Development and steroid regulation of RFamide immunoreactivity in antennal-lobe neurons of the sphinx moth Manduca sexta
1 Department of Biology, Animal Physiology, Philipps-University, 35032
Marburg, Germany
2 Department of Biogeochemistry, MPI for Terrestrial Microbiology, 35043
Marburg, Germany
* Author for correspondence (e-mail: schachtj{at}staff.uni-marburg.de)
Accepted 16 April 2004
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Summary |
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Key words: peptide, RFamide, hormonal regulation, development, olfactory system, Manduca sexta, immunoreactivity
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Introduction |
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Among the peptides found in AL neurons are members of the superfamily of
FMRFamide-related peptides (FaRPs, Homberg
et al., 1990; Homberg and
Müller, 1999
). Members of this family are small peptides of
418 amino acids ending with -RFamide at the C terminus
(Greenberg and Price, 1992
;
Taghert, 1999
;
Orchard et al., 2001
).
FMRFamide itself was first isolated as a cardioexcitatory peptide from the
Venus mussel Macrocalista nimbosa
(Price and Greenberg, 1977).
Since then, FaRPs have been described in the nervous systems of all major
animal phyla, including vertebrates (for reviews: general, see
Greenberg and Price, 1992
;
cnidarians: Grimmelikhuijzen et al.,
1996
; nematodes: Maule et al.,
1996
; Nelson et al.,
1998
; Li et al.,
1999
; molluscs: Santama and
Benjamin, 2000
; insects:
Taghert, 1999
;
Orchard et al., 2001
;
Homberg, 2002
;
Nässel, 2002
;
vertebrates: Dockray et al., 1983; Wright
and Demski, 1996
; Perry et
al., 1997
). To date, a wide range of physiological effects of
FaRPs has been demonstrated. In arthropods various neuromodulatory effects
have been described on skeletal and visceral musculature
(Worden et al., 1995
;
Skerrett et al., 1995
;
Orchard et al., 2001
;
Merte and Nichols, 2002
). In
the mollusc Aplysia californica, FMRFamide has inhibitory effects on
sensory neurons and motoneurons mediating the gill and siphon withdrawal
reflex and is involved in synaptic modification and learning
(Small et al., 1992
;
Pieroni and Byrne, 1992
;
Peter et al., 1994
;
Wu and Schacher, 1994
;
Zhu et al., 1995
;
Sun et al., 1996
;
Santarelli et al., 1996
;
Belkin and Abrams, 1998
;
Keating and Lloyd, 1999
). In
vertebrates, FaRPs affect central serotonergic transmission (Muthal and
Chopde, 1994
,
1995
;
Muthal et al., 1997
), opioid
systems and pain regulation (Kavaliers,
1990
; Vilim et al.,
1999
).
In the nervous system of M. sexta three different FaRPs have been
identified (Manse FLRFamides: F10, F7G, F7D; Kingan et al.,
1990,
1996
). To date, various
physiological activities on peripheral target organs such as skeletal muscles,
gut and heart have been attributed to the three Manse FLRFamides (Kingan et
al., 1990
,
1996
;
Lee et al., 1998
;
Miao et al., 1998
). All three
peptides have an -SFLRFamide at the C terminus but different N-terminal
extensions. Quantitative analysis of homogenates showed that the relative
concentrations of the three peptides in various ganglia change during
postembryonic development (Kingan et al.,
1996
; Miao et al.,
1998
). A transient decline of peptide levels in thoracic and
abdominal ganglia and an accompanying increase in peripheral neurohemal sites
(the transverse nerves) correlates with the time of ecdysis and suggests that
Manduca FaRPs might be involved in modulation of skeletal and
visceral muscles that facilitate ecdysis
(Miao et al., 1998
).
Recent cloning of the F10 gene revealed a single copy gene, and northern
blot analysis showed a developmental regulation of F10 expression in the CNS
(Lu et al., 2002). In
situ hybridization revealed specific expression of the F10 gene in the
brain and ventral nerve cord of M. sexta. In the brain, new neurons
expressing the F10 gene during metamorphosis mainly belong to two cell
clusters located in the optic (about 100 neurons) and in the antennal (about
12 neurons) lobes (Lu et al.,
2002
).
