First published online June 16, 2004
Journal of Experimental Biology 207, 2565-2575 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01069
Dopaminergic and serotonergic innervation of cockroach salivary glands: distribution and morphology of synapses and release sites
Otto Baumann1,*,
Dana Kühnel1,2,
Petra Dames1 and
Bernd Walz1
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
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Fig. 1. Schematic representation of the organization of the cockroach salivary
gland and of its serotonergic and dopaminergic innervation pattern. (A) The
salivary glands are paired and consist of several lobules of secretory acini.
The ducts of each gland unite to a single efferent salivary duct (4) that
fuses with the opposite duct to form the main salivary duct. Innervation of
the salivary gland is via the salivary duct nerve containing a single
dopaminergic and several serotonergic axons and via branches of the
oesophageal nerve containing serotonergic axons. Dopaminergic nerve fibres
(blue) reside on the surface of the acinar tissue (1), ramify extensively in
nerves that interlink adjacent acinar lobules (2) and extend to the various
sections of the duct system. Serotonergic nerve fibres (red) reside on the
acinar tissue (1), penetrate deeply into the acinar lobules and are also
associated with sections of the duct system adjacent to the acinar tissue (3).
(B) Each acinus consists of two peripheral cells (P-cells) with long
microvilli and several central cells (C-cells) with numerous secretory
granules. Triple black lines indicate the position of septate junctions
(Just and Walz, 1994 ). Each
acinus is covered by dopaminergic (blue) and serotonergic (red) nerve fibres.
Serotonergic nerve fibres (red) extend deep into the acini between the
C-cells. The apical surface of the C-cells is covered by a sheath of flattened
fenestrated centroacinar cells. The duct cells have basal and apical
infoldings and form a simple tubule. Dopaminergic nerve fibres (blue) extend
into the epithelial layer of duct cells.
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Fig. 2. Cross-reactivity of anti-Drosophila-synapsin with cockroach
synapsin. (A) Western blot analysis of Drosophila head (lane 1),
cockroach brain (lane 2) and cockroach salivary gland (lane 3) with
anti-synapsin. A broad band with an electrophoretic mobility of about 80 kDa
is intensely labelled in all preparations. In Drosophila, an
additional synapsin isoform is detected at about 140 kDa. (B) In
vitro binding of the synapsin-positive cockroach protein to actin
filaments. An extract of cockroach brain was incubated with F-actin; actin
filaments were then pelleted by high-speed centrifugation. In the absence of
F-actin, the anti-synapsin-positive proteins remain entirely in the
supernatant (S), whereas in the presence of F-actin, a substantial fraction of
the anti-synapsin-positive proteins is detected in the pellet (P) together
with F-actin. The actin-binding protein  -spectrin provides a positive
control, and tubulin a negative control.
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Fig. 3. Immunolocalization of synapsin in serotonergic nerve fibres at the release
sites for neurotransmitters or neurohormones. (A–C) Nerve fibres in a
cockroach muscle double-stained with anti-serotonin and anti-synapsin.
Anti-synapsin immunoreactivity is highly concentrated at the sides of the
nerve terminals (arrowheads in C). (D–F) A salivary duct nerve costained
with anti-serotonin and anti-synapsin. The salivary duct nerve contains two
thick, centrally localized axons that are non-serotonergic and homogeneously
stained with anti-synapsin (E, arrows). These axons are surrounded by several
thin serotonergic axons that ramify and have numerous short sidebranches
forming a neurohaemal organ. Anti-synapsin is highly concentrated at the
distal ends of these sidebranches (arrowheads in F). Bars, 50 µm.
