Dopaminergic and serotonergic innervation of cockroach salivary glands: distribution and morphology of synapses and release sites
1 Institut für Biochemie und Biologie, Zoophysiologie, Universität
Potsdam, Postfach 601553, D-14415 Potsdam, Germany
2 Institut für Ernährungswissenschaft, Ernährungstoxikologie,
Universität Potsdam, Arthur-Scheunert-Allee 114-116, D-14558
Potsdam-Rehbrücke, Germany
* Author for correspondence (e-mail: obaumann{at}rz.uni.potsdam.de)
Accepted 4 May 2004
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Summary |
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Key words: serotonin, 5-hydroxytryptamine, dopamine, synapsin, innervation, synapse, immunocytochemistry, salivary gland, insect, cockroach, Periplaneta americana
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Introduction |
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The paired salivary glands consists of three main cell types with different
functions (Fig. 1). The
grape-like acini are composed of central cells (C-cells) and peripheral cells
(P-cells). C-cells secrete proteins, whereas P-cells secrete ions and water
into the acinar lumen (Ginsborg and House,
1980; Just and Walz,
1996
). The iso-osmotic NaCl-rich primary saliva then passes
through the duct system and is modified by the duct epithelial cells,
resulting in the hypo-osmotic final saliva
(Gupta and Hall, 1983
;
Lang and Walz, 2001
;
Rietdorf et al., 2003
).
Physiological studies of isolated salivary glands have further shown that
these cell types differ in their sensitivity to aminergic secretagogues.
C-cells are responsive only to serotonin, duct cells to dopamine, and P-cells
to both serotonin and dopamine (Just and
Walz, 1996
; Lang and Walz,
1999a
,
2001
).
|
In order to determine whether the different responsiveness of above cell
types to serotonin and dopamine is paralleled by their innervation pattern, we
have examined the spatial relationship of serotonergic and dopaminergic nerve
fibres to these cells. In a previous study, we began to address this question
by immunofluorescence labelling of nerve fibres with anti-dopamine or
anti-tyrosine hydroxylase (anti-TH), both probes for dopaminergic neurons, and
with anti-serotonin (Baumann et al.,
2002). The study showed that acini are entangled in a dense plexus
of dopaminergic and serotonergic fibres
(Fig. 1); the former reside on
the surface of the acini next to P-cells, whereas the latter invade the acini
and form a dense meshwork between C-cells. Duct segments close to the acini
are locally associated with both dopaminergic and serotonergic fibres, whereas
the duct segments further downstream have only dopaminergic innervation.
Moreover, numerous varicosities can be detected along the nerve fibres, either
in close proximity to the acinar cells or along fibre segments at a distance
from the acinar tissue, indicating that serotonin and dopamine are released
either as neurotransmitters at synapses or as neurohormones into the
haemolymph. However, the presence of fibre varicosities can only be an
indication of neurosecretion, as thickened fibre sites might also result from
the accumulation of organelles.
In this study, we complement and complete our description of the
serotonergic and dopaminergic innervation of the cockroach salivary gland by
characterizing the release sites (synapses and varicosities). To determine the
distribution of release sites by immunofluorescence confocal microscopy, we
used an antibody against Drosophila synapsin on whole-mount
preparations of salivary glands. Synapsins are actin-binding proteins that are
associated with the cytoplasmic surface of synaptic vesicles and universally
found at conventional chemical synapses (e.g.
Valtorta et al., 1992;
Südhof, 1995
). Moreover,
we have examined the morphology of the release sites and their spatial
relationship to P- and C-cells by transmission electron microscopy.
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Materials and methods |
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Antibodies
Monoclonal mouse antibody SYNORF1 against Drosophila synapsin
(Klagges et al., 1996) was
provided by Erich Buchner (Universität Würzburg, Germany). Rabbit
anti-serotonin and mouse anti-tubulin were obtained from Sigma (Taufkirchen,
Germany). Rabbit antiserum against rat tyrosine hydroxylase (TH) was purchased
from Chemicon (Temecula, CA, USA). Rabbit antiserum against
Drosophila
-spectrin was a gift from Daniel Branton (Harvard
University, Cambridge, MA, USA). Secondary antibodies conjugated to Cy3 or Cy5
were obtained from Rockland (Gilbertsville, PA, USA) and Dianova (Hamburg,
Germany).