In this paper we describe the occurrence and hormonal regulation of RFamide immunoreactivity during development of the antennal lobe of M. sexta. Analysis of the cellular occurrence of FaRPs is the first step to understanding the role of this peptide family during metamorphosis of the AL. The presence of FaRPs at defined stages of AL development makes them candidate molecules for having developmental effects during these periods.
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Materials and methods |
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Immunocytochemistry
Anti-RFamide antiserum raised in rabbit (#671, used at dilutions of 1:4 000
to 1:10 000) was kindly provided by Dr E. Marder (Brandeis University, USA). A
polyclonal anti-GABA antiserum (#4TB, diluted 1:50 000) was kindly provided by
Dr H. Dircksen (University of Bonn, Germany), and a monoclonal
anti-synaptotagmin antibody raised in mouse (diluted 1:2500) was kindly
provided by Dr K. Menon (Caltech, USA). The RFamide antiserum recognizes
FMRFamide and FLRFamide peptides (Marder
et al., 1987; Kingan et al.,
1990
), including the three FaRPs identified in M. sexta
(Kingan et al., 1990
,
1996
;
Miao et al., 1998
). The
specificity of the anti-GABA antiserum has been characterized by Homberg et
al. (1999
). Specificity of the
anti-synaptotagmin antibody for M. sexta nervous tissue is described
by Dubuque et al. (2001
). The
antibody against the ubiquitous synaptic vesicle protein synaptotagmin has
been shown to label synapse-rich neuropil areas and can be used to label
glomeruli through development (Dubuque et
al., 2001
).
Secondary antibodies, goat anti-rabbit or goat anti-mouse conjugated to
Cy2, Cy3, Cy5 or horseradish peroxidase (all Jackson ImmunoResearch,
Westgrove, PA, USA), were used at 1:300 dilution. After dissection in cold
saline (Weevers, 1966), brains
from various developmental stages of M. sexta were fixed in 4%
formaldehyde in PBS (phosphate-buffered saline, pH 7.4) for 2 h at room
temperature or overnight at 4°C. After fixation, brains were embedded in
gelatin/albumin, postfixed overnight in 8% buffered formaldehyde, and cut at
40 µm with a vibrating blade microtome (Leica VT 1000S; Nussloch, Germany)
in the frontal or horizontal plane. Sections were rinsed in 0.1 mol
l-1 Tris HCl (Sigma-Aldrich Chemie, Munich, Germany)/0.3 mol
l-1 NaCl (SST, pH 7.4) containing 0.1% Triton X-100 (SST-TX 0.1)
for 1 h at room temperature, then preincubated for another hour with 5% normal
goat or donkey serum (Jackson ImmunoResearch) in SST-TX 0.5 (SST containing
0.5% Triton X-100). Primary antibodies were diluted in SST-TX 0.5 with 1%
normal goat or donkey serum. After incubation with the primary and secondary
antisera, sections were rinsed 3 times 10 min in SST-TX 0.1 at room
temperature. For double labeling, the anti-RFamide and the anti-synaptotagmin
antibodies were applied simultaneously and, likewise, the corresponding
secondary antisera. For double labeling using the anti-GABA and the
anti-RFamide antiserum, which were both raised in rabbits, the anti-GABA
antiserum was applied first as described above. Then Fab fragments raised in
goat against rabbit immunoglobulins (1:50; Jackson ImmunoResearch, in SST-TX
0.5 including 1% normal donkey serum) were applied for 2 h at room temperature
(protocol modified from Dianova, Hamburg, Germany). After washing 3 times for
10 min, secondary donkey anti-goat antiserum coupled to Cy3 was applied for 1
h at room temperature and, after washing (3 times for 10 min) the anti-RFamide
antiserum and the corresponding secondary antiserum coupled to Cy2 were
applied as described above. Horseradish peroxidase was visualized with
3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) using the
glucose oxidase (Sigma-Aldrich) technique according to Watson and Burrows
(1981
). Sections were mounted
on chromalaun/gelatin-coated microscope slides, dehydrated in ethanol, cleared
in xylene and mounted in Entellan (Merck, Darmstadt, Germany).
