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First published online August 18, 2005
Journal of Experimental Biology 208, 3303-3319 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01787

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Identification and characterization of a tachykinin-containing neuroendocrine organ in the commissural ganglion of the crab Cancer productus

Daniel I. Messinger^1,2, Kimberly K. Kutz³, Thuc Le⁴, Derek R. Verley⁴, Yun-Wei A. Hsu¹, Christina T. Ngo^1,2, Shaun D. Cain², John T. Birmingham⁴, Lingjun Li^3,5 and Andrew E. Christie^1,2,*

¹ Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA
² Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, WA 98250, USA
³ Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue, Madison, WI 53706-1369, USA
⁴ Department of Physics, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053-0315, USA
⁵ School of Pharmacy, University of Wisconsin–Madison, 777 Highland Avenue, Madison, WI 53705-2222 USA

View larger version (25K): [in a new window]   Fig. 1. Schematic representation of the Cancer productus stomatogastric nervous system (STNS), showing the distribution of tachykinin-related peptide (TRP)-immunopositive structures, including the anterior commissural organs (ACOs). The STNS of the crab C. productus consists of four ganglia as well as a number of interconnecting and motor nerves. The four ganglia are the paired commissural ganglia (CoGs), the single oesophageal ganglion (OG) and the single stomatogastric ganglion (STG). The inferior oesophageal (ion), oesophageal (on), superior oesophageal (son) and stomatogastric (stn) nerves link these ganglia, while motor nerves, including the labral (ln), dorsal posterior oesophageal (dpon), anterior cardiac (acn), anterior lateral (aln), medial ventricular (mvn) and dorsal ventricular (dvn) nerves, provide innervation to the foregut musculature. The inferior ventricular nerve (ivn) and the circumoesophageal connectives (cocs) link the STNS with the supraoesophageal (SoG) and fused thoracic ganglia (TG), respectively. The distribution of TRP immunoreactivity in the STNS of C. productus is shown in red. Here, immunopositive somata are schematized with filled circles, while axons within nerves are represented by thick lines, and immunopositive neuropil by tangles of thinner lines. The locations of the left and right anterior commissural organs (ACOL and ACOR) are indicated with arrows. The nomenclature of ganglia and nerves is per Maynard and Dando (1974).

View larger version (83K): [in a new window]   Fig. 2. Distribution of substance P-like labeling in the commissural ganglion (CoG) of the crab Cancer productus. (A) Within each CoG, substance P-like immunoreactivity is present in seven neuronal somata (three denoted by arrows) as well as in neuropilar processes (star) and a large club-shaped plexus (boxed). This plexus is located in the anterior medial quadrant of the ganglion and originates from a fascicle of small-diameter axons that project from the circumoesophageal connective (coc) connecting the supraoesophageal (SoG) and thoracic ganglia (TG) to the CoG. This micrograph is a brightest pixel projection of 24 optical sections taken at 2.0 µm intervals. (B) A higher magnification view of the plexus boxed in A. As can be seen from this micrograph, the plexus is composed of tightly aggregated, flocculent varicosities. The aggregated varicosities appear to cluster around unlabeled tubular structures, particularly in the posterior portion of the plexus. This micrograph is a brightest pixel projection of 37 optical sections taken at 1.0 µm intervals. (C) A projection of three optical sections taken at 1.0 µm intervals from the boxed region in B, showing aggregated substance P-immunopositive terminals enveloping several unlabeled tubular structures (indicated by asterisks). Scale bars, 200 µm in A, 75 µm in B and 25 µm in C.

View larger version (139K): [in a new window]   Fig. 3. Visualization of hemolymph sinuses/lacunae in the commissural ganglion (CoG) using India ink. India ink injected into the pericardial chamber is incorporated into the circulating fluid and fills blood vessels and sinuses with an easily visualized substrate. As this micrograph shows, the CoG is one of the region of the nervous system possessing vascularization. Here, India ink filling reveals an extensive array of hemolymph lacunae/sinuses within the CoG, including many in the anterior medial quadrant of the ganglion. This image is a digital micrograph of a living ganglion. Abbreviations: coc, circumoesophageal connective; ion, inferior oesophageal nerve; son, superior oesophageal nerve; SoG, supraoesophageal ganglia; TG, thoracic ganglia. Scale bar, 200 µm.

View larger version (110K): [in a new window]   Fig. 4. Hemolymph lacunae are coincident with areas of immunolabel avoidance in the anterior commissural organ (ACO). In the commissural ganglion, the substance P-immunopositive plexus is fenestrated by what appears to be a network of branched tubes. To determine if these tubular structures are hemolymph lacunae, several India ink-filled ganglia were immunoprocessed with the substance P antibody, and the ink and immunolabel were simultaneously imaged via confocal microscopy. As can be seen in this set of micrographs, ink-filling (A) was evident in numerous lacunae in the portion of the ganglion containing the substance P-immunopositive plexus (B). When micrographs of the individual labels were merged (C), it became apparent that the plexus is fenestrated by hemolymph vessels. As the merged micrograph shows, the substance P-immunopositive nerve terminals that form the plexus envelop the hemolymph lacunae. This organization, with nerve terminals in direct apposition to the hemolymph space, is considered the defining characteristic of a crustacean neuroendocrine site. We hypothesize that this plexus is a neuroendocrine organ and we have named it the anterior commissural organ based on its location. Each micrograph is a brightest pixel projection of nine optical sections taken at 0.5 µm intervals. A and B are shown at the same scale. Scale bars, 20 µm in B and C.

