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Allatostatin-like immunoreactivity in the stomatogastric nervous system and the pericardial organs of the crabCancer pagurus the lobster Homarus americanus and the crayfishCherax destructor andProcambarus clarkii

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THE JOURNAL OF COMPARATIVE NEUROLOGY 403:85–105 (1999)
Allatostatin-Like Immunoreactivity in the
Stomatogastric Nervous System and the
Pericardial Organs of the Crab Cancer
pagurus, the Lobster Homarus
americanus, and the Crayfish Cherax
destructor and Procambarus clarkii
PETRA SKIEBE*
Institut für Neurobiologie, Freie Universität Berlin, Berlin, Germany
ABSTRACT
The distribution of allatostatin (AST)-like immunoreactivity was studied in the stomatogastric nervous system (STNS) and the neurosecretory pericardial organs (PO) of four
decapod crustacean species by using wholemount immunocytochemical techniques and
confocal microscopy. AST-like immunoreactivity was found within the STNS of all four
species; its distribution in each was unique. In all four species, AST-like immunoreactivity
was present in the paired commissural ganglia (CoG), in the esophageal ganglion (OG), in the
stomatogastric ganglion (STG), and in their connecting nerves. Within the CoGs, numerous cell
bodies and neuropil were stained. In the OG, two cell bodies were immunoreactive, although their
branching pattern varies between species. In the STG of C. pagurus and H. americanus, neuropil
was stained extensively, but no labeled cell bodies were found. Surprisingly, in C. destructor and
P. clarkii, cell bodies were stained in the STG, one brightly stained cell body in both species and an
additional two to five weakly stained cell bodies in P. clarkii. In all four species, stained gastropyloric
receptor cells were present. In contrast to the variable staining within the STNS, all four species
have a similar pattern of AST-like immunoreactivity within the PO. Only in C. destructor,
AST-immunoreactive varicosities occur on the surface of the circumesophageal connectives and on
the postesophageal commissure and suggest another neurohaemal source for AST-like peptides in
this species. The pattern of this staining suggests that AST-like peptides are likely utilized as both
neurohormones and as neuromodulators in the STNS of decapod crustacea. J. Comp. Neurol.
403:85–105, 1999. r 1999 Wiley-Liss, Inc.
Indexing terms: neuropeptides; neuromodulators; hormones; confocal microscopy; decapod
crustaceans; immunocytochemistry
The allatostatins (AST) are a family of peptides which
were isolated in the process of finding substances which
inhibit juvenile hormone synthesis by the corpora allata, a
major endocrine organ of insects (Stay et al., 1994). The
first AST to be identified were isolated in the cockroach
Diploptera punctata (Woodhead et al., 1989; Pratt et al.,
1989). Since then, AST have been isolated in a number of
other insects including other cockroaches, moths, flies,
crickets (reviewed in: Stay et al., 1994; Bendena et al.,
1997), and locusts (Veelaert et al., 1996a,b). The majority
of these AST peptides share a conserved C-terminal sequence (-Y-X-F-G-L-NH2). Taking advantage of this conserved structure in insects, Duve et al. (1997b) were
recently able to isolate a family of AST from a crustacean,
r 1999 WILEY-LISS, INC.
the crab Carcinus maenas. Twenty different peptides were
isolated in this species including the shortest and the
longest AST isolated so far, making it the largest AST
family.
Anatomical studies performed in a variety of insect
species (Stay et al., 1992; Duve et al., 1993, 1997a; Bellés
et al., 1994; Duve and Thorpe, 1994; Petri et al., 1995; Ude
Grant sponsor: DFG; Grant number: SFB 515, C1.
*Correspondence to: Dr. Petra Skiebe, Institut für Neurobiologie, Freie
Universität Berlin, Königin-Luise-Str. 28–30, D-14195 Berlin, Germany.
E-mail: skiebe@zedat.fu-berlin.de
Received 19 May 1998; Revised 10 August 1998; Accepted 19 August 1998
86
P. SKIEBE
and Agricola, 1995; Veelaert et al., 1995; Würden and
Homberg, 1995; Yoon and Stay, 1995; Vitzthum et al.,
1996) show that AST-like immunoreactivity is present in
many neurons throughout the nervous system, indicating
that AST might have other functions in addition to its
inhibitory effect on juvenile hormone synthesis. Additional
anatomical studies include crustaceans (Abel et al., 1994;
Skiebe and Schneider, 1994; Christie et al., 1995a), spiders
(Agricola et al., 1995), mollusks (Rudolph and Stay, 1997),
and several non-arthropod invertebrates (Smart et al.,
1994) and demonstrate that AST-like peptides are common
in invertebrates.
In insects, an additional function of the AST has been
shown for two muscle systems, the hindgut and the
antennal pulsatile organ muscle. D. punctata AST is able
to inhibit the spontaneous contractions of the hindgut in a
dose-dependent manner (Lange et al., 1993, 1995; Duve et
al., 1995, 1996, 1997a; Veelaert et al., 1996a). In contrast,
application of D. punctata AST to the antennal heart
muscle in vitro did not alter the contractions either in
P. americana (Hertel and Penzlin, 1992) or in D. punctata.
But when D. punctata AST was applied before the excitatory peptide proctolin, the normal enhancement of the
contractions produced by proctolin in P. americana (Hertel
and Penzlin, 1992) could be reduced.
The effect of D. punctata AST on muscles has been
studied in detail in the crustacean, C. borealis (SkiebeCorrette et al., 1993; Jorge-Rivera and Marder, 1997) by
using muscles innervated by the stomatogastric nervous
system (STNS), which was known to contain AST-like
peptides (Skiebe and Schneider, 1994). These stomach
muscles receive excitatory glutamatergic and cholinergic
innervations from the motoneurons of the STNS. JorgeRivera and Marder (1997) could show that D. punctata
AST-1 to 4, but in particular D. punctata AST-3, were able
to reduce the amplitude of nerve-evoked contractions,
excitatory junctional potentials, and excitatory junctional
currents at both cholinergic and glutamatergic neuromus-
Abbreviations
AGR
AST
ASTir
CoG
coc
d-pon
dvn
GPR
ion
ivn
lgn
ln
lvn
mgn
NGS
OG
on
PB
PB-X
pdn
PO
poc
pyn
son
SOG
STG
stn
STNS
anterior gastric receptor neuron
allatostatin
allatostatin-like immunoreactive
commissural ganglion/circumesophageal ganglion
circumesophageal connective
dorsal posterior esophageal nerve (anterior esophageal
nerve in crayfish)
dorsal ventricular nerve
gastropyloric receptor neuron
inferior esophageal nerve
inferior ventricular nerve
lateral gastric nerve
labial nerve
lateral ventricular nerve
median gastric nerve
normal goat serum
esophageal ganglion
esophageal nerve
sodium phosphate buffer
PB containing 0.5% Triton X-100
pyloric dilator nerve
pericardial organs
postesophageal commissure
pyloric nerve
superior esophageal nerve
subesophageal ganglion
stomatogastric ganglion
stomatogastric nerve
stomatogastric nervous system
cular junctions. Their data suggest that D. punctata AST-3
decreases the postsynaptic action of both neurally released
acetylcholine and glutamate.