Dextran application
Crystals of biotinylated dextran (lysine-fixable, molecular mass 3000;
Molecular Probes, Eugene, OR, USA) were placed on the cut ends of one antenna
of immobilized prepupae (W2). The antennal stump was sealed with vaseline. The
animal was kept in a humid chamber overnight at 4°C to allow the dextran
to diffuse through the antennal nerve into its target area in the brain. The
next day animals were dissected, and the brains were processed for
immunocytochemistry as described above. Dextran was visualized using
Cy2-coupled streptavidin (1:300, Jackson Immuno Research), which was applied
for 1 h at room temperature.
Hormone manipulation
20-hydroxyecdysone (20E, Sigma-Aldrich) was dissolved in saline
(Ephrussi and Beadle, 1936) to
a final concentration of 1 µg µl-1. Pupae were chilled on ice
for 2 min and then injected with 15 µg 20E g-1 body mass
(Schachtner et al., 1999
).
Control animals were injected with 15 µl saline g-1 body mass.
All injections were performed using 100 µl Hamilton syringes
dorso-laterally into the pupal thorax. Wounds were sealed immediately with
melted wax, and animals were returned to the walk-in environmental chambers.
Animals were dissected 26 days later and processed according to the
immunocytochemistry protocol described above.
Data processing
Diaminobenzidine-treated sections were photographed using a Polaroid DMCe
digital camera mounted on a Zeiss Axioskop (Jena, Germany). Images were
imported into Adobe Photoshop 6.0 and annotated in Microsoft's PowerPoint or
CorelDraw 10. Fluorescence was analyzed using a confocal laserscan microscope
(Leica TCS sp2). Cell counts were performed at a magnification of x400.
We used two strategies to obtain numbers of labeled cell bodies in the lateral
cell group. (1) For up to 35 cell bodies in total, we compared section by
section to ensure that each cell was only counted once. (2) For more than 35
cells in total, we counted every stained cell body, including fragmented
somata in each section, and used the Abercrombie correction factor to obtain
real cell numbers (Abercrombie,
1946). For a section thickness of 40 µm and mean cell body
diameter of 16.2 µm [obtained from measuring the diameters of 32 cell
bodies in the lateral cell group in a total of four antennal lobes at stages
P9, P10, P12 and pharate adult (P18)], the correction factor used was 0.712.
Comparison of both counting methods in the left and right antennal lobes of a
P10 and a P13 pupa resulted in a maximal difference of four cell bodies
counted, which corresponded to a maximal difference of about 10%.
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Results |
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Most immunostained neurons (type I) belonged to a mixed population of local
interneurons and projection neurons in the lateral cell group (LC), as
described by Homberg et al.
(1990) (Figs
1AC,
2). Type I cells gave rise to
dense RFamide immunostaining in all glomeruli and in the coarse neuropil of
the AL (Figs 1,
3). Fibers of type I cells also
occurred in the outer antenno-cerebral tract, which carried their axons to
higher brain centers (Fig. 1B; Homberg et al., 1990
). Among
the cell bodies in the LC we could not distinguish between local and
projection neuron somata and thus decided to treat them collectively as type
I.
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A single neuron (type II) with a large cell body (diameter about 50 µm) in the LC had no arborizations within the AL but projected its neurite out of the AL (Fig. 1C). In some preparations we could trace the neurite into the tritocerebrum toward the subesophageal ganglion before it intermingled with numerous other RFamide-ir processes (Fig. 1D). Type III neurons consisted of two labeled cell bodies in the LC. Their neurites projected toward the AL neuropil but staining in the neurites faded out on their course to the AL neuropil so that projections in the AL could not be determined (Figs 1D; 3A). Up to stage P2, a single neurite (type IV) gave rise to a tree of arborization in the larval antennal center (LAC)/AL (Fig. 1D). This neurite entered the LAC/AL neuropil baso-laterally and in one preparation we could identify its cell body in a cell group below the LC (Fig. 1D). As we never observed a labeled neurite leaving the LAC/AL from the type IV arborizations, the neuron is most likely to be a larval local interneuron of the AL. Another single neuron (type V) gave rise to a sparse meshwork of varicosities, mainly in basal parts of the AL. The location of its soma was not evident, but was probably outside the AL, since a neurite connecting to the meshwork from outside the AL could clearly be observed in many preparations (Figs 1D, 3B,E,F).
