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Immunohistochemical and electrochemical detection of serotonin in the nervous system of the blood-feeding bug Rhodnius prolixus.

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Archives of Insect Biochemistry and Physiology 8187-201 (1988)
lmmunohistochemical and Electrochemical
Detection of Serotonin in the Nervous System
of the Blood-Feeding Bug, Rhodnius prolixus
Angela B. Lange, Ian Orchard, and Robert J. Lloyd
Department of Zoology, University of Toronto, Toronto, Ontario, Canada
The distribution of serotonin throughout the nervous system of the bloodfeeding bug Rhodnius prolixus has been studied using immunohistochemistry
and reversed-phase high-performance liquid chromatography coupled to
electrochemical detection.
Approximately 150 serotonin-like immunoreactive neurons are distributed
throughout the central nervous system. These neurons are distributed over
both ventral and dorsal surfaces and are found in all ganglia. Several of these
neurons appear to be homologous to previously described serotonin-like
immunoreactive neurons in insects. These include a large pair of bilaterally
symmetrical neurons that project axons out of the suboesophageal ganglion,
and some serially homologous neurons which project contralaterally and
appear to be interneurons.
lmmunoreactive branches and varicosities are found in the neuropile of
all ganglia, and immunoreactive axons are found in all interganglionic
connectives. Several of the peripheral nerves are covered in a plexus of
immunoreactive processes. These processes result in a meshwork of fine
varicose branches lying superficially over the peripheral nerves, which
resemble neurohemal areas.
The central nervous system of Rhodnius contains about 5.5 pmol serotonin
unequally distributed between the ganglia. The highest content is found in
the brain and optic lobes, and the lowest content is found in the prothoracic
ganglion. Substantial amounts of serotonin are present in the corpus
cardiacum and in the peripheral nerves possessing the serotonin-like
neurohemal areas. Serotonin is released from these latter neurohemal areas
in a calcium-dependent manner in response to high-potassium saline.
It is concluded that serotonin plays an important central and peripheral
role in this insect.
Key words: serotonin-like immunoreactivity, serotonin content, central nervous system,
neurohemal area, serotonin release, insect
Acknowledgments: This work was supported by the Natural Sciences and Engineering Research Council of Canada. We are grateful to Dr. Michael Barrett for advice, encouragement,
and provision of Rhodnius throughout this study.
Received January 9,1988; accepted April 25,1988.
Address reprint requests to Dr. Angela B. Lange, Dept. of Zoology, University of Toronto,
Toronto, Ontario, Canda MSS 1Al.
01988 Alan R. Liss, Inc.
188
Lange et al.
INTRODUCTION
The insect CNS" has been shown to contain the biogenic amines dopamine, norepinephrine, octopamine, and serotonin [l-31. Relatively little,
however, is known about the physiological roles for these amines, with the
exception of octopamine. The discovery of identified octopaminergic neurons
in insects has led to a detailed understanding of many of the functional roles
for octopamine [see Ref. 41. Thus, as a prelude to physiological studies of
these other amines, it is important to have a detailed map as to their distribution within the nervous system, with the ultimate goal of characterising
identified neurons.
Serotonin appears to have important regulating functions within insects.
It has been identified by biochemical analysis in the CNS of a variety of
insects [see Ref. 31 where it is assumed to act as a neuroactive substance.
Serotonin also exerts pharmacological effects on a variety of insect peripheral
tissues such as the heart [5,6], salivary glands [7-91, Malpighian tubules [lo],
epidermal cells [ll],and visceral muscles [V]. Some of these observations
suggest that serotonin may act as a neurohormone; others indicate direct
delivery of serotonin to its target tissue.
Serotonin has been detected with fluorescence histochemistry such as the
Falck-Hillarp or glyoxylic acid techniques [see Ref. 131. More recently,
the production of highly specific antibodies to serotonin [13,14] has allowed
the use of immunohistochemical localisation of serotonin in both vertebrates
and invertebrates [3,13-161. This technique is more sensitive and specific than
the others for serotonin [3,17l.
