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First published online February 20, 2004
Journal of Experimental Biology 207, 1163-1182 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00856

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The anterior cardiac plexus: an intrinsic neurosecretory site within the stomatogastric nervous system of the crab Cancer productus

Andrew E. Christie^1,2,*, Shaun D. Cain², John M. Edwards^1,2, Todd A. Clason¹, Elena Cherny¹, Minhui Lin², Amitoz S. Manhas², Kirsten L. Sellereit², Nicholas G. Cowan², Kellen A. Nold², Hans-Peter Strassburg² and Katherine Graubard^1,2

¹ Department of Biology, University of Washington, Box 351800, Seattle, Washington 98195-1800 USA
² Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, Washington 98250 USA

View larger version (10K): [in a new window]   Fig. 1. Schematic representation of the stomatogastric nervous system, including the location of the anterior cardiac plexi (ACPs). The paired ACPs are located on the anterior cardiac nerves (acns) which overlie the cardiac sac region of the foregut. aln, anterior lateral nerve; coc, circumoesophageal connective; CoG, commissural ganglion; dgn, dorsal gastric nerve; dlvn, dorsal lateral ventricular nerve; dpon, dorsal posterior oesophageal nerve; dvn, dorsal ventricular nerve; ion, inferior oesophageal nerve; ivn, inferior ventricular nerve; lgn, lateral gastric nerve; ln, labral nerve; lpn, lateral pyloric nerve; lvn, lateral ventricular nerve; mvn, medial ventricular nerve; OG, oesophageal ganglion; on, oesophageal nerve; pdn, pyloric dilator nerve; psn, pyloric sensory nerve; pyn, pyloric nerve; son, superior oesophageal nerve; STG, stomatogastric ganglion; stn, stomatogastric nerve; vlvn, ventral lateral ventricular nerve.

View larger version (82K): [in a new window]   Fig. 2. Incident light micrographs of a portion of the anterior cardiac nerve (acn) and stomatogastric nerve (stn). (A) Incident light micrograph of the acn. This image, taken approximately 400 µm from the junction of the acn and the stn (see Fig. 1), shows numerous iridescent bluish-white profiles. These profiles appear superficially located and cover an approximately 1000 µm stretch of the nerve. (B) Incident light micrograph of the stn. In contrast to the acn, no iridescent profiles are seen in this nerve or in any other nerves (other than the acns) or ganglia that comprise the stomatogastric nervous system. Scale bar, 100 µm.

View larger version (205K): [in a new window]   Fig. 3. Gross structure of the iridescent portion of anterior cardiac nerve (acn) and electron microscopy of its central core. (A–C) Light micrographs of Toluidine Blue-stained sections of the acn. These micrographs show that the iridescent portion of the acn can be divided into two parts, a central core containing large diameter axons (indicated numerically in A–C) and a peripheral region (see Figs 4 and 5). At the proximal end of the acn (A), five axons (labeled 1–5) are present. Regardless of preparation or fixation protocol, these axons are approximately 10 µm in major cross-sectional diameter. As one moves distally through the nerve (B), four of the five axons terminate into numerous smaller diameter axons (asterisks). The remaining axon (labeled 1 in B and C) maintains a constant diameter through the medial portion of the acn, ultimately exiting the iridescent portion of nerve (C). (D) Transmission electron micrograph of one of the five axons present in the central core of the acn. While taken from a distal section of an acn segment, the ultrastructure of this axon is typical of the ultrastructure of all axons present in the entire iridescent portion of the acn. Like all acn axons, the axon shown in this panel contains filamentous axoplasm (ap), mitochondria (m) and occasionally dense-core (DCV) and electronlucent vesicles. In this micrograph, a single DCV is evident. This, and all other acn axons, is ensheathed by a thick glial wrap (gw). The glia contain a relatively homogeneous cytoplasm, often with mitochondria present. In this example, structures within the axon are labeled with black lettering while those associated with the glial wrap are labeled with white lettering. A, B and C are taken from different preparations. D is taken from the same preparation as C. Scale bars, 30 µm (A–C); 1 µm (D). It should be noted that the difference in appearance of the tissue in A versus B and C is due to the type of plastic used for embedding, namely LRWhite and EMBed, respectively.