Beside the reduction of muscle contractions in C. borealis, AST has also been shown to play an important
modulatory role within the STNS. D. punctata AST-1 to 4
have been shown to inhibit the pyloric (Skiebe and
Schneider, 1994) and the gastric rhythms (Skiebe-Corrette
et al., 1993) generated by the STNS in a dose-dependent
manner. For the crayfish Cherax destructor and Orconectes
limosus, injection of D. punctata AST-3 into the hemolymph reduces the number of spikes per cycle of some of
the neurons and the frequency of the pyloric rhythm
(Heinzel et al., 1997), suggesting that AST released from
neurohemal organs into the hemolymph might be able to
change the rhythms generated by the STNS, as implied for
other peptides (Weimann et al., 1997).
These effects of D. punctata AST on the crustacean
STNS are of particular interest because the effects attributed to AST are the first inhibitory effects described for
peptides in this system, although numerous peptides have
been studied physiologically including Phe-Met-Arg-Phe
(FMRF)amide-like peptides, proctolin, red pigment-concentrating hormone, cholecystokinin-like peptides, crustacean cardioactive peptide, and tachykinin-like peptides
(Weimann et al., 1993, 1997; Blitz et al., 1995; Christie et
al., 1997; Tierney et al., 1997; reviews: Harris-Warrick et
al., 1992; Marder et al., 1997).
The demonstrated physiological action of the AST on the
muscles and the motor pattern-generating network and its
distribution within the crustacean STNS is only known for
one species (C. borealis). Because it is known that distribution of peptides can vary among different species (Mortin
and Marder, 1991; Turrigiano and Selverston, 1991) and
given the complexity of the AST family of peptides, a
comparative anatomical study of the distribution of AST
within the STNS of several species of crustaceans would be
useful to determine the degree to which the AST peptide
family has a conserved distribution in the STNS, and to lay
the groundwork for physiological studies on identifiable
AST-immunoreactive neurons.
I have used immunocytochemical techniques to compare
the distribution of AST-like immunoreactivity within the
Fig. 1. Schematic drawings showing the stomatogastric nervous
system (STNS), stomach, heart, and pericardial organs. A: Location of
the STNS, stomach, heart, and a pericardial organ (PO) in C.
destructor. C. destructor was chosen as an example, because its body
shape is similar to that of H. americanus and P. clarkii. B: Enlarged
view of the STNS, stomach, heart, and PO. The STNS consists of four
ganglia, the paired CoGs, the OG, and the STG, and their connecting
and motor nerves. The STNS is located between the brain and the
SOG, which are connected by the coc surrounding the esophagus. The
poc links both cocs close to the SOG. The paired CoGs lie next to the
esophagus. The STG lies on the stomach within the ophthalmic artery.
The PO are major neurohemal organs located in the pericardial
chamber. Neurohormones released by the PO are pumped through the
opthalmic artery to the STG. C: Schematic of an isolated STNS. To
isolate the STNS, the connections with the brain and the SOG via the
cocs are cut on either side of the CoGs, the poc is cut in the middle, and
the motor nerves are removed from the muscles. The CoGs are
connected with the OG by the ions. The STG is coupled to the CoG by
the sons and the stn. The dvn is the major motor nerve originating
from the STG, which splits into two lvns, from which the lgn and mgn
branch. Branching from the tip of the lvn are pdn and pyn. Not drawn
to scale. Additional nerves not discussed in the paper are not shown.
For definitions of abbreviations in this and subsequent figures, see list.
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
Figure 1
87
88
P. SKIEBE
Figure 2
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
STNS and the pericardial organs (PO) of four decapod
crustaceans: the crab Cancer pagurus, the lobster Homarus americanus, and the crayfish Cherax destructor and
Procambarus clarkii. The PO were included because the
data from Heinzel et al. (1997) also suggested a neurohemal role for AST. Two particular factors influenced the
choice of animals for comparison. First, Casasnovas (1995)
suggested a role for an inhibitory modulator in the maturation from embryonic to adult rhythms generated by the
STNS, and much information is available on the development of the nervous system for H. americanus (Factor,
1981; Charmantier et al., 1991; Helluy and Beltz, 1991;
Beltz et al., 1990, 1992) and for C. destructor (Sandeman
and Sandeman, 1991; Scholtz, 1992; Helluy et al., 1993).
Second, the distribution of several other peptides within
the STNS of P. clarkii (Mortin and Marder, 1991; Turrigiano and Selverston, 1991; Tierney et al., 1997) is known,
and C. pagurus is closely related to Cancer borealis, for
which the immunocytochemical distribution of other peptides has been intensely studied (Marder et al., 1994).
The results of the present study indicate an overall
pattern of AST-like immunoreactivity within the crustacean STNS which is unique for each species, and suggest
that AST-like peptides function as circulating hormones
and as modulators released by interneurons, by sensory
neurons and, possibly, by motoneurons. Preliminary reports of some of these data have appeared in abstract form
(Skiebe and Dietel, 1996; Skiebe, 1997).
MATERIALS AND METHODS
Animals
Four species of adult decapod crustaceans of both sexes
were used in this study: the crab Cancer pagurus (n ⫽ 7),
the lobster Homarus americanus (n ⫽ 6), and the freshwater crayfish Cherax destructor (n ⫽ 10) and Procambarus
clarkii (n ⫽ 10). C. pagurus were purchased from a marine
biological station (Biologische Anstalt Helgoland, Germany), H. americanus were purchased from Lindenberg &
Fig. 2. Wholemount immunocytochemical staining of the STNS of
C. pagurus. A: The highest density of AST-immunoreactive (ASTir)
cell bodies and neuropil is seen in the paired CoGs. There are ASTir
fibers entering or leaving the CoG through the ion and son. In
addition, ASTir fibers are present in the coc, which enter, exit, or
bypass the CoG. B: The unpaired OG contains two large labeled cell
bodies with processes projecting into the ivn. One pair of neurites
enters the OG through each ion (asterisks) and both pairs leave the
OG via the ivn. Another pair connects both CoGs (arrows). C: ASTir
processes are present in the neuropil of the STG. Cell bodies are not
labeled. A pair of descending neurites (asterisk) enters this ganglion
via the stn and ramifies close to the ganglion and into the neuropil.
Ascending axons (arrows) enter the STG via the dvn and leave via the
stn. The area of entry into the STG via the dvn is magnified in the
inset. Two normal and two weakly stained axons are marked by the
arrows. These weakly stained fibers were only seen in one out of seven
preparations. D: ASTir axons at the stn-son junction. A single, brightly
stained axon is present in each son (asterisks) which continue into the
stn. In addition to this pair of strongly stained axons, two weakly
stained axons can be seen bifurcating into each son (arrows). The
branching points of these weakly stained fibers are enlarged in the
inset. E: ASTir axons at the dvn-lvn junction. A pair of stained axons
are present in each lvn (arrows), which enters the dvn. One of the
fibers seen in the lvn is weakly stained and was only seen in one out of
seven preparations. F: ASTir cell body in the lvn, which due to its
location and branching pattern is assumed to be a GPR neuron. Scale
bars ⫽ 100 µm in A,C, 50 µm in B,E,F, 25 µm in D.