The use of serotonin antibodies has enabled the mapping of neurons
showing serotonin-like immunoreactivity in a variety of insects [3,16,18-231.
Of some interest is the conservation of apparently homologous neurons
between insect species and the demonstration of an extensive plexus of
immunoreactive varicose fibers in the neural sheath of peripheral nerves
[16,22,24] and ganglia 1221, indicative of neurohemal areas for the release of
serotonin into the hemolymph. Few studies, however, have combined immunohistochemical mapping with direct assays of serotonin content, and no
reports have demonstrated the release of endogenous serotonin from the
apparent neurohemal areas.
The present paper describes a study of serotonin throughout the nervous
system of the blood-feeding bug, Rhodnius prolixus. We have chosen Rhodnius
because of an earlier report of serotonin-like immunoreactivity in ventral
nerve cord of the fifth instars of this insect [25] and the demonstration of a
plexus of immunoreactive fibers in the neural sheath of some abdominal
nerves. Our interest lies in the central and peripheral actions of serotonin
and the possible involvement of serotonin as a neurohormone released from
the apparent neurohemal areas. However, our preliminary immunohisto-
*Abbreviations: BSA = bovine serum albumin; CNS = central nervous system; FlTC =
fluorescein isothiocyanate: HEPES = N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid;
HPLC = high-performance liquid chromatography; NGS = normal goat serum; PBS =
phosphate-buffered saline.
Serotonin in Rhodnius
189
chemical studies revealed more stained neurons then previously shown by
Flanagan [25], and so we have re-evaluated the distribution of serotonin-like
immunoreactivity throughout the nervous system of Rhodnius, including the
brain, which had not previously been examined. In addition we have assayed
the content of serotonin in the same structures by HPLC coupled with
electrochemical detection, and we have confirmed that the serotonin-like
immunoreactivity is probably due to endogenous serotonin. Finally, we have
demonstrated for the first time in an insect that endogenous serotonin can
be released from neurohemal areas, and this release is calcium-dependent.
MATERIALS AND METHODS
Adults of Rhodnius prolixus Stil, obtained from a long-established colony
at the University of Toronto, were used throughout the present study. The
bugs were maintained at 25°C in high relative humidity and were used,
unfed, within 30-40 days of emergence as adults.
Serotonin-Like Immunoreactivity
Immunohistochemistry was performed on whole mounts of the central
nervous system, corpus cardiacum, and stretches of peripheral nerve, which
were fixed for 2 h at room temperature in 2% paraformaldehyde in Millonigs
buffer (120 mM NaH2P04buffered with NaOH to pH 7.4, containing 1% Dglucose and 0.005% CaC12).Following fixation the tissues were washed for 2
hr at room temperature in PBS (10 mh4 phosphate buffer (pH 7.2) containing
0.9% NaC1) and then incubated for 1h in PBS containing 2% BSA, 4% Triton
X-100, and 2% NGS. The anti-serotonin antiserum (Immuno Nuclear Corp,
Stillwater, MN) was diluted 1:1,000 with PBS containing 2% BSA, 0.4%
Triton X-100, and 2% NGS, and incubated at 4°C for 18 h. The diluted
antiserum was then incubated with the tissues for 48 h at 4°C with constant
shaking. The tissues were subsequently washed for 5 h at room temperature
in PBS and then incubated in 1:200 FITC-labeled sheep anti-rabbit IgG (Daymar Laboratories, Toronto) in 10% NGS in PBS for 18 h at 4°C. Following a
wash of 4-18 h in PBS the tissues were mounted in 5% n-propyl gallate in
glycerol, pH 7.3, and observed for serotonin-like immunoreactivity with epiillumination fluorescence microscopy. The specificity of the serotonin-like
immunoreactivity was verified by preabsorbing the antiserotonin antiserum
with either 1 mgiml serotonin or 100 pglml serotonin-conjugated BSA (Immuno Nuclear Corp.) for 18 h at 4°C.