View larger version (138K): [in a new window]   Fig. 4. Transmission electron microscopy of the peripheral portion of the anterior cardiac nerve (acn). (A) A low magnification micrograph of the periphery of the acn. An epineurium (ep) separates the nerve from the hemolymph space. Directly under the epineurium lie numerous nerve terminals, three of which are indicated with asterisks. Glial protuberances (g) are also present in the periphery of the acn. (B) A high magnification micrograph of the periphery of the acn. The epineurium is composed of a moderately dense, amorphous material that is fenestrated with minute open spaces (three of the larger fenestrations are indicated with black arrowheads). All or portions of five nerve terminals (asterisks) are present below the epineurium in this image. These terminals contain numerous dense-core vesicles (DCVs) and often mitochondria (m) and a small number of electron-lucent vesicles (ELVs). Morphological correlates of vesicle secretion, including vesicles docked to the plasma membrane and omega ()-figures, are common on the nerve terminals. In this micrograph four -figures are clearly visible. A and B are taken from the same preparation. Scale bars, 1 µm (A); 500 nm (B).

View larger version (198K): [in a new window]   Fig. 5. Morphological correlates of hormone secretion are present in the anterior cardiac plexus. This transmission electron micrograph shows portions of several nerve terminals at high magnification. Both dense-core (DCV) and electron-lucent (ELV) vesicles are present in these terminals. In this image, one DCV is docked to the plasma membrane. Likewise, several DCVs have fused with the plasma membrane and are in the process of exocytosing their contents. This exocytosis creates characteristic ultrastuctural features on the plasma membrane commonly referred to as omega ()-figures. The docked DCV and the three -figures visible in this micrograph create a pseudo-time course of hormone secretion. First, a DCV docks to the plasma membrane (1) and subsequently fuses with it, releasing its dense-core and forming an -figure (1). The membrane of the DCV rapidly is incorporated into the plasma membrane of the terminal and the -figure subsides (2 and 3). Note that this micrograph is from a different preparation to that shown in Fig. 4. Scale bar, 200 nm.

View larger version (76K): [in a new window]   Fig. 6. FLRFamide immunoreactivity in the anterior cardiac plexus (ACP) is derived from four axons which project to the structure through the superior oesophageal (son), stomatogastric (stn) and anterior cardiac (acn) nerves. (A) Montage of seven confocal micrographs showing the projection pathway of the axons that give rise to the ACPs. In this preparation, the axons (denoted by the arrows) travel through much of the nervous system as tightly associated fascicles. These fascicles can be followed unambiguously from the sons, through the stn and acns to the ACPs. In this image, the beginning and end of the right ACP are defined by asterisks. The left ACP is not shown. In this montage, the individual micrographs are brightest pixel projections of 30–55 optical sections taken at 2.0 µm intervals. (B) Confocal micrograph showing four FLRFamide labeled axons projecting into the acn. In this preparation, the four FLRFamide immunopostive axons that arborize into the ACPs are clearly visible entering the left acn. Each of these axons (arbitrarily designated 1–4) is indicated with an arrow. The branch point of axon 4 is marked with an asterisk and the left and right projecting branches labeled 4L and 4R, respectively. This image is a brightest pixel projection of 22 optical sections taken at 2.0 µm intervals. Scale bars, 200 µm (A); 100 µm (B). on, oesophageal nerve; STG, stomatogastric ganglion.

View larger version (113K): [in a new window]   Fig. 7. FLRFamide labeling in the anterior cardiac plexus consists of peripherally located varicosities derived from large diameter fibers. (A) Confocal micrograph showing the distal termination of the left ACP. Here, several immunolabeled fibers can be seen to project fine processes toward the periphery of the anterior cardiac nerve. These fine neurites arborize, producing numerous clusters of varicosities which are located within or just below the acn sheath. This image is a brightest pixel projection of 26 optical sections taken at 2.0 µm intervals. (B) A higher magnification view of a portion of the acn boxed in A. Note that many of the varicosities are connected together by fine neurites, giving rise to a `beads on a string'-like conformation. This image is a brightest pixel projection of 24 optical sections taken at 1.0 µm intervals. (C) A further magnified view of one cluster of FLRFamide containing varicosities boxed in B. Note the grape-like cluster of terminal varicosities. This image is a brightest pixel projection of 44 optical sections taken at 0.5 µm intervals. Scale bars, 100 µm (A,B); 25 µm (C).