89
Co. GmbH (Berlin, Germany) and the crayfish C. destructor were purchased from Langbein & Co. GmbH (Hamburg, Germany). The crayfish P. clarkii were a gift from Dr.
Barbara Schmitz (Technische Universität München, Germany). C. pagurus were maintained in aerated seawater,
and C. destructor and P. clarkii in fresh water, at temperatures between 8 and 14°C. H. americanus were used on the
day of purchase. All animals were anesthetized by packing
in ice for 20–80 minutes, depending on size, prior to
dissection. The stomach, including the STNS, was then
removed from the animal. The rest of the dissection was
performed in chilled physiological saline.
Physiological salines
Cancer saline (mM): NaCl, 440; MgCl2, 26; CaCl2, 13;
KCl, 11; Trizma base 10; maleic acid, 5; pH 7.4–7.5
(Heinzel et al., 1993); Homarus saline (mM): NaCl, 462;
MgCl2, 8; CaCl2, 26; KCl,16; Trizma base 10; maleic acid, 5;
pH 7.4 (Marder et al., 1986); crayfish saline (mM): NaCl,
205; MgCl2, 2.6; CaCl2, 13.6; KCl, 0.54; HEPES 7.6; pH
7.4–7.5. (Van Harreveld, 1936).
Wholemount immunocytochemistry
Wholemount immunocytochemistry was performed by
using standard techniques for this system (Beltz and
Kravitz, 1983; Marder et al., 1986; Skiebe and Schneider,
1994). STNSs were fixed overnight in 4% paraformaldehyde in sodium phosphate buffer (PB, 0.1 M, pH 7.3–7.4).
In the case of C. pagurus and H. americanus, 15% sucrose
was added. Following fixation, the preparations were
rinsed six times at 1-hour intervals in 0.1 M PB and then
were incubated in PB containing 0.5% Triton X-100 (PB-X)
and 10% normal goat serum (NGS) for 1 hour. Antisera
against each of two principal ASTs, the tridecapeptide
AST-1 (provided by Dr. Hans Agricola, Friedrich-SchillerUniversität, Jena, Germany), and the octadecapeptide
AST-5 (provided by Dr. René Feyereisen, University of
Arizona) were used. It has been shown that the antibody
against AST-1 recognizes Diploptera AST-1 through 5, but
it is almost two orders of magnitude more sensitive to
AST-1 (Vitzthum et al., 1996), indicating that it recognizes
the common C-terminus. The antibody against AST-1 was
applied in a final dilution of 1:10,000 and the antibody
against AST-5 in a final dilution of 1:500 in PB-X containing 10% NGS for 2 days. The AST-5 antibody served as a
control and was only applied to one preparation per
species, because no differences in the intensity and pattern
of staining relative to the AST-1 antibody were able to be
detected. Secondary anti-rabbit antibodies labeled with
Cy3 (1:400, Dianova, Hamburg, Germany) were applied
for 1 day in PB-X containing 10% NGS. After each incubation with antibody, preparations were rinsed six times at
1-hour intervals and finally were mounted in glycerol. The
preparations were viewed by using a Leica microscope
equipped for confocal microscopy (Leica TCS-4D with a
krypton/argon laser). Computer images of the wholemounts were generated from optical sections (2 µm) by
using software included with the microscope. The final
figures of the micrographs were asssembled with Photoshop (version 2.5) and FreeHand (version 5.0.1) on a
Macintosh computer and printed with an Epson Stylus
Photo color inkjet printer. The staining of the preparations
showed a wide range of intensities, and some lightly
stained cell bodies and axons marked by arrows in the
figures may not be well reproduced.
90
P. SKIEBE
Preadsorption controls were performed by preincubating the primary antibody with 10⫺4 M AST-3 (Bachem
Biochemical GmbH, Heidelberg, Germany) for 1 hour at
room temperature before adding the combined solution to
the nervous system and processing as described above.
Lucifer yellow backfills
Lucifer yellow (Sigma, Deisenhofen, Germany) backfills
of the stomatogastric nerve (stn) were carried out by
placing a vaseline well around it. After replacing the saline
in the well with a 15% Lucifer yellow solution (diluted in
distilled water), the stn within the well was cut and the
preparation incubated at 4°C for 24 hours. Then the tissue
was fixed in 4% paraformaldehyde and processed for
wholemount immunocytochemistry. After 24 hours, the
dye traveled to the junction of the stn and the superior
esophageal nerve (son) and also into the STG, but did not
reach the commissural ganglia or the esophageal ganglion.
Backfills were performed on the stn of C. destructor (n ⫽ 3)
and P. clarkii (n ⫽ 3) to verify which neurons send their
axons into this nerve.
RESULTS
Figure 1A shows the location of the stomach with the
STNS, the heart and one of the two PO within the thorax of
C. destructor. A more detailed view of these organs is
shown enlarged in Figure 1B. This schematic is also useful
for the other three species used in this study, because the
relationships of these structures to one another remains
essentially the same. The STNS is composed of four
ganglia together with connecting and motor nerves (Fig.
1B,C). The paired commissural ganglia (CoGs) lie between
the brain and the subesophageal ganglion (SOG), which
are interconnected by the circumesophageal connectives
(coc). The two cocs are linked by the postesophageal
commissure (poc). The esophageal ganglion (OG) is located
between the CoGs and is connected with them by the
inferior esophageal nerves (ions, Fig. 1C). The OG is also
connected with the brain via the inferior ventricular nerve
(ivn). The STG is connected with the CoGs by the superior
esophageal nerves (sons) and the stomatogastric nerve
(stn, Fig. 1B,C). The major nerve which branches from the
son is the dorsal posterior esophageal nerve (d-pon, Fig.
1C) in crabs and lobsters, referred to as the anterior
esophageal nerve in crayfish. The STG lies within the
ophthalmic artery and substances released by the PO, a
pair of neurohaemal structures located in the pericardial
chamber, are pumped by the heart through the ophthalmic
artery to the STG.
Nearly all the motoneurons present in the STG send
axons to the muscles of the forgut by way of the dorsal
ventricular nerve (dvn), which branches into two lateral
ventricular nerves (lvn, Fig. 1B,C), from which the lateral
gastric nerve (lgn), the medial gastric nerve (mgn), the
pyloric dilator nerve (pdn), and the pyloric nerve originate
(pyn). The STG receives descending input from the paired
CoGs by way of the ions and the sons, which converge at
the stn, the sole input nerve to the STG (Coleman et al.,
1992). The CoGs contain a few hundred neurons and are
thought to be important centers for coordination of the
foregut motor pattern (Selverston et al., 1976). Some of the
neurons in the OG project to the STG through the esophageal nerve (on) and some project to the brain through the
ivn. Because the AST antibodies stain a large number of
axons connecting the ganglia of the STNS, the results will
be presented separately for each species to simplify describing these interconnections.