Electrochemical Detection of Serotonin
Serotonin was assayed by HPLC using electrochemical detection [26] according to slight modifications of the procedure of Orchard et al. [2]. Appropriate tissues were dissected under physiological saline, immediately placed
into polypropylene tubes containing 25 pl of ice-cold 0.2 N perchloric acid,
and subsequently maintained in the dark to avoid UV inactivation [27]. After
approximately 15 min, 225 pl of HPLC buffer (see later) was added to the
tubes. The mixture was sonicated and, following centrifugation at 30,OOOg
190
Lange et al.
for 30 min or filtration through a 0.22-pm nylon filter, 100 pl was injected
onto a Brownlee RP-18 Spheri-5 HPLC column (4.6mm X 22cm). The mobile
phase, pumped at 0.8 mllmin, contained 62.5 mM NaH2P04,1.5 mM sodium
dodecyl sulfate, 1pM EDTA, 15% methanol, and 16.2% acetonitrile and was
adjusted to pH 3.3 with concentrated perchloric acid. Detection of eluted
compounds was achieved electrochemically using an ESA model 5100 A
detection system coupled to an ESA model 5010 dual coulometric detector
(ESA, Inc., Bedford, MA). The first detector was set at 0.05 V to act as a
screen, and serotonin was detected using the second detector set at 0.35 V.
A guard cell inserted before the injector valve was set at 0.4 V to preoxidize
possible contaminants in the mobile phase. The output of the second detector
was recorded on a Spectra Physics 4270 integrator (Spectra Physics, San Jose,
CA), and serotonin was quantified using the external standard method.
Tissues were pooled to provide suitable content for quantification (three to
five tissues per pool) and spiked with serotonin to confirm the identity of the
oxidizable substance and to check for losses.
Release of Serotonin
Stretches of abdominal nerves were dissected under saline, and their
lengths measured with an eye-piece micrometer. Pooled nerves were washed
and incubated for 10 min in 20 pl of various experimental salines at room
temperature. The salines were individually collected, and 90 pl of HPLC
buffer was added. This solution was injected directly onto the HPLC column,
and the serotonin content was quantified by reference to an external standard. Under the conditions of the present study, the sensitivity of the assay
for serotonin was found to be 3 pg (14 fmol). At the end of the experiment
the nerves were collected into 20 pl of 0.2 N perchloric acid, and serotonin
was measured as described earlier for the nervous tissue. The composition
of the salines was as follows: normal saline, NaCl 150 mM, KC1 8.6 mM,
CaC12 2.0 mM, MgC12 8.5 mM, HEPES buffer (pH 7.0) 10 mM, glucose 34
mM; high potassium saline, normal saline in which 100 mM KC1 was substituted for 100 mM NaCl; zero calcium saline, normal saline in which CaC12
was removed and MgC12 was elevated to 17 mM; high potassium zero
calcium saline, zero calcium saline in which 100 mM KC1 was substituted for
100 mM NaCl.
HPLC grade reagents (American Burdick and Jackson, Canlab, Toronto),
were used throughout for the HPLC work. All other chemicals were obtained
from Sigma Chemical Co. (St. Louis), unless otherwise stated.
RESULTS
Serotonin-Like Immunoreactivity
The CNS of Rhodnius consists of a series of ganglia comprising the brain,
suboesophageal ganglion, prothoracic ganglion, and the mesothoracic ganglionic mass (a fused ganglion composed of the mesothoracic, metathoracic,
and abdominal ganglia). The serotonin-antiserum revealed numerous immunoreactive neurons and processes within the nervous system of Xhodnius.
Serotonin in Rhodnius
191
The staining observed was reduced, or for most cells eliminated, by preabsorption of the antiserum with serotonin. All staining was eliminated by
preabsorption with serotonin conjugated to BSA. Figures 1and 2 summarise
the data obtained for cells that stained with high-to-moderate intensity in a
consistent manner. Neurons showing serotonin-like immunoreactivity occur
on both dorsal and ventral aspects of all ganglia of the CNS and usually exist
as bilaterally symmetrical clusters, although a small number in the midline
are not so obviously paired.
