder and Richards,
1999). CCAP also plays a role in modulating the stomatogastric
system of crustaceans during the molt cycle. As many crustaceans progress
through their molt cycle, they swallow water in order to swell their bodies
and rupture their old carapace. In at least two species of decapod
crustaceans, transient increases in CCAP titers coincide with an increase in
anterior foregut activity and the swallowing of water
(Phlippen et al., 2000). The
target of CCAP during the molt cycle of these Crustacea is thought to be the
stomatogastric motor system, but this has yet to be confirmed. The recent
identification of the CCAP receptor from Drosophila represents a key
step in identifying the potential targets of CCAP
(Cazzamali et al., 2003;
Park et al., 2002).
A factor from the brain is also necessary for the initiation of MF
ingestion (Table 1).
Predictably, molting larvae in which the connectives between the brain and the
rest of the CNS were transected or the brain itself removed, failed to
initiate ecdysis. The neurons that release eclosion hormone, one of the
peptide hormones essential for triggering ecdysis, reside in the brain and
send projections the length of the nerve cord to release sites in the
proctodeal nerve of the terminal ganglion
(Copenhaver and Truman, 1986;
Truman and Copenhaver, 1989).
Animals in which the brain connectives were severed failed to initiate
ecdysis, but they were still capable of ingesting MF. However, animals in
which the brain was completely removed also failed to ingest their MF. At this
point there is no information available on the nature or the target of the
brain-derived signal controlling anterior foregut peristaltic activity. The
possibilities include direct inputs from the brain to the larval anterior
foregut motor system through, for example, the many fine nerves of the
retrocerebral complex. Alternatively, the brain itself could be the source of
a hormonal signal targeting the SEG or the anterior foregut directly.
In summary, arthropod molts are characterized by the strict coordination of
a series of behavioral events including the swallowing of MF and swallowing of
air or water in order to inflate the new cuticle
(Carlson and O'Gara, 1983;
Hughes, 1980;
Miles and Booker, 1998;
Park et al., 2003). During the
larval–larval molts of the moth M. sexta, the ongoing activity
of the anterior foregut is modulated to control the timing of MF ingestion.
The data outlined above suggest that M. sexta uses a combination of
hormones and neuromodulators to regulate anterior foregut motility during a
larval–larval molt. It also appears that it is the presynaptic endings
on the anterior foreguts that are the primary target of these modulators. The
regulation of the anterior foregut motor system of molting M. sexta
larvae offers an experimentally accessible system to study the cellular basis
of the modulation of complex behaviors.
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Bell, R. A. and Joachim, F. G. (1976). Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Ann. Entomol. Soc. Am. 69,365 -373.
Bestman, J. E. and Booker, R. (2003).
Modulation of foregut synaptic activity controls resorption of molting fluid
during larval molts of the moth Manduca sexta. J. Exp.
Biol. 206,1207
-1220.
Betz, W. J. and Bewick, G. S. (1992). Optical
analysis of synaptic vesicle recycling at the frog neuromuscular junction.
Science 255,200
-203.
Breidbach, O., Dircksen, H. and Wegerhoff, R. (1995). Common general morphological pattern of peptidergic neurons in the arachnid brain: crustacean cardioactive peptide-immunoreactive neurons in the protocerebrum of seven arachnid species. Cell Tissue Res. 279,183 -197.[CrossRef][Medline]
Carlson, J. R. and O'Gara, B. A. (1983). The ecdysis of the cricket, Teleogryllus oceanicus: generation of the pharyngeal air-swallowing motor program by the isolated frontal ganglion. Comp. Biochem. Physiol. 75A,579 -587.[CrossRef]
Cazzamali, G., Hauser, F., Kobberup, S., Williamson, M. and Grimmelikhuijzen, C. J. (2003). Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide. Biochem. Biophys. Res. Commun. 303,146 -152.[CrossRef][Medline]
Christie, A. E., Skiebe, P. and Marder, E. (1995). Matrix of neuromodulators in neurosecretory structures of the crab Cancer borealis. J. Exp. Biol. 198,2431 -2439.