Cancer pagurus
In the STNS of C. pagurus, staining for AST was found
in all ganglia. In the CoGs, numerous cell bodies and
densely stained neuropil were found (Fig. 2A). Immunostained fibers enter or leave the CoGs through the ions, the
sons and through the coc. Within the coc, fibers can also be
seen which bypass the CoG. In the d-pon and son are
immunoreactive fibers connecting the d-pon with the CoG
(not shown). In two out of seven preparations, immunoreactive cell bodies were stained which lie in the d-pon or in
the son between the d-pon and the CoG. Two pairs of fibers
were found in each ion (Fig. 2B), the polarity and origin of
which could not be determined. One pair passes through
the OG and connects the CoGs (Fig. 2B, arrows). The other
pair from each ion projects into the ivn (Fig. 2B, asterisks),
where they join the neurites of two large, monopolar cell
bodies stained in the OG. These six fibers reach the brain
via the ivn. No neuropil was labeled in the OG. In the STG,
the neuropil was strongly stained, but no cell bodies were
labeled (Fig. 2C). The neuropil staining can be attributed
to two sources: a set of two fibers which descend from the
CoGs through the son (Fig. 2D, asterisks) into the stn and
branch at the entrance to the STG (Fig. 2C, asterisk), and
two faintly stained fibers which enter the STG by the dvn
(Fig. 2C, arrows). In one out of seven preparations, the two
labeled fibers in the dvn were accompanied by two additional very faintly stained fibers (arrows in the inset of Fig.
2C). The faintly stained fibers in the dvn can be traced into
the lvns (Fig. 2E, arrows). These faintly stained fibers
originate from bipolar cell bodies located either in the lvn
(n ⫽ 12, Fig. 2F) or in the mgn (n ⫽ 1). Due to both their
morphology and their location, these cells are assumed to
be the gastropyloric receptor neurons (GPRs, Katz et al.,
1989). In a previous study, it was shown that the GPR cells
in C. borealis (identified by serotonin immunoreactivity)
contain AST-like peptides (Skiebe and Schneider, 1994). In
the seven preparations, no more than one pair of GPR cell
bodies was found: one pair was found in six preparations,
and in one preparation, only a single GPR cell body was
present. In one of the seven preparations, an additional
axon next to a GPR cell body was stained, but for which no
cell body could be found (Fig. 2F, arrows). The fibers of the
GPR neurons run through the dvn, the STG, the stn, and
branch at the stn-son junction into each son (Fig. 2D,
arrows and inset).
Figure 3 shows stained fibers connecting the CoGs with
the SOG, the poc, and the brain. The fibers entering or
leaving the CoG by way of the coc are schematically drawn
in Figure 3A. These fibers run between the CoG and the
SOG (marked with an arrow), the CoG and the brain
(marked with an double arrow) or run between the CoG
and the poc (marked with a asterisk). In addition to the
fibers drawn in Figure 3A, other allatostatin-like immunoreactive (ASTir) fibers pass the CoG and connect the brain
with the SOG (Fig. 3B, marked by two open arrows).
Strong AST-like immunostaining is present in each PO.
The surface of a PO is covered by strongly stained varicosities (Fig. 4A,C), whereas within a PO, the staining is not as
dense and stained fibers are visible (Fig. 4B).
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
91
Fig. 3. ASTir fibers in one of the circumesophageal connectives
(coc). A: Drawing of ASTir fibers that enter or exit the CoGs. Fibers
bypassing the CoGs were not drawn. The outline was drawn from a
montage of eight confocal micrographs. Fibers connect the CoG with
the brain (double-headed arrows), and the CoG with the SOG (arrow).
The asterisk marks fibers running from the CoG through the poc. The
box marks the area which is shown in the confocal micrograph in B,
which also shows numerous ASTir fibers in the coc that bypass the
CoG (outlined arrows). Scale bars ⫽ 250 µm in A, 100 µm in B.
Fig. 4. AST-like staining in the pericardial organ of C. pagurus.
A: A confocal micrograph of a part of a PO showing that its surface is
covered by strongly immunoreactive varicosities. This micrograph is a
composite of all 71 confocal sections separated by 2 µm. B: Composite
micrograph of the same area produced by using only the inner confocal
sections (22 to 57). Only stained fibers are seen within the PO, the
strongly stained varicosities being confined to the surface. C: Enlargement of the framed area in A. Scale bars ⫽ 100 µm in A,B, 50 µm in C.
Homarus americanus
Staining was present in all four ganglia of the lobster
STNS, but the pattern of staining for AST is much simpler
than that found for C. pagurus. In the CoGs, numerous cell
bodies and densely stained neuropil were found (Fig. 5A).
Immunostained fibers enter or leave the CoGs by the ions,
the sons, and the coc. In one out of six preparations,
immunoreactive cell bodies were stained which lie in the
son and project to the CoG (not shown). Some fibers in the
coc connect the CoG with the SOG, the brain or the poc,
whereas other coc fibers bypass the CoG and connect the
brain with the SOG (not shown). In each ion, two pairs of
fibers were found (Fig. 5B). One pair of axons in each ion
(Fig. 5B, arrowheads) can be traced to the two large cell
bodies stained in the OG, from which only one pair of fibers
project into the ivn. The other neurite pair from each ion
also joins the ivn (Fig. 5B, asterisks), connecting the brain
with the CoGs. The staining of these fibers is much
92
P. SKIEBE
Figure 5
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
93
Fig. 6. AST-like staining in the pericardial organ of H. americanus. A: A confocal micrograph of a part
of a PO showing ASTir fibers and strongly stained varicosities on the surface. Montage of three confocal
micrographs. B: Enlarged view of the framed area of A. Scale bars ⫽ 100 µm in A, 25 µm in B.
stronger. All six fibers reach the brain via the ivn. In the
OG, neuropil was not labeled. In the STG, the neuropil, but
no cell bodies, was strongly stained (Fig. 5C). In the
lobster, the source of this AST-like peptide within the STG
seems to be exclusively the GPR neurons, because no
descending fibers were found to be stained in the stn. This
particular animal had seven stained GPR cell bodies and
axons (see also Fig. 14). In general, the number of stained
GPR cell bodies varied between six and eight in a given
animal and are all located in the lvn (Fig. 5F, arrows). In
Fig. 5. Wholemount immunocytochemical staining of the STNS of
H. americanus. A: AST-like immunoreactivity in cell bodies and
neuropil within the CoG. Fibers from the coc, the ion, and son enter or
exit the ganglion. Montage assembled from four confocal micrographs.
B: The unpaired OG contains two large labeled cell bodies with
processes projecting into the ivn and both ions (arrowheads). Four
additional fibers run in the ivn, two entering or exiting from each ion
(asterisks). C: The STG shows densely stained neuropil but no stained
cell bodies. A bundle of ASTir axons runs through the STG (arrows).