Labelling specificity has been demonstrated previously for antibodies
against serotonin and TH (Baumann et al.,
2002). Anti-tubulin and anti-Drosophila
-spectrin
are known to cross-react with their corresponding antigens in various insects
(Baumann and Lautenschläger,
1994
; Baumann,
1997
,
1998
) and also identify a
single protein of the appropriate molecular weight on western blots of
cockroach tissues (data not shown).
Immunofluorescence labelling
Salivary glands were fixed and processed for immunofluorescence labelling
as described previously (Baumann et al.,
2002). Anti-synapsin was applied at a dilution of 1:25 (v/v),
anti-serotonin at 1:10 000, and anti-TH at 1:200. In order to locate the
various acinar cells and to provide a spatial reference for the position of
the immunoreactive structures, specimens were colabelled with BODIPY-FL
phallacidin (Molecular Probes, Eugene, OR, USA), a phallotoxin that binds
specifically to F-actin. Fluorescence images were recorded using a Zeiss LSM
510 confocal microscope (Carl Zeiss, Jena, Germany) equipped with a 488 nm
argon laser, a 543 nm heliumneon laser and a 633 nm heliumneon
laser.
Quantitative analysis of colocalization
Cryostat sections of salivary glands or entire glands were colabelled with
anti-synapsin and either anti-serotonin or anti-TH. By using the software
MetaMorph (Universal Imaging Corp., Downingtown, PA, USA), confocal
fluorescence images of the specimens were thresholded and binarized. In order
to obtain the number of putative release sites for neurotransmitters and/or
neurohormones, structures <10 pixels (1 pixel=0.11 µmx0.11 µm)
and >100 pixels were eliminated within the anti-synapsin image, and the
total number of immunoreactive structures was counted; structures <10
pixels (<0.12 µm2) were smaller than the limit of optical
resolution of the microscope and may have resulted from image noise or from
synapsin-positive foci out of focus, and structures >100 pixels (>1.2
µm2) represented homogeneously labelled axons or autofluorescent
tracheoles. Although we did not place any constraints on the shape of
structures selected by these procedures, comparison of the resulting images
with the original fluorescence images confirmed that all and only
synapsin-positive foci were selected by this method. Costained structures were
then identified by multiplication of each pixel value in the binarized
anti-synapsin image with the value of the corresponding pixel in the binarized
anti-serotonin or anti-TH image. This procedure resulted in the elimination of
structures that exhibited fluorescence in a single channel (pixel value in
channel 1x value of the corresponding pixel in channel 2: 1x0=0)
whereas structures that exhibited fluorescence in both channels (pixel value
in channel 1x pixel value in channel 2: 1x1=1) were present in the
resulting image. The number of colabelled structure was then counted by
MetaMorph.
Biochemical methods
For western blot analysis, specimens (cockroach brains, cockroach salivary
glands, Drosophila heads) were homogenized on ice in reducing sample
buffer (Carl Roth, Karlsruhe, Germany). The preparations were boiled for 5 min
and then centrifuged for 10 min at 16 000 g to remove
non-solubilized material. Samples were loaded on 12% sodium dodecyl sulphate
(SDS)-polyacrylamide gels, electrophoresed, and transferred onto
nitrocellulose sheets. Western blotting was then carried out as described in
detail previously (Baumann,
2001), by using anti-synapsin at a dilution of 1:100 (v/v) and
enhanced chemiluminescence for antibody detection.
Cosedimentation assays with F-actin were performed with proteins isolated
from nervous tissue of the cockroach. Two brains were dissected and
homogenized in extraction buffer containing 25 mmol l1 KCl,
10 mmol l1 Tris, 1 mmol l1 EGTA, 5 mmol
l1 MgCl2, 1 mmol l1 DTT, 0.1
mol l1 sucrose, 0.5% Triton X-100 (pH 7.4), supplemented
with a protease inhibitor cocktail (Sigma). The preparation was centrifuged at
200 000 g for 30 min at 4°C and the supernatant was
incubated for 1 h on ice with 6.5 µg ml1
phalloidin-stabilized F-actin, purified from rabbit muscle
(Pardee and Spudich, 1982).