Time course of RFamide immunoreactivity in the developing AL
Cell bodies of type I neurons were not labeled with the RFamide antiserum
in pupal stages earlier than P5 (N=11). At stage P5, four (from two
animals) of 11 ALs analyzed still showed only type II and III cells. The
numbers of type I neurons increased from P5 to P7/8 in a first step to about
25 strongly labeled cell bodies (Fig.
4). This number remained relatively constant up to P10/11 and
increased from P11 to P16 in a second step to about 60 cells (Figs
3,
4). As early as P5 we observed
faint RFamide immunostaining in the developing AL neuropil. With the beginning
of synaptogenesis and the appearance of protoglomeruli and glomeruli at stage
P7/8 (Dubuque et al., 2001)
the relatively weak staining became confined to the basal parts of all
developing glomeruli (Fig. 3C).
In later stages (from P12) immunostaining in the glomeruli strongly increased
in intensity and extended finger-like protrusions from basal to more distal
parts of each glomerulus (Figs
3D,E). RFamide labeling in the outer antenno-cerebral tract (OACT)
occurred around pupal stage P7/8. Double immunostaining with the anti-RFamide-
and an anti-GABA antiserum at stage P10 revealed about ten (10±1,
N=3) cell bodies in LC, which were only immunopositive for RFamide
and not for GABA, suggesting that these ten cells are the source of the fibers
in the OACT (Fig. 2).
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The arborization pattern of the type IV neuron dramatically changed during metamorphosis. From the larva up to pupal stage P2/3 the arborizations in the LAC and in the developing AL were strongly labeled (Fig. 1D). In the following two stages the arborizations were reduced and had completely disappeared from the AL by stage P4 (Fig. 3B).
The varicose meshwork of the type V neuron in basal regions of the AL was found in fifth instar larvae up to the adult (Figs 1D, 3). During and after glomeruli formation (starting at P7/8), arborizations of type V neurons extended into distal areas of the glomeruli, which are densely packed with endings of incoming olfactory receptor neurons, while the projections of type I neurons were confined to the basal part of the glomeruli (Fig. 3C). Up to P12/13, staining of the type V meshwork was much more intense than that of the glomerular arborizations originating from type I cells (Fig. 3C). In later stages and in the adult, however, the staining intensity of type I neurons reached the intensity level of the type V meshwork, which was therefore hardly distinguishable from the type I arborizations (Fig. 3D).
Hormone manipulation
The increasing number of RFamide-ir type I cell bodies in the LC parallels
the increasing titer of 20-hydroxyecdysone (20E) in the hemolymph from P4 to
P9 (Fig. 4;
Warren and Gilbert, 1986). To
test whether 20E is responsible for the increasing number of type I neurons,
we injected 15 µg g-1 body mass of 20E into the hemolymph of
stage P1 pupae (Schachtner et al.,
1999
). Pupae were dissected 28/9 days later and processed
for immunocytochemistry. Cell counts in the LCs of these animals already
showed slightly higher numbers of RFamide-ir cell bodies 2 days after 20E
injection (P3, Fig. 5A). 3 and
4 days after injection (P4, 5) the numbers of RFamide-ir cells had increased
to a level that during normal development was reached 2 days later
(Fig. 5A). 58/9 days
after injection (P6P9/10) the increase of LC cell numbers in 20E
treated versus control animals was even more marked, corresponding to
a difference of at least 5 days (Fig.
5A, compare with Fig.
4). In the 20E injected animals no obvious plateau niveau in LC
cell numbers occurred (Fig.
5A). Instead, the numbers of RFamide-ir cells steadily
increased.