Montage of two confocal micrographs. D: These ASTir axons from the
STG bifurcate at the stn-son junction and due to their location and
branching pattern are likely to be from the GPR neurons. E: These
presumed GPR axons also bifurcate at the lvn-mgn junction. F: The
cell bodies of GPR neurons are stained within the lvn. Scale bars ⫽ 200
µm in A, 100 µm in B,C,E, 25 µm in D, 50 µm in F.
contrast to the staining in C. pagurus, the cell bodies and
axons of the GPR neurons are strongly stained in
H. americanus. The axons of the bipolar GPR cells enter
posterior to the pyn and run anterior through the lvn. In
the lvn, axons branch into the mgn (Fig. 5E). The axon of
the GPR neurons from both sides join in the dvn, and run
through the STG and stn. At the stn-son junction, the
axons bifurcate into each son running further to the COGs
(Fig. 5D).
In H. americanus, strong ASTir varicosities and fibers
are also present in the PO (Fig. 6A,B).
Cherax destructor
The STNS of C. destructor shows staining for AST in all
ganglia. In the CoGs, numerous cell bodies and densely
stained neuropil were found (Fig. 7A). Immunostained
fibers enter or leave the CoGs through the ions and the
sons. Within a coc, stained fibers connect the CoG with the
SOG, the brain and the poc and additional fibers in the coc
bypass the CoG (not shown). In each ion, six axons were
found, four of them strongly stained and two of them
weakly (Fig. 7B). The weakly stained axons belong to two
weakly stained cell bodies within the OG and run within
the ivn and both ions. The first branching point of these
weakly stained axons are marked by arrowheads in Figure
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Figure 7
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
95
Fig. 8. Wholemount of the STG and the son-stn junction following
double-labeling by backfilling the stn with Lucifer yellow and subsequent AST immunocytochemistry in C. destructor. A: The ASTir cell
body in the STG (A1, arrow) also shows Lucifer yellow staining (A2,
arrow), suggesting that its axon projects into the stn. In addition, nine
other cell bodies were labeled by the backfill including the AGR cell
body in the dvn. Each image is a montage from three confocal
micrographs. B: Lower magnification of the son-stn junction, which
shows that the ASTir cell bodies in this junction (B1, asterisks) are
also labeled by Lucifer yellow (B2, asterisks), indicating that their
axons project into the stn. C: The double-labeling is more prominent at
higher magnification (asterisks). Scale bars ⫽ 100 µm in A,B, 25 µm in C.
7B. The four strongly stained axons also run within the ivn
and both ions (Fig. 7B, asterisks), connecting the brain
with both CoGs. The polarity and origin of these four axons
could not be determined. In the other five preparations,
there was sparse neuropil-like staining in the OG. In the
STG, the neuropil was strongly stained and one cell body
was also labeled (Fig. 7C, thick arrow). This cell body
appears to send its axon through the stn, bifurcating at the
son-stn junction into both sons (Fig. 7D, thick arrow). In
addition, one axon from each son runs in the stn (Fig. 7D,
double-headed arrows) connecting the CoGs with the STG.
Two pairs of bipolar neurons, assumed to be GPR neurons,
were stained in C. destructor. Their cell bodies lie within
the lvn (Fig. 7E, arrows) and their axons run through the
lvn, the dvn, the STG (Fig. 7C, small arrows) and the stn,
where they bifurcate into each ion (Fig. 7D, small arrow).
In the stn-son junction, two cell bodies were stained (Fig.
7D, asterisk) that did not stain in either C. pagurus or
H. americanus. These cells send their axons into the stn,
but because they are very thin and faintly stained, it was
not possible to trace them further. In the stn-son junction,
neuropil-like structures were also ASTir (Fig. 7D, hollow
ellipse). The structures were not only present at the
stn-son junction, but also within the stn and sons, themselves. These structures were prominently stained in
C. destructor, but were visible in the other three species as
well.
To confirm whether the ASTir neuron in the STG and the
neurons within the stn-son junction send their axons into
the stn, Lucifer yellow backfills of the stn were combined
with wholemount immunocytochemistry in C. destructor.
The ASTir neuron in the STG was double-labeled in all
experiments (n ⫽ 3, Fig. 8A1,2 thick arrows), indicating
that it has an axon in the stn. Within the STG, 10 (n ⫽ 1) or
11 (n ⫽ 2) cell bodies were labled by Lucifer yellow,
although with different intensities. Among them was the
anterior gastric receptor neuron (AGR), which was identified by its position and by the fact that it is bipolar (Fig.
8A2). The two cell bodies in the stn-son junction which
exhibit AST-like immunoreactivity were also labeled in
two of the three Lucifer yellow backfills of the stn (Fig.
8B,C, asterisks), although they have thin axons.
Fig. 7. Wholemount immunocytochemical staining of the STNS of
C. destructor. A: Within the COG, cell bodies and neuropil were
stained. Fibers from both the ion and son enter or exit the ganglion.
B: In the OG, two cell bodies are stained, which send their axons
through the ivn and both ions. First branching points of the axons of
these cells are marked with arrowheads. Four additional stained
fibers run in the ivn, which interconnect the CoGs (asterisks). C: One
stained cell body (large arrow) and densely stained neuropil are
present in the STG. In addition, a fiber bundle presumably from the
GPR cells enter the STG via the dvn and exit via the stn (small
arrows). Montage of three confocal micrographs. D: Two cell bodies
(asterisks) and axons are ASTir at the stn-son junction. One brightly
stained axon bifurcates at the stn-son junction (thick arrow), probably
originating from the stained cell in the STG. In addition, two other
axons enter the stn, one from each son (double-headed arrows). The
remaining fibers marked with the arrow are likely from the GPR
neurons. In addition to cell bodies and axons, neuropil-like structures
stained (hollow ellipses). E: C. destructor has two pairs of GPR-like cell
bodies, located in the lvn. Scale bars ⫽ 100 µm in A,C,D, 50 µm in B,E.
96
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Fig. 9. AST-like staining in the pericardial organ of C. destructor.
A: A confocal micrograph of a PO showing ASTir fibers and strongly
stained varicosities on the surface. Montage from six confocal micrographs. B: Magnified view of ASTir fibers within a PO within the
framed area in the middle of A. C: Magnified view of ASTir varicosities
within the area framed at the top of A. Scale bars ⫽ 100 µm in A, 50 µm in
B,C.