The ventral suface of the CNS is conspicuous for a series of apparently
homologous, bilaterally symmetrical neurons lying in a posterior lateral position in each ganglion of the ventral nerve cord. These neurons were originally described by Flanagan [25]. The neurites of these cells characteristically
project transversely in the neuropile and then extend as contralateral axons,
which in some cases appear to ascend and descend the ventral nerve cord.
However, it is difficult to trace the axons with any certainty, and we do not
know if all of these ventral contralateral neurons are interganglionic. They
do not, however, appear to leave the CNS, and, therefore, are apparently
interneurons. The ventral contralateral neurons produce numerous branches
and varicosities in the anterior quandrant of the neuropile, extending over
both ventral and dorsal surfaces. The suboesophageal ganglion and prothoracic ganglion each contain three bilaterally symmetrical ventral contralateral
neurons, although the cell body diameters in the prothoracic ganglion are
larger than those in the suboesophageal ganglion (15 pm versus 10 pm
diameter). The mesothoracic ganglionic mass has two ventral contralateral
neurons (20 pm diameter) on each side of the mesothoracic segmental neuromere, one on each side of the metathoracic neuromere (10 pm diameter), and
what appears to be one on each side of the remaining abdominal neuromeres
(10 pm cell body diameter).
The remaining cells on the ventral surface consist of a pair of large ventral
lateral neurons in the prothoracic ganglion (23 pm diameter), a pair of small
neurons (7 pm diameter) lying bilaterally in a medial position in both the
metathoracic neuromere and prothoracic ganglion (the ventral medial bilateral neurons), and a cluster (four or five) of ventral medial bilateral neurons
in the suboesophageal ganglion (15 pm diameter). These latter neurons have
been described earlier [25]. Processes are hard to trace from the cell bodies of
these neurons. In addition, the suboesophageal ganglion also contains a pair
of large bilaterally symmetrical cells whose axons leave the CNS via the
ipsilateral anterior peripheral nerve root. These ventral efferent neurons,
which have been described earlier [25], have large cell bodies (20 pm diameter) and are the only neurons in the CNS whose axons can be traced with
certainty leaving the CNS (Figs. 1, 2). Their axons project anteriorly into the
head region along the peripheral nerve root.
The brain has only four pairs of cells on the ventral surface, lying in a
medial position: the ventral medial bilateral neurons. The most anterior pair
are large neurons (35 pm diameter), while the other three pairs are smaller
(10-15 pm diameter). A neurite can be seen leaving each cell body but cannot
be traced any great distance. Lying at the base of the optic lobes are a cluster
of four or five cells located in a posterior position: the ventral optic neurons.
Again, we have been unable to trace the neurites leaving their cell bodies.
Lange et al.
192
DORSAL
*;
DAMB
VENTRAL
.
w
vo
DPO
3%
BRAIN
SOG
PRO
MTGM
200 pln
Fig. 1. Serotonin-like irnrnunoreactivity in the central nervous system of Rhodnius. Cornposite of camera lucida drawings of neurons that consistently reacted with the serotonin antiserum. Positively staining neurons and peripheral nerves containing serotonin-like
irnrnunoreactive processes are shown as solid. Faintly staining neurons are shown as dotted.
Neurons as follows: DAL, dorsal anterior lateral: DAMB, dorsal anterior medial bilateral; DAO,
dorsal anterior optic; DL, dorsal lateral; DM, dorsal medial; DPL, dorsal posterior lateral;
DPMB, dorsal posterior medial bilateral; DPO, dorsal posterior optic; VC, ventral contralateral; VE, ventral efferent; VMB, ventral medial bilateral; VL, ventral lateral; VO, ventral optic;
MTGM, mesothoracic ganglionic mass; PRO, prothoracic ganglion; SOG, suboesophageal
ganglion.
