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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

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Dopaminergic and serotonergic innervation of cockroach salivary glands: distribution and morphology of synapses and release sites

Otto Baumann^1,*, Dana Kühnel^1,2, 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

View larger version (75K): [in a new window]   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.

View larger version (32K): [in a new window]   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.

View larger version (17K): [in a new window]   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.

View larger version (66K): [in a new window]   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).

View larger version (96K): [in a new window]   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).

View larger version (10K): [in a new window]   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).

View larger version (172K): [in a new window]   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).