Actin filaments and bound proteins were sedimented by centrifugation at 200
000 g and the pellet and the supernatant were processed for
western blot analysis as described above.
Electron microscopy
Salivary glands were isolated, fixed and embedded in Epon as described in
detail previously (Just and Walz,
1994). Ultrathin sections were treated with uranyl acetate and
lead citrate and viewed in a CM100 electron microscope (Philips, Eindhofen,
The Netherlands) operated at 80 kV.
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Results |
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Synapsins have the ability to bind to filamentous actin
(Benfenati et al., 1992;
Valtorta et al., 1992
). To
examine whether the anti-synapsin-positive (ASP) cockroach polypeptides also
displayed this property, we incubated an extract of brain tissue with actin
filaments, pelleted the actin filaments, and probed the pellet and the
supernatant fraction for the presence of ASP proteins. As shown in
Fig. 2B, the ASP proteins
copelleted with F-actin, similar to the F-actin-binding protein
-spectrin. Tubulin, used as a negative control, remained entirely in
the supernatant, demonstrating the specificity of the assay.
Provided that the immunoreactive cockroach proteins are synapsins, they
would be expected to be enriched within neurons at presynaptic sites.
Fig. 3 demonstrates that this
is the case. In serotonergic nerve fibres that innervate the reservoir muscle
(Baumann et al., 2002),
anti-synapsin immunoreactivity was highly concentrated at varicosities and in
short processes extending laterally from the axons, whereas the axons proper
displayed only faint immunoreactivity (Fig.
3AC). Within these varicosities, anti-synapsin
immunoreactivity was not evenly distributed but was enriched at one side,
possible the region around the active zone
(Fig. 3C). To examine whether
anti-synapsin immunoreactivity is not only concentrated at conventional
synapses but also at neurohaemal release sites, we colabelled salivary nerves
with anti-synapsin and anti-serotonin (Fig.
3DF). In addition to two thick, centrally localized,
non-serotonergic axons, each salivary nerve contains several thin serotonergic
nerve fibres that form a neurohaemal structure on the surface of the nerve
(Davis, 1985
;
Ali 1997
;
Baumann et al., 2002
). Here,
intense immunoreactivity for anti-synapsin was concentrated within spine-like
structures that extended from the serotonergic fibres towards the surface of
the salivary nerve. Anti-synapsin immunoreactivity was faint within the
serotonergic axons proper but was relatively intense and homogeneous within
the two thick axons at the nerve centre. We thus conclude that
anti-Drosophila-synapsin crossreacts with cockroach synapsins and
that foci of intense anti-synapsin immunoreactivity (=ASP foci) represent
release sites for neurotransmitters or neurohormones.
|
Distribution of anti-synapsin immunoreactivity over and within the salivary gland
Using anti-synapsin, we analysed the distribution of putative release sites
for neurotransmitters and/or neurohormones in whole-mount preparations of
salivary glands (Fig. 4). ASP
foci were detected throughout the various parts of the salivary gland complex,
viz. the acinar tissue, the nerves interconnecting the acinar lobules
and the salivary ducts. The alignment of ASP structures in rows suggested that
they were distributed along the length of individual axons.
|
In acinar lobules, numerous ASP foci were detected both on the surface of and deep within the acinar tissue (Fig. 4AF). Every pair of P-cells, visualized by a bow-tie-like staining pattern with phallotoxin, had at least one row of ASP foci at its periphery (Fig. 4B,C). In some cases, P-cell pairs were almost completely surrounded by ASP structures. Numerous rows of ASP foci also extended deep into the acinar tissue, residing among C-cells. In nerves that interconnected adjacent acinar lobules, individual axons were stained for synapsin homogeneously over their entire length (Fig. 4G). In addition, the nerves contained numerous foci of intense ASP immunoreactivity. Generally, homogenously stained axons had a central position, whereas ASP foci resided close to the surface of the nerves. Along the salivary duct system, ASP structures were sparse (Fig. 4HK). Rows of ASP structures resided on the outer surface of the duct epithelium or they extended deep into the epithelial layer, being localized close to the apical surface of the epithelial cells.