Serotonin in Rhodnius
193
Fig. 2. Whole-mount preparations showing serotonin-like immunoreactive neurons in Rhodnius nervous system. Ventral surface of selected areas of A, brain and suboesophageal
ganglion; B, prothoracic ganglion; and C, mesothoracic ganglionic mass. Labelling as for
Figure 1. lmmunoreactive processes associated with peripheral nerves are shown in D-F. D:
Two axons from the VE neurons of the suboesophageal ganglion as they project in the
anterior peripheral nerve root. E,F: lmmunoreactive branches and varicosities lying superficially on abdominal nerves. Scale bar: A, 40 pm; B,C, 50 pm; D,13 pm; E, 29 pm; F, 50 pm.
194
Lange et al.
The dorsal surface of the CNS also contains numerous serotonin-like
immunoreactive neurons, although processes are rarely seen leaving their
cell bodies. Lying at the posterior dorsal surface of the mesothoracic ganglionic mass are a cluster of bilaterally symmetrical cells, the dorsal posterior
lateral neurons, whose number varies from six to nine. Their cell body
diameters range from 5 pm to 20 pm. As well, the mesothoracic ganglionic
mass contains a cluster (three to four) of bilaterally symmetrical neurons
lying in a midlateral position, the dorsal lateral neurons, whose cell body
diameters range from 10 pm to 30 pm. Also within this ganglion are some
lightly staining cells. A group of dorsal medial neurons with very large cell
bodies (40 pm diameter) lie medially in a posterior region and are not
obviously paired. Lightly staining bilaterally symmetrical neurons are also
consistently observed in an anterior lateral position of both the mesothoracic
ganglionic mass and prothoracic ganglion. These dorsal anterior lateral neurons also have large cell bodies (20-30 pm diameter), with a single neuron
occurring on each side of both the mesothoracic ganglionic mass and prothoracic ganglion.
The suboesophageal ganglion contains bilaterally symmetrical neurons,
the dorsal lateral neurons, which lie as triplets on each side of the ganglion.
These have neurites passing toward the midline of the ganglion and projecting posteriorly.
Numerous serotonin-like immunoreactive neurons lie as bilaterally symmetrical clusters at the posterior dorsal surface of the brain. There are approximately nine to twelve cells in this dorsal posterior medial bilateral
cluster. In addition, there are two sets of bilateral pairs of neurons in the
brain, the dorsal anterior medial bilateral neurons, and the dorsal anterior
lateral neurons. Finally, the dorsal surface of the optic lobes contains two
clusters of neurons: the dorsal posterior optic neurons and the dorsal anterior
optic neurons.
Serotonin-Like Immunoreactive Neurohemal Area
The corpus cardiacum, a well-defined neurohemal organ in insects, contains processes that are serotonin-like immunoreactive in Xhodnius. These
processes consist of branches and varicosities throughout the corpus cardiacum, while other processes continue through the corpus cardiacum to
project along the aorta.
Several peripheral nerves associated with the ventral nerve cord contain a
plexus of immunoreactive processes lying superficially along their length.
These nerves are identified in Figure 1. Most notably the four pairs of thin
abdominal nerves projecting posteriorly from the abdominal neuromere of
the mesothoracic ganglionic mass contain extensive arborizations of serotonin-like immunoreactive processes, which result in a meshwork of fine,
varicose branches over much of their surface (Fig. 2). The neurohemal areas
lying on these four pairs of abdominal nerves have been described previously
[24]. The large pair of posterior median nerve trunks do not in themselves
possess this plexus, although fine abdominal nerves that branch from them
more posteriorly do. (It is worth mentioning, however, that we have observed this plexus on the large posterior median nerve trunks in fifth instar
Serotonin in Rhodnius
195
larval Rhodnius.) Other nerves noted in Figure 1also contain serotonin-like
immunoreactive processes which are located along their surface, although
the plexus on each nerve is less extensive than that observed on the thin
abdominal nerves. We have not observed serotonin-like immunoreactive
processes connecting each plexus with cells within the CNS. Each plexus
terminates close to where the peripheral nerve enters the CNS.
Electrochemical Detection of Serotonin
We have used HPLC coupled with electrochemical detection to quantify
the content of serotonin within the nervous system of Rhodnius (Table 1).