Cochilla, A. J., Angleson, J. K. and Betz, W. J. (1999). Monitoring secretory membrane with FM1-43 fluorescence. Annu. Rev. Neurosci. 22,1 -10.[CrossRef][Medline]
Copenhaver, P. F. and Truman, J. W. (1986). Identification of the cerebral neurosecretory cells that contain eclosion hormone in the moth Manduca sexta. J. Neurosci. 6,1738 -1747.[Abstract]
Davis, N. T., Dulcis, D. and Hildebrand, J. G. (2001). Innervation of the heart and aorta of Manduca sexta.J. Comp. Neurol. 440,245 -260.[CrossRef][Medline]
Dircksen, H. and Keller, R. (1998). Immunocytochemical localization of CCAP, a novel crustacean cardioactive peptide, in the nervous system of the shore crab, Carcinus maenas L.Cell Tissue Res. 254,347 -360.
Donini, A., Ngo, C. and Lange, A. B. (2002). Evidence for crustacean cardioactive peptide-like innervation of the gut in Locusta migratoria. Peptides 23,1915 -1923.[CrossRef][Medline]
Ewer, J. and Truman, J. W. (1996). Increases in cyclic 3', 5'-guanosine monophosphate (cGMP) occur at ecdysis in an evolutionarily conserved crustacean cardioactive peptide-immunoreactive insect neuronal network. J. Comp. Neurol. 370,330 -341.[CrossRef][Medline]
Ewer, J. and Truman, J. W. (1997). Invariant association of ecdysis with increases in cyclic 3',5'-guanosine monophosphate immunoreactivity in a small network of peptidergic neurons in the hornworm, Manduca sexta. J. Comp. Physiol. A 181,319 -330.[CrossRef][Medline]
Ewer, J., De Vente, J. and Truman, J. W. (1994). Neuropeptide induction of cyclic GMP increases in the insect CNS: resolution at the level of single identifiable neurons. J. Neurosci. 14,7704 -7712.[Abstract]
Ewer, J., Gammie, S. C. and Truman, J. W. (1997). Control of insect ecdysis by a positive-feedback endocrine system: roles of eclosion hormone and ecdysis triggering hormone. J. Exp. Biol. 200,869 -881.[Abstract]
Fuse, M. and Truman, J. W. (2002). Modulation
of ecdysis in the moth Manduca sexta: the roles of the suboesophageal
and thoracic ganglia. J. Exp. Biol.
205,1047
-1058.
Groome, J. R. and Lehman, H. K. (1995). Characterization of crustacean cardioactive peptide-like immunoreactivity in the horseshoe crab, Limulus polyphemus. J. Comp. Neurol. 357,36 -51.[CrossRef][Medline]
Hughes, T. D. (1980). The imaginal ecdysis of the desert locust, Schistocerca gregaria. IV. The role of the gut. Physiol. Entomol. 5,153 -164.
Kingan, T. G., Gray, W., Zitnan, D. and Adams, M. E. (1997). Regulation of ecdysis-triggering hormone release by eclosion hormone. J. Exp. Biol. 200,3245 -3256.[Abstract]
Klukas, K. A., Brelje, T. C. and Mesce, K. A. (1996). Novel mouse IgG-like immunoreactivity expressed by neurons in the moth Manduca sexta: developmental regulation and colocalization with crustacean cardioactive peptide. Microsc. Res. Tech. 35,242 -264.[CrossRef][Medline]
Loi, P. K., Emmal, S. A., Park, Y. and Tublitz, N. J. (2001). Identification, sequence and expression of a crustacean cardioactive peptide (CCAP) gene in the moth Manduca sexta. J. Exp. Biol. 204,2803 -2816.
Marder, E. and Richards, K. S. (1999). Development of the peptidergic modulation of a rhythmic pattern generating network. Brain Res. 848,35 -44.[CrossRef][Medline]
McNeil, G. P., Zhang, X., Genova, G. and Jackson, F. R. (1998). A molecular rhythm mediating circadian clock output in Drosophila. Neuron 20,297 -303.[CrossRef][Medline]
Miles, C. I. and Booker, R. (1994). The role of the frontal ganglion in foregut movements of the moth, Manduca sexta.J. Comp. Physiol. A 174,755 -767.