The surface of the PO in C. destructor is also covered
with strongly stained varicosities (Fig. 9). In Figure 9B,
some of the ASTir fibers are enlarged, and in Figure 9C,
the varicosities are enlarged. Surprisingly, strongly stained
varicose structures were also present on the surface of the
coc and the poc in C. destructor (Fig. 10A) which were not
seen in the other three species. The intensity of this
staining was extremely strong, compared with the staining in other parts of the STNS such as the CoG seen in
Figure 10C. Figure 10A shows a drawing of these structures on the surface of the coc and the poc, excluding
stained axons, neuropil, and cell bodies to better show the
web-like appearance of this structure. Especially on the
surface of the poc, these structures look like a tightly
woven web with swollen varicosites (Fig. 10B), sending out
branches away from the middle of the poc. Within the poc,
fibers were stained, some of them connecting the poc with
both CoGs. This web-like structure extends over the
surface of the coc close to the CoGs (Fig. 10C). The
superficial location and the intensity and varicosal nature
of the staining of these structures suggests that they are
neurohemal. The fibers from the CoGs did not seem to be
the source of the neurohemal-like structure because the
fibers did not show any branching points. No cell bodies
were found to be connected to this neurohemal-like structure.
fibers enter or leave the CoGs through the ions and the
sons. Within the coc, stained fibers connect the CoG with
the SOG, the brain and the poc, and additional fibers
bypass the CoG (not shown). In each ion, six axons were
found (Fig. 11B). Two of the axons belong to two stained
cell bodies within the OG and run within both ions and the
ivn. The first branching point of these weakly stained
axons are marked by arrowheads. The other four stained
axons also run within the ivn and both ions, connecting the
brain with both CoGs. The polarity and origin of these four
axons were not determined. No neuropil was stained in the
OG. In the STG, the neuropil and one cell body (Fig. 11C,
thick arrow) were strongly stained. In addition to the one
strongly stained cell body, two to five additional weakly
stained cell bodies were present. In four out of eight STGs,
a total of six cell bodies were stained (Fig. 11C); in one
STG, five cell bodies were stained, and in the other three
STGs, only three cells were marked. The axon of the
strongly stained cell body appears to project through the
stn, bifurcating at the son-stn junction into both sons. Two
additional axons connect the CoGs with the STG: one axon
in each ion runs in the stn (Fig. 11D, double-headed
arrows). In only three of 10 preparations, weakly ASTir
cell bodies were able to be detected within the son-stn
junction (Fig. 11D, asterisks; this example represents the
strongest staining obtained).
The weakly stained cell bodies in the STG likely send
their axons through the dvn, in which weakly stained
axons are visible (Fig. 11E, arrows). These axons bifurcate
into both lvns. In two out of ten preparations, four axons
were weakly stained in the dvn; in four preparations, three
Procambarus clarkii
Staining for AST was found in all ganglia of the STNS of
P. clarkii. In the CoGs, numerous cell bodies and densely
stained neuropil were found (Fig. 11A). Immunostained
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
97
Fig. 10. AST-like staining suggests that the surface of the connectives and of the postesophageal commissure are neurohemal in
C. destructor. A: Drawing of strongly stained structures on the surface
of the cocs and the poc. Drawn from a montage of 20 confocal
micrographs. Other stained structures, including axons in the coc and
cell bodies and neuropil in the CoG, were not drawn. B: AST-like
staining on the surface of the poc. The picture is a composite of four
confocal micrographs. C: Confocal micrograph showing the detailed
structure on the surfaces of the connectives. Composite of four confocal
micrographs. Scale bars ⫽ 500 µm in A, 200 µm in B,C.
axons; in two preparations, two axons; in one preparation,
one axon; and in another preparation, none.
Six additional strongly stained axons were always present in the dvn, three of which running in each lvn (Fig.
11E) and later sending branches into the mgn. Based on
this branching pattern, these axons likely belong to the
GPR neurons. Although there were always three pairs of
strongly labeled axons, there were never more than two
pairs of stained bipolar cell bodies in the lvn (Fig. 11F),
suggesting that one of the three pairs did not belong to
GPR neurons, or that one pair of cell bodies was hard to
locate, or that one pair of GPR cell bodies did not stain.
These presumed axons of the GPR neurons run through
the lvn, the dvn, the STG and the stn, where they bifurcate
into each ion (Fig. 11D, small arrow). Additional axons
were also labeled in the lvn, perhaps those of the weakly
stained cell bodies in the STG.
As in C. destructor, Lucifer yellow backfills of the stn
were combined with wholemount immunocytochemistry to
further examine the stained cells with axons in the stn.
The strongly stained ASTir neuron in the STG was doublelabeled in all experiments (n ⫽ 3, Fig. 12A, thick arrow),
strongly suggesting that it sends an axon into the stn. In
addition, the AGR neuron was also double-labeled (doubleheaded arrow), identified by its position and by the fact
that it is bipolar. In all backfill experiments, nine (n ⫽ 3)
cell bodies were labeled by Lucifer yellow in the STG,
although with different intensities. The two cell bodies in
the stn-son junction which exhibit AST-like immunoreactivity were also labeled in two of the three Lucifer yellow
backfills of the stn (Fig. 12B, asterisks), although they
have thin axons.
Similar to the other three species, immunostained fibers
and strongly stained varicosities were present in the PO of
P. clarkii (Fig. 13).
DISCUSSION
The results presented here demonstrate that AST-like
peptides are present in both the PO and the STNS of the
four different species of decapod crustaceans examined.
Both AST-1 and AST-5 antisera used for these studies
(antibodies from Agricola and Feyereisen) were found to
stain the same structures with the same relative intensity
in each species. This similarity of staining and the fact
that the antibody against AST-1 recognizes Diploptera
AST-1 to 5 (Vitzthum et al., 1996) indicate that both
antibodies recognize the common C-teminal pentapeptide
sequence -Y-X-F-G-L-NH2. Because the AST found in both
in the crab C. maenas (Duve et al., 1997b) and in the
crayfish O. limosus (Dircksen et al., 1998) also share this
C-terminal sequence, it is likely that these antibodies bind
98
P. SKIEBE
Figure 11
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
to many if not all members of the crustacean AST peptide
family. The different intensities of immunoreactivity found
in this study could result both from differences in the
amounts of peptide present in particular cells and/or in the
different binding affinities of members of the peptide
family with the antibody. That the distribution of AST
could be complex is suggested by the recent work of Duve
et al. (1997b) in the crab, C. maenas, where a family of at
least 20 different AST peptides were isolated.
Pericardial organs
AST-like peptides are present in the PO of all four
species of decapod crustacea investigated. This is not
surprising, because many peptides have been localized in
the PO (Dircksen et al., 1987; Dircksen, 1990; Christie et
al., 1995a) and the rhythms of the STG can be influenced
by injections of AST into the heart chamber (Heinzel et al.,
1997). The presence of AST-like immunoreactivity within
the PO suggests that AST also functions as a neurohormone in decapod crustacea.
Putative neurohaemal organs on the surface
of the connectives and the post oesophageal
commissure of C. destructor
A surprising finding was the presence of ASTir structures on the surfaces of coc and poc of C. destructor,
because similar staining was not found in the other species
investigated (Figs. 10, 14C, black). These web-like structures on the coc (Fig. 10C) and on the poc (Fig. 10B) of
C. destructor are superficial and have varicosity-like swellings. Due to their superficial location and varicosal staining, these structures appear to be neurohemal, and electron microscopic experiments support this hypothesis
(Skiebe et al., 1998). ASTir fibers running in the middle of
the poc connecting the CoGs are present in all investigated
species and might not therefore be the origin of the
web-like structure. Fibers interconecting the CoGs via the
poc have been earlier demonstrated by methylene blue
staining for Penaeus braziliensis (Knowles, 1953) and by
gamma aminobutyric acid (GABA) immunocytochemistry
for the crayfish Pacifastacus leniusculus (Mulloney and
Hall, 1990).