The CNS of adult males contains about 5.5 pmol serotonin unequally distributed between the ganglia. The highest content is found in the brain and optic
lobes, with lower values in the suboesophageal ganglion and mesothoracic
ganglionic mass, and the lowest content in the prothoracic ganglion. The
content of serotonin throughout the nervous system tends to parallel the
distribution and number of serotonin-immunoreactive neurons.
Substantial amounts of serotonin are present in the corpus cardiacum and
in lengths of peripheral nerves dissected from the four pairs of thin abdominal nerves projecting posteriorly from the mesothoracic ganglionic mass
(Table 1).Thus again, content of serotonin parallels the distribution of serotonin-like immunoreactivity.
Release of Serotonin
The meshwork of serotonin-like immunoreactive processes lying over the
abdominal and other peripheral nerves resembles neurohemal tissue. In an
attempt to provide evidence for a possible hormonal role for serotonin we
examined for the release of serotonin from these structures. Incubation of
abdominal nerves containing this meshwork in high-potassium saline resulted in the release of serotonin. During a 10-min incubation in normal
saline, 40.4 28.7 fmol serotonin (n = 8) was released compared to 119.4
28.7 fmol serotonin (n = 8) released in high-potassium saline. The tissue
content of serotonin after the experiments was 426.4 k 99.6 fmol (n = 8)
indicating that 14.5% of the total store of serotonin had been released by
elevated potassium. In a different series of experiments, the high-potassium-
+
TABLE 1. Serotonin Content of Rhodnius Nervous Tissue*
Nervous tissuea
+
Brain optic lobes
Suboesophageal ganglion
Prothoracic ganglion
Mesothoracic ganglionic mass
Corpus cardiacum
Abdominal nerves (per cm)
nb
4
4
4
4
4
4
pmolitissue
(mean i SEM)
2.79 &
1.46 &
0.53 &
1.37 &
0.78
0.39
0.05
0.28
0.45 & 0.19
0.77 & 0.31
*Serotonin content determined by HPLC with electrochemical detection.
'Peripheral nerves were removed from the ganglia close to their exit. Tissues were pooled
(three to five) for each assay.
bNo. of replicates.
196
Lange et al.
induced release of serotonin was shown to be calcium-dependent (Fig. 3).
The effects of calcium-free saline were reversible, with 11%of the total store
of serotonin released by a subsequent 10-min incubation in elevated
potassium.
DISCUSSION
The serotonin antiserum used in the present study has been reported to
have high specificity with no serious problems of crossreactivity [17,19,28,29].
We believe that the immunoreactivity observed in the present study is probably due to serotonin since preabsorption of the antiserum with serotonin
conjugated to BSA abolishes all staining, and the distribution of serotoninlike immunoreactivity tends to parallel the content of serotonin determined
by HPLC.
The content of serotonin throughout the CNS of Rhodnius reveals levels of
a similar order of magnitude (though lower) to those described for other
insects. For example, the cerebral ganglia of honeybee [30], cockroach [31],
and blowfly [9] contain 21.4, 23.2, and 6.5 pmol, respectively, while the
suboesophageal ganglion of honeybee [30] contains 5.6 pmol and the thoracic
ganglia of blowfly [9] contains 2.2 pmol. The number of neurons staining
with the antiserum in each ganglion correlates fairly well with the content of
serotonin. Thus, the brain contains the highest number of immunoreactive
I'
Zero Ca
2mM Ca
m
Time (min)
Fig. 3. Release of serotonin from abdominal nerves. Pooled abdominal nerves were incubated in calcium-free saline, calcium-free high-potassium saline, normal saline and highpotassium saline for 10 min, and serotonin release into the medium was quantified by HPLC
with electrochemical detection. High-potassium saline resulted in the release of serotonin.
This release did not occur in the absence of calcium ions. Following the experiment the
abdominal nerves were found to contain 450 & 140 fmol serotonin, thereby illustrating that
approximately 11 % of the total store was released by high-potassium saline.