Double labeling of vibratome sections of 20E injected animals with the
RFamide antiserum and an antibody against synaptotagmin
(Dubuque et al., 2001) was
used to determine the developmental stage of glomeruli formation. By 3 days
after 20E injection we found an advancement of glomerulus formation to a stage
P7/8 AL, which is determined by the beginning of glomeruli formation
(Fig. 6B, Dubuque et al., 2001
).
Compared to the 2 days advancement in the development of RFamide
immunostaining seen 3 days after hormone injection, formation of glomeruli was
accelerated even more and was 34 days ahead of normal development.
|
Interestingly, 3 days after hormone treatment, the basal type V meshwork seemed to contain much more branching than was normally observed around this time of development (Fig. 6C). The large type II neuron did not change its arborization pattern after 20E treatment.
The second increase in the number of type I cells between P11 and P16
(Fig. 4) coincided with the
decreasing 20E hemolymph titer (Warren and
Gilbert, 1986). To test whether the falling 20E titer might cause
the increase in RFamide-ir LC neurons, we injected 20E (15 µg
g-1 body mass) into P9 and P10/11 pupae. After 6/7 days (P15/16, P9
injection), 3/4 (P14, P10/11 injection) and 5/6 days (P16, P10/11 injection)
the animals were dissected and subjected to immunocytochemistry. As judged by
external developmental markers such as scale pigmentation on the thorax or on
the wings (Schwartz and Truman,
1983
; Jindra et al.,
1997
), the hormone treatment delayed metamorphic development for
23 days, respectively (inset in Fig.
5B). However, counts of RFamide-ir cells in LC did not reflect
this external developmental delay (Fig.
5B). Also, the developmental state of the glomeruli revealed no
obvious differences between the ALs of hormone-treated animals and control
animals, as determined by synaptotagmin labeling
(Dubuque et al., 2001
).
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Discussion |
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RFamide immunoreactivity in larval and in adult-specific neurons
The antennal lobe (AL) of M. sexta is newly built during
metamorphosis. Its cellular components (local interneurons and projection
neurons) derive from a few neuroblasts, which produce neurons during all
larval instars up to pupal stage P3 when the last AL neurons are born
(Hildebrand et al., 1997). In
addition, an unknown number of interneurons of the larval antennal center
(LAC), the progenitor of the AL in the larva, survives through metamorphosis
and becomes part of the adult AL (Homberg
and Hildebrand, 1994
).
We describe RFamide immunoreactivity in five different types of AL neurons.
Types IIV are already present in the fifth instar larva (L5). Of these
neurons types II and V could be unequivocally followed into adulthood.
Interestingly, neither in the larva nor in the adult did the large type II
neuron project into the LAC/AL neuropil. Thus the type II neuron might not be
directly involved in olfactory signal processing in the larva or in the adult.
The neuron sends its primary neurite toward the subesophageal ganglion and,
therefore, is not identical with the single serotonin-immunoreactive neuron of
the AL, which extends its primary neurite to the protocerebrum
(Kent et al., 1987). The type
V neuron very likely has its cell body outside the AL and might thus account
for a peptidergic centrifugal neuron. Due to the morphological changes
observed during AL development, the type V meshwork clearly seems to be
remodeled to fit the requirements of the adult AL. Also, the pattern of the
type V meshwork seems to be under hormonal control as early 20E injection
leads to a much denser meshwork than is normally observed in any of the stages
examined (Fig. 6C).
Immunostaining in type III neurites typically fades out after a short
distance, possibly owing to low amounts of antigen, so that we could never
observe whether they were connected to the LAC/AL neuropil. The characteristic
arborization of the type IV neuron gradually disappears from the developing AL
neuropil after stage P2/3, which could mean that the arborizations are
retracted during early metamorphosis. A later regrowth could not be detected,
but would probably be masked by the increasing arborizations from type I
neurons.
Type I neurons are the most prominent RFamide-ir cell group, consisting of
about 60 neurons in the adult LC. Homberg et al.
(1990) counted about 80
Rfamide-positive neurons in paraffin and in vibratome sections of adult ALs.
The difference of about 20 cells may be caused by different counting methods
(counts of nuclei by Homberg et al. vs. counts of somata in this
study) or by differences in the specificities of the two antisera used in the
two studies.