In summary, these results suggest that numerous release sites for neurotransmitters and/or neurohormones are associated with both P-cells and C-cells of the acinar tissue. Moreover, the nerves interconnecting acinar lobules may function as neurohaemal organs as they contain a large number of synapsin-enriched foci. Finally, there appears to be a low number of release sites for neurotransmitters/neurohormones along the salivary ducts, both on their outer surface and between epithelial cells.
Colocalization of synapsin-enriched structures with serotonergic and dopaminergic fibres
By triple-labelling with phallotoxin, anti-synapsin and either
anti-serotonin or anti-TH, we examined whether the above-mentioned release
sites were serotonergic or dopaminergic
(Fig. 5). Anti-TH was used as a
probe for dopaminergic neurons, because the fixation methods required for
staining with anti-dopamine (see Baumann et
al., 2002) interfered with the staining for synapsin.
|
On the outer surface of the acinar lobules, colabelling of ASP foci was observed with anti-serotonin and anti-TH (Fig. 5AF,GL). Dopaminergic ASP foci were sparsely distributed on the acinar tissue, whereas serotonergic ASP foci were relatively frequent and detected next to almost every pair of P-cells. ASP foci within the acinar tissue, suggesting a location between C-cells, colocalized exclusively with serotonergic nerve fibres. In nerves interconnecting acinar lobules, few ASP foci appeared to be serotonergic, whereas most foci were costained with anti-TH (Fig. 5M,N). Finally, on initial segments of the duct system, ASP foci were observed along serotonergic and dopaminergic nerve fibres (Fig. 5O,P). The segments of the duct system further downstream exhibited ASP foci exclusively along dopaminergic nerve fibres (data not shown), whereas the large salivary ducts (no. 4 in Fig. 1A) has most ASP foci along dopaminergic nerve fibres and few ASP foci along serotonergic nerve fibres (Fig. 5Q,R).
In addition to serotonergic and dopaminergic axons, the salivary nerve
contains a thick axon of unknown neurotransmitter content
(Ali, 1997;
Baumann et al., 2002
). We thus
wanted to determine whether a fraction of ASP foci on or within the salivary
gland neither colocalized with anti-serotonin nor anti-TH. Unfortunately,
technical reasons prevented triple-labelling with anti-synapsin,
anti-serotonin and anti-TH. Morphometric analyses of specimens double-labelled
with anti-synapsin and either anti-serotonin or anti-TH suggested, however,
that the relative number of non-serotonergic/non-dopaminergic release sites
must be small. In cryostat sections through acinar lobules, 88.8±7.4%
of the ASP foci on or within the acinar tissue were serotonergic, and
11.3±7.4% of the foci were colabelled with anti-TH
(Fig. 6A). In nerves
interlinking adjacent acinar lobules, 22.6±19.1% of the ASP foci were
serotonergic, and 74.7±14.0% of ASP foci were costained with anti-TH
(Fig. 6B).
|
Ultrastructural characteristics of release sites
The morphology of the release sites for neurotransmitters/neurohormones was
investigated by transmission electron microscopy. Structures considered to be
release sites satisfied the following criteria: (1) an accumulation of
synaptic vesicles, (2) the absence of a glial sheath, and (3) the presence of
an active zone with an electron-dense area on the cytoplasmic face of the
plasma membrane. In cases of ideal planes of sectioning, this electron-dense
area was seen as a ribbon that extended 80100 nm into the cytoplasm
(Fig. 7C, insets).
|
In accordance with the results of anti-synapsin immunohistochemistry, release sites were observed (1) within the acini embedded between C-cells, (2) within nerves running along the surface of acinar lobules and thus residing close to P-cells, and (3) within nerves that interconnected acinar lobules. Although we also examined cross-sections through salivary ducts, we could only detect axonal profiles (data not shown) but no release sites. As the number of ASP foci in the ducts is low, release sites may be very rare and extensive serial sectioning is required to detect such structures by electron microscopy.