Miles, C. I. and Booker, R. (1998). The role of the frontal ganglion in the feeding and eclosion behavior of the moth Manduca sexta. J. Exp. Biol. 201,1785 -1798.[Abstract]
Mulloney, B., Namba, H., Agricola, H. J. and Hall, W. M.
(1997). Modulation of force during locomotion: differential
action of crustacean cardioactive peptide on power-stroke and return-stroke
motor neurons. J. Neurosci.
17,6872
-6883.
Park, J. H., Schroeder, A. J., Helfrich-Forster, C., Jackson, F.
R. and Ewer, J. (2003). Targeted ablation of CCAP
neuropeptide-containing neurons of Drosophila causes specific defects
in execution and circadian timing of ecdysis behavior.
Development 130,2645
-2656.
Park, Y., Kim, Y. J. and Adams, M. E. (2002).
Identification of G protein-coupled receptors for Drosophila PRXamide
peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor
coevolution. Proc. Natl. Acad. Sci. USA
99,11423
-11428.
Phlippen, M. K., Webster, S. G., Chung, J. S. and Dircksen, H. (2000). Ecdysis of decapod crustaceans is associated with a dramatic release of crustacean cardioactive peptide into the haemolymph. J. Exp. Biol. 203,521 -536.[Abstract]
Predel, R., Nachman, R. J. and Gade, G. (2001a). Myostimulatory neuropeptides in cockroaches: structures, distribution, pharmacological activities, and mimetic analogs. J. Insect Physiol. 47,311 -324.[CrossRef][Medline]
Predel, R., Rapus, J. and Eckert, M. (2001b). Myoinhibitory neuropeptides in the American cockroach. Peptides 22,199 -208.[CrossRef][Medline]
Stangier, J., Hilbich, C., Beyreuther, K. and Keller, R.
(1987). Unusual cardioactive peptide (CCAP) from pericardial
organs of the shore crab Carcinus maenas. Proc. Natl. Acad. Sci.
USA 84,575
-579.
Stangier, J., Hilbich, C., Dircksen, H. and Keller, R. (1988). Distribution of a novel cardioactive neuropeptide (CCAP) in the nervous system of the shore crab Carcinus maenas.Peptides 9,795 -800.[CrossRef][Medline]
Trimmer, B. A. and Weeks, J. C. (1989). Effects
of nicotinic and muscarinic agents on an identified motoneurone and its direct
afferent inputs in larval Manduca sexta. J. Exp. Biol.
144,303
-337.
Truman, J. W. and Copenhaver, P. F. (1989). The
larval eclosion hormone neurones in Manduca sexta: identification of
the brain-proctodeal neurosecretory system. J. Exp.
Biol. 147,457
-470.
Truman, J. W., Gammie, S. C. and McNabb, S. (1998). Role of neuroendocrine cascades in orchestrating complex behavioral programs in insects. In New Neuroethology on the Move: Proceedings of the 26th Gottingen Neurobiology Conference (ed. N. Elsner and R. Wehner), pp. 65-85. Stuttgart: Georg Thieme Verlag.
Weimann, J. M., Skiebe, P., Heinzel, H. G., Soto, C., Kopell,
N., Jorge-Rivera, J. C. and Marder, E. (1997). Modulation of
oscillator interactions in the crab stomatogastric ganglion by crustacean
cardioactive peptide. J. Neurosci.
17,1748
-1760.
Zilberstein, Y., Fuchs, E., Hershtik, L. and Ayali, A. (2004). Neuromodulation for behavior in the locust frontal ganglion. J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 190,301 -309.[CrossRef][Medline]
Zitnan, D. and Adams, M. E. (2000). Excitatory and inhibitory roles of central ganglia in initiation of the insect ecdysis behavioural sequence. J. Exp. Biol. 203,1329 -1340.[Abstract]
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