From double labeling experiments with antisera against RFamide and GABA,
Homberg et al. (1990)
concluded that about 20 RFamide-ir cells in LC might be projection neurons
responsible for the staining in the outer antenno-cerebral tract (OACT) and in
the isthmus of the AL. RFamide immunoreactivity in the root of the OACT
occurred as early as stage P7/8. Double labeling with the RFamide- and the
GABA antiserum in stage P10 ALs revealed RFamide- but not GABA immunostaining
in about ten somata in LC (including the type II neuron) and in the OACT.
These RFamide-ir cells (of a total of about 25 RFamide cells) that did not
show additional GABA immunoreactivity were probably the projection neurons,
which gave rise to RFamide immunostaining in the OACT. This suggests that
throughout metamorphosis, acquisition of RFamides in local and projection
neurons of the AL occurs in parallel and not sequentially.
Judged from the acquisition of RFamide immunostaining during metamorphosis,
type I neurons are adult-specific neurons that are newly born during
postembryonic development to serve functions in the adult
(Truman, 1996a). In a few
examples, however, it has been demonstrated that neurons can change their
peptide identity (Loi and Tublitz,
1993
; Tubliz and Loi, 1993;
Witten and Truman, 1996
). This
raises the possibility, that some of the type I neurons could be larval
neurons that change their peptide identity during metamorphosis.
Developmental regulation of RFamide immunoreactivity
The developmental increase in the number of type I RFamide-ir cells showed
three phases; a rising phase from P5 to P8, a plateau phase from P8 to P12,
and a second rising phase from P12 to P16
(Fig. 4). This suggests that
two populations of neurons acquire RFamide immunostaining, possibly by two
developmental mechanisms, at different times of metamorphosis. The time course
of RFamide-ir cell numbers shows two parallels with that of the adult 20E peak
in the hemolymph (Fig. 4;
Warren and Gilbert, 1986). The
first increase in numbers of RFamide-ir neurons parallels the increasing 20E
titer, whereas the second increase in cell numbers coincides with a decrease
of the 20E titer.
The steroid hormone 20E acts on target cells by binding to a heterodimeric
nuclear receptor consisting of the ecdysone receptor (EcR) itself and
ultraspiracle, an orphan receptor
(Riddiford et al., 2000). In
M. sexta and D. melanogaster three EcR isoforms are known,
and seem to correlate with different phases of neuronal development
(Truman et al., 1994
;
Truman, 1996b
). Many studies
on 20E action have led to the assumption that steroidal fluctuations in
combination with different EcR receptor isoforms can orchestrate developmental
events during metamorphosis (Thummel,
1996
; Truman,
1996a
).
Experimentally shifting the onset of the pupal 20E peak by 20E injection to
an earlier developmental time point resulted in the precocious appearance of
RFamide-ir cells (Fig. 4A).
This result clearly demonstrates a regulatory role of the 20E rise for the
first phase of RFamide expression in the AL. Interestingly, the increase in
cell numbers in 20E-injected animals did not show a plateau phase as observed
in untreated animals (compare Figs
4 and
5A). This could suggest an
inhibition of RFamide expression occurring during normal development between
P8 and P12, which is absent after early 20E injection. On the other hand the
lack of a plateau phase could simply reflect the fact that a single hormone
injection does not mimic the pupal ecdysteroid peak as well as a continuous
hormone infusion might do (Lehman et al.,
2000). Furthermore, synaptotagmin staining revealed that not only
the presence of RF-amides, but also the formation of glomeruli, occurred
earlier than during normal development. Thus our results strongly suggest an
important role of 20E in organizing the development of the AL with respect to
glomeruli formation and transmitter acquisition.
The rising phase of the pupal 20E peak is associated with promotion of
development, whereas preventing the declining phase seems to retard
development (Schwartz and Truman,
1983; Truman,
1996a
). Accordingly, experiments that prolonged the 20E peak into
later developmental stages delayed pupal development, as judged by external
developmental markers such as scale pigmentation on thorax and wings (inset
Fig. 5B). However, the numbers
of RFamideir neurons in the LC did not differ between treated and control
animals (Fig. 5B). Thus,
enhanced 20E levels at later developmental times did not prevent the second
increase in RFamide-ir cell numbers. This result suggests that the decreasing
20E titer has no direct consequences on the numbers of RFamide-ir cells and
implies the involvement of other unknown mechanisms in the second phase of
FaRP acquisition.