Release sites within the acinar tissue had a uniform appearance (Fig. 7A,B). The structures were without a basal lamina and thus separated only by a narrow extracellular space from the plasma membrane of the C-cells. These release sites can thus be classified as synapses. The synaptic profiles contained numerous small electron-lucent vesicles with a mean diameter of 53.2±6.8 nm (mean ± S.D.; N=164). Dense-core vesicles with a mean diameter of 88.4±14.2 nm (N=27) were also present at low numbers at these release sites. The clear vesicles clustered around the active zones, whereas the dense-core vesicles appeared to be homogeneously distributed throughout the synaptic profiles. No `postsynaptic' specialization could be detected on the adjacent region of the plasma membrane of the C-cells. It should be noted, however, that the cytoplasm of the C-cells was electron-dense and may have prevented the unequivocal identification of postsynaptic densities.
Release sites at the surface of nerves that run superficially on acinar lobules (Fig. 7C) or that interconnecting acinar lobules (Fig. 7DF) were separated from the P-cell surface and the haemolymph space, respectively, by a basal lamina. These release sites can thus be regarded as neurohaemal structures. Here, at least two different types (A and B) of release sites could be distinguished by their vesicle content. Both of them contained numerous electron-lucent vesicles of similar size (Type A: 53.7±11.4 nm, N=301; type B: 50.1±9.5 nm, N=155) that were clustered near the active zones. The size, morphology and proportion of dense-core vesicles, however, differed between the two types of release sites. Type A release sites contained few dense-core vesicles (98.9±19.5 nm; N=31). Type B release sites were observed less frequently and had numerous dense-core vesicles of a larger size (122.8±23.3 nm; N=45) and of lower electron density.
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Discussion |
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The chief findings of these analyses are as follows: (1) varicosities of serotonergic and dopaminergic fibres represent release sites, (2) there are numerous serotonergic synapses within the acini between C-cells, (3) neurohaemal release sites for serotonin were detected next to each pair of P-cells, (4) neurohaemal release sites for dopamine reside occasionally in the immediate vicinity of P-cells, (5) nerves that traverse the acini contain numerous sites for the neurohaemal release of dopamine and few sites for the neurohaemal release of serotonin, (6) few dopaminergic and serotonergic nerve fibres invade the epithelial layer of the salivary ducts and have release sites between the duct cells.
Anti-synapsin as a probe for release sites
The interpretation of our data critically depends on the specificity of
anti-synapsin as a marker for release sites. The monoclonal antibody SYNORF1
used in this study was raised against Drosophila synapsin
(Klagges et al., 1996).
Cross-reactivity of this antibody with cockroach synapsin is demonstrated by
our western blot analysis. Samples of cockroach brains and salivary glands
reveal a single broad band of about 80 kDa that comigrates with the major
isoforms of Drosophila synapsins. Biochemical assays demonstrate
further that the immunoreactive proteins bind to actin filaments, a property
shared with synapsins (Valtorta et al.,
1992
). Finally, the immunoreactive proteins are neuron-specific,
as shown by the immunofluorescence labelling of salivary glands.
Since synapsins are universally found on synaptic vesicles at chemical
synapses, and since synaptic vesicles are abundant at presynaptic sites,
anti-synapsin should provide a good marker for release sites
(Valtorta et al., 1992).
Control studies with defined synapses and neurohaemal structures, showing an
enrichment of anti-synapsin immunoreactivity near the active zones, suggest
that ASP foci detected within the salivary gland also represent release sites.
The fidelity of the colocalization between anti-synapsin and the varicosities
along serotonergic or dopaminergic neurons within the salivary gland endorses
the specificity of immunostaining. Furthermore, electron microscopy has
revealed numerous chemical synapses and neurohaemal release sites at
comparable positions within the salivary gland.
In addition to the punctate staining of varicosities with anti-synapsin,
some axons also display homogeneous labelling. This feature is particularly
evident on axons with a large diameter, such as those in the salivary nerve
(Fig. 3E), and has been noted
previously for this antibody in other preparations
(Skiebe and Ganeshina, 2000).