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Fig. 4. Distribution of anti-synapsin-positive (ASP) structures within the salivary
gland. Whole-mount preparations of salivary glands were costained with
anti-synapsin (red) and BODIPY-FL phallacidin (blue) and imaged by confocal
microscopy. (A–F) A series of confocal sections through an acinar
lobule. Each image shows the sum of 8 consecutive optical sections
(inter-section distance: 0.45 µm) representing a total thickness of 3.6
µm. (A) The sum of all confocal images. The P-cells are arranged in pairs
and their apical arrays of phallotoxin-stained microvilli appear as `bow ties'
(asterisks). The C-cells are identified by short phallotoxin-labelled
microvilli (E,F, open arrows) on their luminal surface. Rows of ASP foci
(arrowheads) reside on the surface of the acinar tissue, next to P-cells (B),
and extend deep into the acinar tissue, residing amongst C-cells (E,F). (G)
Nerves interconnecting adjacent acinar lobules contain rows of ASP foci
(arrowheads) and axons with homogeneous labelling for synapsin (arrows). (H,I)
A small salivary duct (H) and a large efferent duct (I) with ASP foci
(arrowheads) on the surface of the epithelial layer. Arrows indicate
autofluorescent tracheoles. (J,K) Horizontal sections through a small duct (J)
and a large duct (K) demonstrate that the ASP foci also reside within the duct
epithelium, between the apical surface covered with phallotoxin-labelled
microvilli (broad arrows) and the basal surface (broken line). Scale bars, 25
µm (A–I); 10 µm (J,K).
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Fig. 5. Colocalization of synapsin-enriched structures with serotonergic and
dopaminergic fibres. Whole-mount preparations of salivary glands were
triple-labelled with anti-synapsin (red), BODIPY-FL phallacidin (blue), and
either anti-serotonin (green) or anti-TH (green). (B–D,H–L) Series
of confocal images through acinar tissue, each image representing the sum of 6
consecutive optical sections (inter-section distance: 0.4 µm). (A,G) The
sum of all images. ASP foci on the outer surface of the acinar tissue
colocalize with either anti-serotonin (B–D) or anti-TH (H–J). ASP
foci lie deep in the acinar tissue, amongst C-cells that are identified by
short phallotoxin-labelled microvilli (arrowheads) on their luminal surface,
and colocalize with anti-serotonin only (arrows in F,L). (M,N) Within the
nerves that interlink adjacent acinar lobules, most ASP foci (arrows)
colocalize with anti-TH; few foci colocalize with anti-serotonin (insets in
M). (O,P) ASP foci on initial duct segments colocalize with either
anti-serotonin or anti-TH. (Q,R) On the large salivary ducts, ASP foci
colocalize with anti-TH almost exclusively. (Insets in P,R) Horizontal
sections through a duct at the position indicated by the dotted boxes in (P
and R) demonstrate the distribution of ASP foci along dopaminergic fibres
within the duct epithelium. Broad arrows indicate autofluorescent tracheoles.
Scale bars, 10 µm (A–L); 25 µm (M–R).
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Fig. 6. Quantitative analysis of the colocalization of anti-synapsin-positive (ASP)
foci with either anti-serotonin or anti-TH. (A) Most ASP foci on and within
acinar tissue colocalize with anti-serotonin. (B) In neurohaemal structures
between acinar lobules, the majority of ASP foci colocalizes with anti-TH, and
a smaller fraction colocalizes with anti-serotonin. Values means ±
S.D.(N=number of images analysed).
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Fig. 7. Ultrastructure of putative release sites for
neurotransmitters/neurohormones associated with the salivary gland. (A,B)
Axonal profiles embedded between C-cells (asterisks) contain numerous clear
vesicles and a few dense vesicles. Small electron-dense areas on the
cytoplasmic face of the plasma membrane (white arrowheads) may represent
active zones. (C) A putative release site on the outer surface of an acinar
lobule, abutting a P-cell (white asterisk). A thick basal lamina (black
asterisk) separates the presynaptic membrane from the plasma membrane of the
P-cell. (Insets) Ribbon-like electron-dense structures (arrowheads) on the
cytoplasmic face of the axonal membrane at higher magnification, in
cross-section (left) and en face view (right). Rows of vesicles are
adjacent to these electron-dense structures, suggesting that they represent
the structural correlates of active zones. (D–F) Axonal profiles within
nerves that interconnect acinar lobules. On one side, the axonal profiles are
without glial wrapping, face a thick basal lamina (asterisks) and have
electron-densities on the cytoplasmic face of their plasma membrane
(arrowheads). Type A release sites (D,F) have numerous small clear vesicles
and few larger dense vesicles. Type B release sites (E) contain fewer clear
vesicles and numerous dense vesicles. Scale bars, 0.5 µm (A–F); 0.2
µm (inset).
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© The Company of Biologists Ltd 2004