Serotonin in Rhodnius
197
neurons and the highest content of serotonin, whereas the prothoracic ganglion contains the smallest number and smallest content. One anomaly lies
in the almost equal content of serotonin in the suboesophageal ganglion and
mesothoracic ganglionic mass in spite of the fact that the mesothoracic
ganglionic mass possesses more immunoreactive neurons. However, the
serotonin content must reflect not only cell body content but arborisations
and terminals within the neuropile. It is possible, therefore, that more serotonin is stored in the neuropile of the suboesophageal ganglion than in the
neuropile of the mesothoracic ganglionic mass.
The presence of serotonin in the corpus cardiacum of Rhodnius extends
earlier observations of its presence in other insect neurohemal organs [see
Ref. 231. The precise role of serotonin within this organ is not understood,
but it may serve as a neurohormone or as a neuromodulator of peptide
hormone release [32].
The present investigation of adult Rhodnius CNS reveals approximately
150 serotonin-like immunoreactive neurons. These neurons are distributed
over both ventral and dorsal surfaces and are found in all ganglia. A number
of these neurons are large and present the possibility of becoming identified
serotoninergic neurons. Our demonstration of serotonin-like immunoreactivity in the ventral nerve cord of Rhodnius extends an earlier report for this
insect. Flanagan [25] examined the distribution of serotonin-like immunoreactivity in the ventral nerve cord of Rhodnius (the brain was not examined)
and found only 26 cells. It is possible that the differences in number of
stained cells may be genuine biological differences between the batches of
animals used. The differences are unlikely to be due to technique since the
cells described by Flanagan [25] were intensely stained. Nor are they due to
a difference in stage of insect since Flanagan reported similar results for third
instar, fifth instar, and adult Rhodnius, and we have observed no differences
between fifth instar and adult, which could account for the lack of cells. We
are left with the conclusion that the neurons in Flanagan’s study contained a
lower content of serotonin, which resulted in their remaining undetected. It
will be interesting to try to identify the physiological conditions of the
Rhodnius that led to this lower content.
Of some particular interest from the present and previous study [25] are
the neurons which appear to correspond to serotonin-like immunoreactive
neurons described in other insects. Intensely staining neurons with neurites
extending contralaterally are typically found in the posterolateral margin of
ganglia in cockroaches, grasshoppers, locusts, and crickets [16,18,33,35].
These ventral contralateral neurons of Rhodnius project along ganglionic
connectives and, in accord with similar neurons mentioned above, do not
appear to project out of the CNS. They are apparently interneurons.
A second set of serotonin-like immunoreactive neurons that are conserved
between insect species include the large bilaterally paired neurons in the
suboesophageal ganglion that have axons projecting out of the CNS. These
ventral efferent neurons of Rhodnius appear to be homologous to neurons in
cockroach and blowfly [16,22,33]. In these latter insects, such neurons give
rise to an extensive neurohemal complex lying over the surface of the ganglia
andlor peripheral nerves, which innervate the mouthparts. No such complex
198
Lange et al.
was evident close to the suboesophageal ganglion of Rhodnius, although it is
possible that the axons arborise more anteriorly.
The ventral medial bilateral neurons in the suboesophageal ganglion of
Rhodnius and the ventral medial bilateral neurons in the prothoracic ganglion
and mesothoracic ganglionic mass appear to be similar to ones described in
cockroach [33], although without stained neurites further comparisons are
hard to make. The dorsal surface of the ventral nerve cord contains serotoninlike immunoreactive neurons lying in lateral positions, at the base of peripheral nerves, and therefore located in similar positions to neurons in cockroach
[33]. The dorsal anterior lateral neurons, though lightly stained, may be
homologous to anterior dorsal bilateral neurons of cockroaches [33], and the
dorsal medial neurons may be homologous to a medial group of neurons in
the terminal abdominal ganglion of crickets that project to the hindgut [18].
As described for cockroach [19,33], honeybee [30], blowfly [21], locust [23],
and dragonfly [15], numerous serotonin-like immunoreactive neurons are
distributed throughout the brain and optic lobes.