Axonal labelling may be attributable to synapsin molecules that lie within the
cytomatrix or on synaptic vesicle precursors that are en route to
presynaptic sites (Baitinger and Willard,
1987
; Paggi and Petrucci,
1992
). Thus, only foci that exhibit intense anti-synapsin
immunoreactivity and that are associated with a varicosity are indicative of a
release site for neurotransmitter/neurohormone.
Release sites on C-cells are serotonergic
Labelling with anti-synapsin visualizes a high density of release sites in
the interior of the acini, between C-cells. Although the outline of C-cells
could not be resolved in the phallotoxin images, the high density of ASP
structures in the interior of the acini suggests that each C-cell has at least
one release site on its surface. In electron microscopic images, equally
positioned release sites are frequently observed and are separated only by an
extracellular space of a few nanometers in width from the plasma membrane of
the C-cells (Maxwell, 1978;
present study). Based on morphology, release sites on C-cells can thus be
classified as synapses. Colocalization with anti-serotonin but not with
anti-TH and the uniform equipment with synaptic vesicles imply further that
synapses on C-cells are of the same functional type, i.e. serotonergic. This
finding is in accordance with the results of physiological studies,
demonstrating that C-cells respond only to serotonin
(Just and Walz, 1996
). Hence,
we conclude that each C-cell is stimulated by serotonin liberated directly at
its surface. It remains to be determined whether serotonin receptors on
C-cells are clustered in the postsynaptic area or distributed over the plasma
membrane.
P-cells have neurohaemal release sites for serotonin and dopamine in their vicinity
P-cells reside at the surface of the acinar lobules and are thus closely
juxtaposed to the nerves that entangle the glandular tissue. By labelling with
anti-synapsin and by electron microscopy, release sites were identified within
these nerves. These sites are separated by a thick basal lamina from the
plasma membrane of the P-cells. Secretagogues released from these sites must
thus diffuse across an extracellular space of about 0.5 µm in width in
order to reach their putative receptors on the P-cell surface. Release sites
at this position are heterogeneous with respect to their neurotransmitter
content. Serotonergic release sites are detected at each individual pair of
P-cells, whereas dopaminergic release sites are relatively sparse. Electron
microscopy confirmed that evenly positioned release sites are diverse
regarding their equipment with synaptic vesicles
(Maxwell, 1978; present
study), but an explicit classification with respect to their transmitter
content cannot yet be made.
Nerve fibres around the acinar tissue may not be the only source of
secretagogues for the stimulation of P-cells. Since P-cells face the
haemolymph with their basal side, they are freely accessible to secretagogues
released at some distance by neurohaemal structures, viz. the nerves
traversing adjacent acinar lobules. Release sites in these interlinking nerves
resemble those on the surface of the acinar tissue in their morphology and
their equipment with synaptic vesicles
(Maxwell, 1978; present
study), suggesting a similar transmitter content. In interlinking nerves,
however, dopaminergic release sites far outnumber serotonergic release
sites.
We thus conclude that P-cells are exposed to serotonin and dopamine
liberated at some distance from their surface, in the case of serotonin
preferentially at varicosities within nerves on the acinar surface, and in the
case of dopamine preferentially at neurohaemal structures between the acinar
lobules. The presence of both serotonergic and dopaminergic release sites in
the vicinity of P-cells agrees with the view that ion and water transport by
this cell type is stimulated by either serotonin or dopamine
(Rietdorf et al., 2003).
Serotonergic and dopaminergic release sites are rare and differentially distributed along the salivary duct system
The salivary ducts are sparsely innervated by serotonergic and dopaminergic
nerve fibres (Baumann et al.,
2002). Surprisingly, these nerve fibres with their release sites
are not confined to the outer surface of the ducts, as is the case for other
ion-transporting epithelia, but invade deeply into the epithelium. These
release sites are, however, infrequent, suggesting that only a minor fraction
of the duct epithelial cells is in intimate contact with such sites. Since the
duct epithelial cells are extensively coupled by gap junctions
(Lang and Walz, 1999b
), and
since second messenger molecules, such as Ca2+
(Lang and Walz, 1999a
), can
diffuse through gap junctions from an innervated cell to its non-innervated
neighbours, direct stimulation of a few epithelial cells may be sufficient to
activate ion transport over the entire epithelium. Moreover, since the ducts
seem to have free access to the haemolymph, the entire duct system may be
exposed to secretagogues liberated at neurohaemal sites, viz. the
nerves between acinar lobules, and the direct innervation of the duct
epithelium may only serve to trigger the response.