Fig. 11. Wholemount immunocytochemical staining of the STNS of
P. clarkii. A: Within the CoG, cell bodies and neuropil were stained.
Fibers from the coc, the ion, and son enter or exit the ganglion. There
are also ASTir fibers bypassing the CoG. B: In the OG, two cell bodies
are stained, which send their axons through the ivn and both ions. The
first branching point of the axons of these two cells is marked
(arrowheads). Four additional fibers run in the ivn and also connect
both COGs. C: The STG shows densely stained neuropil and one
strongly stained cell body (thick arrow, see also Fig. 12A1) plus five
additional faintly stained cell bodies (thin arrows). Composite of two
confocal micrographs. D: In the son-stn junction are ASTir cell bodies
stained in three out of 10 preparations. One fiber runs in each son
between each of the CoGs and the STG (double-headed arrows).
Putative GPR axons also bifurcate at the son-stn junction (small
arrow). Two cell bodies are also stained (asterisks). E: Nine stained
fibers are present in the dvn. Six of them are strongly stained and
separate at dvn-lvn junction, three of them running in each lvn. These
fibers presumably belong to the GPR cells, although only a maximum
of two pairs of GPR cell bodies were found in a single preparation. The
other three faintly stained fibers in the dvn (arrows) bifurcate at the
dvn-lvn junction, all of them sending an axon into each lvn. F: A GPR
cell body next to a bundle of axons within the lvn. Scale bars ⫽ 100 µm
in A,C,D, 50 µm in B,E,F.
99
Neurohemal structures associated with the poc have
been described by Knowles (1951) for the prawn P. braziliensis and named postcommissure organ. Postcommissure
organs also have been described for the prawn Leander
serratus (Knowles, 1953), the stomatopod Squilla mantis
(Carlisle and Knowles, 1959), and in 12 brachyuran species including Cancer borealis (Maynard, 1961; Fingerman
et al., 1965). In P. braziliensis, L. serratus, S. mantis, and
the two fiddler crab species investigated by Fingerman et
al. (1965), the postcommissure organs are located close to
the poc and are connected to the poc by the a short
postcommissure nerve (Knowles, 1953; Carlisle and
Knowles, 1959). In the 10 brachyuran species investigated
by Maynard (1961) though, they are located further apart,
connected both to the poc by a longer postcommissure
nerve and to the CoG by a nerve which also innervates
esophageal muscles and joins the postcommissure nerve.
Is the ASTir web-like structure a postcommissure organ? From its web-like structure it resembles very much
the ‘‘meshwork of finely branched fibers many of which
have a beaded appearance after methylene blue staining,’’
reported by Carlisle and Knowles (1959, p. 30 and plate
V[b] p. 80). A postcommissure nerve or clear lamellae-like
structure has not been found in C. destructor, although
there was a flap connected with the poc into which some of
the ASTir fibers could be seen to extend. I have examined
only the STNS in this study and have not tried to locate the
postcommissural organs in C. destructor or in the other
investigated species. It is interesting to find a neurohemal
structure so close to the STNS, especially because Carlisle
and Knowles (1959) suggested that its innervation comes
from the brain. The AST-like immunoreactivity within
these structures further supports a neurohormonal role for
AST, but more detailed studies are necessary.
STNS
In the STNS, a detailed comparison of the distribution of
AST-like peptides reveals a number of distinct differences
from species to species, as shown in the colored schema of
Figure 14.
In all four species, both neuropil and numerous cell
bodies are stained in the CoGs (Fig. 14, black). Due to the
density of staining in the CoGs, the branching patterns of
single neurons were not able to be traced. It was therefore
not possible to identify the cell bodies of CoG neurons
which project to the STG in the crab and both crayfish.
Even homologous neurons like the GPR cells and the pair
of ASTir neurons in the OG show strong differences in the
intensity of their immunoreactivity between species. Variations in staining intensity between different preparations
of a species also occur, and make it difficult to obtain an
exact count of stained cell bodies. Therefore, no attempt
was made to count the stained cells in the CoGs or to
homologize these neurons. All investigated species also
have immunostained fibers which enter or leave the CoGs
through the ions, the sons, and the coc (Fig. 14, black). All
species have two cell bodies in the OG, although the exact
branching pattern differs (Fig. 14, orange). In C. pagurus,
the axons run exclusively in the ivn (Fig. 14A, orange),
whereas in the other three species the cells have collaterals that run through both ions.
100
P. SKIEBE
Fig. 12. Double-labeled whole mount of the STG and the son-stn
junction following a Lucifer yellow backfill of the stn and subsequent
AST-like immunocytochemistry in P. clarkii. A: The strong ASTir cell
body in the STG (A1, arrow) as well as the ASTir AGR neuron
(double-headed arrow) also show Lucifer yellow staining (A2), suggesting that both these neurons send their axons into the stn. In addition,
eight other cell bodies were labeled by the backfill. Each image is a
montage from three confocal micrographs. B: Higher magnification of
the son-stn junction, showing that the ASTir cell bodies in this
junction (B1, asterisks) are also labeled by Lucifer yellow (B2,
asterisks), indicating that their axons project into the stn. Scale
bars ⫽ 100 µm in A, 25 µm in B.
GPR cells
putative third pair is marked in light blue. Katz and
Tazaki (1992), though, describe only two pairs of GPR cells
in P. clarkii. This means that either the third pair of GPR
cell bodies is hard to find or that the third pair of axons
does not stem from GPR cells, but additional experiments
are necessary to clarify this point. All GPR axons run
through the lvn, dvn, STG into the stn, where they
bifurcate into the sons and are strongly stained, with the
exception of those of C. pagurus, where the axons are
weakly stained. A relatively weak staining of these axons
was also found for C. borealis (Skiebe and Schneider,
1994), suggesting a possible difference in the type or
amount of AST-like peptides in the GPR cells of these
related crabs compared to lobster and crayfish.
All four species have ASTir GPR cells, although the
number of stained cells and their intensity varies between
species. A similar variation in number of stained cells and
in cotransmitter content is known for other GPR cotransmitters (Katz, 1991; Katz and Tazaki, 1992). In C. pagurus, one pair of weakly ASTir GPR cells is definitely
present (Fig. 14A, dark blue), whereas in C. borealis, two
pairs of weakly staining GPR cell bodies were able to be
found (Skiebe and Schneider, 1994). In C. pagurus, one of
seven preparations had an additional pair of weakly
stained axons, indicating that there might be an additional
pair (Fig. 14A, light blue). In H. americanus, six to eight
GPR cells are ASTir (Fig. 14B, dark blue), and in
C. destructor, two pairs of GPR cells are clearly stained;
three pairs of strongly stained axons are present in the dvn
and lvn, but only two pairs of cell bodies (Fig. 14D, dark
blue), suggesting two or three pairs of GPR cells. The
ASTir neurons in the stn
In both crayfish, two ASTir cell bodies were stained in
the stn-son junction, which send an axon into the stn. In
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
101
Fig. 13.AST-like staining in the pericardial organ of P. clarkii. A: A confocal microgragh of a PO showing
ASTir fibers and strongly stained varicosities on the surface. The picture is a composite of six confocal
micrographs. B: Magnification showing details of ASTir varicosities within the area framed in A. Scale
bars ⫽ 100 µm in A, 50 µm in B.