View larger version (83K): [in a new window]   Fig. 8. Clustering of FLRFamide-immunopositive terminals in the anterior cardiac plexus (ACP) often gives rise to a bark-like appearance of this structure. (A) A brightest pixel projection of 32 optical sections taken at 2.0 µm intervals through a portion of the anterior cardiac nerve (acn) containing the ACP. (B–D) Single optical sections from the projection shown in A. These images were selected as representative of the labeling seen near the top (B; section 6 of 32), center (C; section 18 of 32) and bottom (D; section 29 of 32) of the acn. Note that the FLRFamide immunolabeled terminals that comprise the ACP are concentrated in the peripheral portion of the acn, with essentially no terminals present in the core of the nerve. Scale bar, 100 µm.

View larger version (109K): [in a new window]   Fig. 9. On some anterior cardiac plexi (ACPs), blister-like protuberances are evident. In some preparations, the ACPs contain multiple blister-like protuberances of the sheath that are densely packed with FLRFamide-immunopositive profiles. In this example, numerous protuberances are present, several of which are indicated with arrows. This image is a brightest pixel projection of 36 optical sections taken at 2.0 µm intervals. Scale bar, 100 µm.

View larger version (80K): [in a new window]   Fig. 10. Lucifer Yellow-CH dye (LY) backfill of an anterior cardiac nerve (acn) produces dye-filling in axons that project posteriorly from the superior oesophageal nerves (sons) and anteriorly from the stomatogastric ganglion (STG). (A1–3) LY backfilling of single acns produces dye-filling in two large diameter axons in each of the paired sons. These axons are FLRFamide immunopositive. Several smaller diameter FLRFamide-immunopositive axons are also present in each son. (B1–3) LY backfilling of single acns also produces dye-filling in a single large diameter axon that projects via the stomatogastric nerve (stn) from the STG. This axon is not FLRFamide immunopositive. As is seen in the son, several small FLRFamide-immunopositive axons are present in the stn. (A1–3) Brightest pixel projections of 19 optical sections taken at 1.0 µm intervals through the son. (A1) LY dye-filled axons pseudocolored green. (A2) FLRFamide immunoreactivity pseudocolored red. The optical sections used to produce A1 and A2 were simultaneously collected from the same focal planes. (A3) A merged image of A1 and A2. Profiles exhibiting only LY dye-filling or FLRFamide immunolabeling, appear green or red, respectively. Structures showing colocalization of LY dye and FLRFamide label appear yellow (or shades thereof). (B1–3) Brightest pixel projection of 22 optical sections taken at 1.0 µm intervals through the stn. Organization and pseudocoloring of B1–3 is identical to that of A1–3. A and B are from the same preparation. Scale bar, 50 µm

View larger version (77K): [in a new window]   Fig. 11. Synapsin-like labeling is restricted to FLRFamide-immunopositive terminals in the anterior cardiac plexus (ACP). Five axons are present in the portion of the anterior cardiac nerve containing the ACP. Four of the five axons are FLRFamide immunopositive and contribute innervation to the ACP. To assess whether the remaining axon contributes to the innervation of the ACPs, double-immunolabels pairing FLRFamide and synapsin antibodies were conducted. In all preparations, the synapsin label was found localized in FLRFamide-immunopositive terminals. Most FLRFamide labeled terminals exhibited some degree of synapsin staining. In no preparation were any terminals found that contained only synapsin immunoreactivity. Interestingly, within a given terminal, the FLRFamide and synapsin labels are often non-uniformly segregated. (A1–3) and (B1–3) show examples of this localization from two different preparations. (A1–3) Brightest pixel projections of 19 optical sections taken at 1.0 µm intervals. (A1) FLRFamide immunoreactivity pseudocolored red. (A2) Synapsin immunoreactivity pseudocolored green. The optical sections used to produce A1 and A2 were simultaneously collected from the same focal planes. (A3) A merged image of A1 and A2. Profiles exhibiting only FLRFamide or synapsin immunolabeling appear red or green, respectively. Structures showing coincidence of FLRFamide and synapsin labels appear yellow (or shades thereof). (B1–3) Brightest pixel projection of 12 optical sections taken at 1.0 µm intervals. Organization and pseudocoloring of B1–3 is identical to that of A1–3. Scale bar, 50 µm.