Physiological studies on the salivary ducts have shown that dopamine
effects ion transport across the epithelial cells, whereas serotonin has no
obvious effect (Lang and Walz,
1999a). These observations seem to contradict our finding that the
initial segments of the duct system contain both serotonergic and dopaminergic
release zones. However, all the physiological studies were performed on areas
further downstream and, thus, on duct segments exclusively innervated by
dopaminergic fibres. The presence of serotonergic release sites suggests that
duct segments next to the acini differ in their physiological properties from
other segments. This hypothesis is in agreement with morphological data
demonstrating the presence of secretory granules in duct cells next to acini
and the lack of such granules in all other duct cells (Raychaudhuri and Gosh,
1963; Bland and House, 1971
).
This putative functional diversity of various duct segments warrants further
investigation.
Are other neurotransmitters/neurohormones involved with the control of salivation?
Serotonin and dopamine may not be the only secretagogues involved in the
control of salivation. In addition to several thin serotonergic axons and a
thick dopaminergic axon of salivary neuron 1 (SN1), the salivary nerve
contains another thick axon whose cell body is termed SN2 and that resides in
the suboesophageal ganglion (Ali,
1997). In locusts, which possess a salivary gland of similar
morphology, there is evidence that SN2 contains serotonin
(Ali et al., 1993
;
Ali, 1997
) and/or
-amino
butyric acid (GABA; Watkins and Burrows,
1989
). In cockroaches, however, SN2 contains neither serotonin nor
dopamine (Davis, 1985
,
1987
;
Gifford et al., 1991
;
Baumann et al., 2002
), and
whether cockroach SN2 is GABAergic has not yet been examined. The transmitter
content and innervation pattern of SN2 is still unknown for the cockroach. If
SN2 has varicosities and terminals on or within the glandular tissue, then
these should have been labelled with anti-synapsin, and a population of
release sites should not have colocalized with anti-serotonin or anti-TH. In
our quantitative analysis, release sites that colocalized with anti-dopamine
or anti-serotonin on and within the acinar lobules and within interlinking
nerves made up roughly 100% of the total number of release sites. We suppose,
however, that our quantitative analysis slightly overestimates the number of
colocalizing release sites. Provided that an ASP foci is not labelled for
serotonin or dopamine but sits next to or in a plane just above a nerve fibre
labelled for the secretagogue, the fluorescence signals from both structures
may overlap due to the limits of spatial resolution of light microscopy, and
such ASP foci may appear in our analysis in the category `colabelled'.
Nevertheless, the above data suggest that release sites of SN2 represent only
a minor fraction of the total number of release sites on or within the
glandular tissue.
The finding that axonal profiles associated with the salivary gland contain
two types of vesicles, viz. small clear vesicles and large dense-core
vesicles (Whitehead, 1971;
Maxwell, 1978
; present study)
may indicate some additional complexity in the control of salivation. Small
clear vesicles are generally considered to contain classical
neurotransmitters, whereas large dense-core vesicles may serve the release of
neuropeptides (Richmond and Broadie,
2002
). It is thus reasonable to assume that neuropeptides are
coreleased with serotonin and dopamine in the vicinity of the gland cells.
Although the occurrence of various neuropeptides has been reported in the
salivary glands of the locust and the blood-feeding bug, Rhodnius
prolixus (Myers and Evans,
1985
; Tsang and Orchard,
1991
; Veelaert et al.,
1995
; Fusé et al.,
1996
; Ali, 1997
;
Te Brugge and Orchard, 2002
),
our knowledge of the role of neuropeptides in the control of salivation is far
from complete. In particular, evidence for the peptidergic innervation of
cockroach salivary glands is still lacking and awaits detailed
investigation.
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Acknowledgments |
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References |
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