Effects of 20E on metamorphic development of the nervous system of M.
sexta have been shown in several studies. The pupal 20E peak regulates
the fusion of thoracic and abdominal ganglia
(Amos et al., 1996), it
controls cell proliferation during genesis of the optic lobes and the retina
but also programmed cell death of optic lobe neuroblasts (Champlin and Truman,
1998a
,b
,
2000
). Specifically, 20E also
regulates the pupal expression of tyramine ß-hydroxylase, an essential
enzyme for octopamine biosynthesis (Lehman
et al., 2000
). We recently showed that 20E injections early in
metamorphosis lead to elevated concentrations of the second messenger molecule
cyclic guanosine monophosophate in LC neurons, which during normal development
does not occur before stages P7/8 (Schachtner et al.,
1998
,
1999
). Fluctuations of 20E
other than the pupal peak have also been shown to be involved in aspects of
nervous system development including reorganization of neuronal networks
(Levine et al., 1986
;
Levine, 1989
;
Truman and Reiss, 1995
) and
regulating alterations in neuroactive substances
(Loi and Tublitz, 1993
;
Tublitz and Loi, 1993
;
Witten and Truman, 1996
;
it
an et al.,
1999
).
it
an et al.
(1999
) even established in an
elegant study that rising 20E levels can directly influence expression of a
gene that encodes for two peptides which are needed for preecdysis and ecdysis
behavior in M. sexta.
Possible roles of FaRPs during nervous system development
Several recent studies suggest possible roles of neuropeptides during
neuronal development. During embryonic and larval development of the lobster
stomatogastric nervous system, defined neurons express various neuropeptides
(e.g. FLRFamides; Fenelon et al.,
1998,
1999
;
Kilman et al., 1999
). These
neuropeptides are thought to play a role in activity dependent tuning of
intrinsic and synaptic properties of the developing stomatogastric ganglion
pattern generator (Marder and Richards,
1999
). In molluscs, neurons labeled with an FMRFamide antiserum
occur very early in embryonic development and it is speculated that they might
be involved in neurogenesis (Voronezhskaya
and Elekes, 1996
; Croll,
2000
). Recent data from the developing or regenerating mouse
olfactory epithelium demonstrated a role of two neuropeptides (neuropeptide Y
and pituitary adenylate cyclase-activating polypeptide) in initiating
proliferation of basal cells (Hansel et al.,
2001a
,b
).
In vertebrates there is increasing evidence that peptides exert trophic
actions during embryonic development
(Strand et al., 1991
;
De Felipe et al., 1995
;
Hökfelt et al., 2000
).
FaRPs also play a role in learning and neuronal plasticity, which are thought
to imply similar mechanisms as used during development
(Carew et al., 1998
). In
Aplysia FMRFamide induced long-term depression (LTD) leads to
downregulation of a neuronal cell adhesion molecule (apCAM;
Peter et al., 1994
). During AL
development in M. sexta, fasciclin II, an insect homologue of apCAM
is expressed by developing olfactory receptor neurons and serves a role in
guiding the growing axons to the correct glomeruli
(Rössler et al., 1999
;
Higgins et al., 2002
).
The present study revealed for the first time in an insect an exact analysis of the cellular appearance of neuropeptide expression during the development of a defined brain area, the AL of M. sexta. We showed by using immunocytochemistry that FaRPs occur in several types of AL neurons at certain times of metamorphosis. Manipulating the pupal 20E titer revealed that one group of AL neurons expresses FaRPs under the developmental control of 20E. Furthermore, the timing of glomerular formation is under the control of the same 20E peak. The parallel time courses of certain phases of AL development with the acquisition of RFamide immunoreactivity are consistent with the hypothesis that RFamides might be involved in certain aspects of AL development.
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