C. destructor, these cells were clearly and strongly stained;
in P. clarkii the staining was weak. These cells also stain in
P. clarkii (Tierney et al., 1997) and in C. destructor (Skiebe
et al., 1998) with an antibody against FMRFamide.
Cell bodies in the STG
Surprisingly, ASTir cell bodies were also found in the
STG of C. destructor and P. clarkii. To date, neuropeptideimmunoreactive cell bodies have only been found within
the STG of the shrimp Palaemon serratus (Meyrand and
Marder, 1991). Using two different antibodies against
FMRFamide, Meyrand and Marder (1991) found one large
and two small immunoreactive cell bodies in the STG. In
P. serratus, two immunoreactive axons leave the STG
through the dvn, one of which can be traced into each lvn
and to the nerves innervating the cardiopyloric valve
muscle 1, the dilator muscle of the pyloric chamber. This
muscle is innervated by the pyloric dilator neurons, but
their cell bodies do not show FMRFamide immunoreactivity, leaving the identity of those cell bodies undetermined.
The presence of these immunoreactive cells, though, could
relate to the simplicity of the stomach in P. serratus
compared with the other decapod crustaceans. In P. serratus, for example, both the gastric teeth and their extrinsic
muscles and the muscles and the neurons of the cardiopyloric valve are missing (Meyrand and Moulins, 1986,
1988). The STG, though, has nearly as many neurons (25)
as C. borealis (25–26, Kilman and Marder, 1996), H.
americanus (about 30, Maynard, 1971), Panulirus interrup-
tus (27–32, King, 1976), and O. limosus (25–26, Eitner,
1997).
Other studies investigating FMRFamide immunoreactivity in the STNS including C. borealis (Marder et al., 1987),
P. interruptus (Marder et al., 1987), and the crayfish P.
clarkii (Tierney et al., 1997) and C. destructor (Skiebe et
al., 1998), do not find FMRFamide-immunoreactive cell
bodies in the STG. The study investigating the distribution of AST within C. borealis does not describe ASTir cell
bodies in the STG (Skiebe and Schneider, 1994). All the
other immunocytochemical studies done on the STNS of
decapods with various antibodies against peptides also
found no evidence for peptidergic cell bodies within the
STG (buccalin: Christie et al., 1994; cholecystokinin: Turrigiano and Selverston, 1991; Christie et al., 1995b; myomodulin: Christie et al., 1994; proctolin: Marder et al.,
1994; red pigment-concentrating hormone: Nusbaum and
Marder, 1988; tachykinin: Blitz et al., 1995) including
crayfish (␤-pigment-dispersing hormone: Mortin and
Marder, 1991).
Backfills of the stn labeled nine to eleven cell bodies in
the STG of crayfish, a number which is similar to the
number of neurons backfilled in C. borealis (10, Goldberg
et al., 1988; Coleman et al., 1992). Among these cells with
axons in the stn are six identified neurons including
interneurons (anterior burster neuron, interneuron 1),
motoneurons (anterior median neuron, and in crayfish also
the inferior cardiac neuron) and a sensory neuron (AGR;
Claiborne and Ayers, 1987; Katz and Tazaki, 1992; Eitner,
102
Fig. 14. A–D: Schematic showing the distribution of AST-like
immunoreactivity in the STNS of four different decapod crustaceans.
Color code: black marks cell bodies, axons, and neuropil that is
stained, but unidentified. Red marks axons projecting from the CoG to
the STG. Orange marks cell bodies in the OG and their axons. Green
P. SKIEBE
marks strongly stained cell bodies in the STG and their axons. Light
green marks weakly stained cell bodies in the STG and their axons.
Blue marks GPR cell bodies and their axons. Light blue marks
putative GPR cell bodies and their axons. Purple marks stained cell
bodies in the stn and their axons.
AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS
1997; Weimann, 1992), leaving three to five unidentified
neurons. Can the strongly ASTir cell bodies in the STG of
C. destructor and P. clarkii be one of the identified neurons? The AGR neuron can be excluded, because its
distinct bipolar cell body is located in the dvn close to the
STG and it shows weak ASTir in P. clarkii. The anterior
median neuron can also be excluded because its axons
leaves the stn before the stn-son junction into the anterior
median nerve, and no ASTir axons are present in this
nerve. In crayfish, the cardiac dilator 2 and inferior cardiac
neurons should have their axons in the posterior esopageal
nerve, which does not show ASTir, leaving the anterior
burster neuron and interneuron 1 as the most likely
candidates. The size of this cell body would be consistent
with either of these identified interneurons, but the other
unidentified neurons cannot be excluded. These could
include nonspiking STG neurons which are known to exist
in C. borealis (Weimann, personal communication). To
positively identify the strongly stained ASTir neuron,
intracellular recording in combination with immunocytochemistry has to be done.
In summary, H. americanus has the simplest staining
pattern; the source of all AST-like immunoreactivity within
the STG is the GPR cells (Fig. 14B, dark blue). The next
simplest pattern was found in C. pagurus, in which the
AST-like immunoreactivity within the STG comes from
the GPR cells and two descending axons (Fig. 14A, dark
blue and red). This pattern of staining in C. pagurus is
identical to that previously reported for C. borealis (Skiebe
and Schneider, 1994), with the exception of the GPR cells.
In C. destructor and P. clarkii, the AST-like immunoreactivity within the STG comes from the GPR cells, two descending axons, and the ASTir cell or cells in the STG itself plus
the two cells stained in the stn-son junction (Fig. 14C,D,
dark blue, red, green and purple).
This comparative study has revealed a number of important differences in the pattern of distribution of AST-like
peptides in the STNS of different decapod crustaceans and
should provide a basis for several useful preparations for
further physiological experiments. The strongly stained
GPR cells in H. americanus are the only source of AST-like
immunoreactivity in the STG in this species and provide
an opportunity to study AST in sensory neurons in the
absence of other sources of AST. The role of AST in network
activity can be examined by using the single ASTir cell in
the STG of C. destructor. This neuron also has an ascending axon and could be involved in providing feedback to the
CoGs. The two ASTir neurons in stn-son junction of the
crayfish are also easily accessible, and it would also be
interesting to study their function.
ACKNOWLEGDMENTS
Thanks to Dr. Hans Agricola (Jena, Germany) and Dr.
René Feyereisen (Tucson, AZ) for providing the antibodies
and Dr. Barbara Schmitz for supplying Procambarus
clarkii. I also thank Dr. Brian J. Corrette and Heike
Wolfenberg for technical support and Dr. Brian J. Corrette
for reading the manuscript. My gratitude also to Dr.
Hans-Joachim Pflüger and Dr. Randolf Menzel for their
support.
103
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system, immunoreactivity, organy, like, crayfishcherax, pagurus, lobster, allatostatin, nervous, homarus, stomatogastric, destructor, american, clarkii, crabcancer, andprocambarus, pericardial
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