Dense immunoreactive branches and varicosities are located in the dorsal
and ventral neuropile of all ganglia of the ventral nerve cord and brain, and
immunoreactive axons are found in all interganglionic connectives. Thus,
serotonin must play an important central role within the nervous system of
Rhodnius. The presence of serotonin-like immunoreactivity and serotonin
throughout the optic lobes, brain, ganglia, and connectives indicates a functionally diverse involvement of serotonin within the CNS. Unfortunately, at
the present time, nothing is known about the central role of serotonin or
serotoninergic neurons in the insect CNS.
In addition to its obvious central role, serotonin appears to have a peripheral function as indicated by the meshwork of immunoreactive processes
lying upon peripheral nerves. The morphology of these processes suggests a
neurohemal function for these areas. An earlier study [24] reported the
presence of such neurohemal areas on the four pairs of abdominal nerves.
However, the present study reveals that these areas are more extensive and
lie upon several other peripheral nerves. Although superficial serotonin-like
immunoreactive nerve terminals have been found in neurohemal regions of
peripheral nerve roots of Rhodnius 1241 and other insect species [16,22,24],
serotonin has not previously been localised and quantified in them. This is
an important discovery because it lends credence to the notion that the
immunoreactive terminals are indeed serotoninergic. The present results
reveal substantial amounts of serotonin within the four pairs of thin abdominal nerves that possess the meshwork of serotonin-like immunoreactive
branches and varicosities and, even more importantly, demonstrates the
release of endogenous serotonin from such areas in response to a depolarising stimulus. This release is calcium dependent, giving some confidence to
the actual physiological nature of the release. This is the first report to
demonstrate the release of endogenous serotonin from neurohemal areas in
insects, although [3H] serotonin has been shown to be sequestered and
released by these areas [%I. Thus, serotonin may well possess a neurohormonal function in Rhodnius in addition to its central role. Such is believed to
be the case in crustaceans, in which serotonin-like immunoreactive processes
Serotonin in Rhodnius
199
are found centrally in the neuropile and superficially on peripheral nerve
roots [271. Serotonin also occurs in physiological levels in the blood of
crustaceans [see Ref. 271. While serotonin is yet to be demonstrated in the
hemolymph of insects in response to a physiological event, it has been shown
to be present following injections of high-potassium saline into blowflies [9].
Since high-potassium saline injection results in salivation, a process mimicked by serotonin and inhibited by serotonin antagonists, it has been suggested that serotonin may be a neurohormone stimulating salivation in this
insect [9]. Other investigators have suggested that serotonin may be a neurohormone released at feeding to regulate peripheral events required at this
time [10,11]. This may also be the case in Rhodnius, in which serotonin has
been shown to induce plasticisation of the abdominal cuticle [ll] and to act
upon the Malpighian tubules to stimulate diuresis [lo]-events which occur
at feeding [lO,ll].
We have recently reported an immunohistochemical map and content of
the pentapeptide proctolin in Rhodnius [36]. The distribution of proctolinergic
neurons appears quite distinct from the serotoninergic neurons. For example,
proctolin and proctolin-like immunoreactive neurons are more concentrated
in the ventral nerve cord, whereas serotonin and serotonin-immunoreactive
neurons are more concentrated in the brain. There are no proctolin-immunoreactive neurons in the optic lobes, whereas serotonin-immunoreactive
neurons are present. Indeed, there appears in general to be little overlap
between proctolin and serotonin, i.e., there are few cells that appear to stain
with both antibodies. Thus, as with cockroach [33], proctolin and serotonincontaining neurons are generally distinct neurons.
The distribution of serotonin-like immunoreactive neurons within the CNS
of Rhodnius constitutes a subpopulation of cells with a distinct biochemical
phenotype. We are now continuing our studies in order to establish some of
these neurons as identified neurons and to define their physiological role. In
particular we are interested in the neurohormonal role of serotonin and
possible involvement in the integration of feeding activities.
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bug, nervous, prolixus, immunohistochemical, rhodnius, detection, feeding, serotonin, system, electrochemically, blood
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