View larger version (71K): [in a new window]   Fig. 5. Double-immunolabeling shows that the anterior commissural organ (ACO) does not possess allatostatin-like peptide or serotonin co-transmitters. (A) Co-labeling of the commissural ganglion (CoG) with antibodies to substance P (-Sub P) and allatostatin (-AST). Both the substance P (A1) and allatostatin (A2) antibodies give rise to extensive labeling within the CoG. However, superimposition of micrographs of the two labels (A3) shows that the ACO (as defined by the substance P label) is not allatostatin-immunopositive. All micrographs in A are brightest pixel projections of 25 optical sections taken at 2.0 µm intervals. (B) Co-labeling of the CoG with antibodies to substance P and serotonin (-5HT). As in the previous pairing, both the substance P (B1) and serotonin (B2) antibodies give rise to extensive labeling within the CoG. Again, superimposition of micrographs of the two labels (B3) shows that the ACO (as defined by the substance P label) is not serotonin-immunopositive. All micrographs in B are brightest pixel projections of 30 optical sections taken at 2.0 µm intervals. A1, A2, B1 and B2 are shown at the same scale. Likewise, A3 and B3 are shown at the same scale. It should be noted that in A3 and B3, the apparent co-localization of the immunolabels (yellow coloration) is an artifact of the projection of multiple optical sections spanning the entire thickness of the ganglia shown rather than true co-localization of the immunoreactivities in a common structure. Scale bars, 100 µm.

View larger version (36K): [in a new window]   Fig. 6. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometric (MALDI-FTMS) identification of APSGFLGMRamide (CabTRP Ia) in the anterior medial quadrant of the commissural ganglion (CoG) and hemolymph. (A) CabTRP Ia in the anterior medial quadrant of the CoG. As can be seen in this mass spectrum, taken from a tissue fragment from the quadrant of the CoG containing the anterior commissural organ, a number of peaks corresponding to individual peptides are evident. These include peaks corresponding to three isoforms of orcokinin, corazonin, three FLRFamide-related peptides, two orcomyotropin-related peptides (FDAFTTGFGHS and FDAFTTGFGHN), Gly1-SIFamide, red pigment concentrating hormone (RPCH) and the recently identified peptide HLGSLYRamide (all labeled in blue). Of particular interest is the peak at m/z 934.501 (red arrow). This m/z is identical to that of authentic CabTRP Ia (a mass measurement error of 8.56 p.p.m. from its theoretical m/z of 934.493) and confirms the presence of this peptide in the portion of the CoG containing the ACO. (B) MALDI-FTMS detection of CabTRP Ia in the hemolymph. Hemolymph extract examined via MALDI-FTMS shows an m/z peak at 934.491 (red arrow). Again, this m/z peak is characteristic of authentic CabTRP Ia (mass measurement error 2.14 p.p.m.) and strongly suggests that this peptide is a circulating hormone in C. productus. It should be noted that in both A and B there is an m/z peak that corresponds to a known electrical noise artifact in the MALDI-FTMS system used. This peak is denoted via an asterisk in both spectra.

View larger version (24K): [in a new window]   Fig. 7. The effects of CabTRP Ia on nerve-evoked excitatory junction potentials (EJPs) in six stomatogastric muscles. (A) Nerve-evoked EJP in a gastric mill 8a (gm8a) muscle fiber in control saline (black), after 10 min in 10–7 mol l–1 CabTRP Ia (red), and after rinsing the peptide (green). The membrane potential was –77 mV for all three conditions. Each trace in this panel is the average of four EJPs elicited at 20 s intervals. (B) A bar graph plot of average gm8a EJP amplitude in control saline and after 10 min in 10–7 mol l–1 CabTRP Ia. The increase in amplitude in CabTRP Ia was significant (N=6; paired t-test, **P<0.01). (C) A bar graph showing the average increase in EJP amplitude in 10–7 mol l–1 CabTRP Ia for six stomatogastric muscles: gastric mill 4 (gm4; N=5), gastric mill 5a (gm5a; N=6), gastric mill 6a (gm6a; N=8), gm8a (N=6), pyloric 1 (p1; N=8) and pyloric 2 (p2; N=6). For each individual experiment used to construct this panel, the EJP amplitude was determined by averaging four EJPs, as in A. For each muscle, significance was computed using paired t-tests on the EJP amplitudes (*P<0.05, **P<0.01, ***P<0.001).

View larger version (13K): [in a new window]   Fig. 8. CabTRP Ia increases contraction of the gastric mill 8 (gm8) muscle. (A) The lateral ventricular nerve (lvn) was stimulated at 10 Hz for 3 s, and the resulting contractions of a gm8 muscle in control saline (black), after 10 min in 10–7 mol l–1 CabTRP Ia (red), and after rinsing the peptide (green) are shown. (B) A bar graph of average gm8 peak contraction amplitude in control saline and after 10 min in10–7 mol l–1 CabTRP Ia. The increase in amplitude in peptide was significant (N=7; paired t-test, **P<0.01).

View larger version (25K): [in a new window]   Fig. 9. Schematic representation of hypothetical paracrine actions of the anterior commissural organ (ACO) in the commissural ganglion (CoG). In addition to serving as a source of CabTRP Ia to the hemolymph, some portions of the ACO terminate directly on areas of synaptic neuropil, and this structure may thus also function as a paracrine modulator of intrinsic CoG targets. One potential role for this paracrine signaling is the coordination of multiple neuroendocrine systems. The soma of the large (L)-cell, which projects to and innervates the pericardial organ (PO), is located within the CoG and is known to arborize in the vicinity of the ACO. Likewise, anterior commissural neurons 1 and 2 (ACN1/2), which are the sole source of innervation to the anterior cardiac plexus (ACP), are also located in the CoG and arborize near the ACO. If these neurons are modulated by CabTRP Ia, then elements of at least two other neuroendocrine centers could be modulated/synchronized locally within the CoG, concurrent with the release of CabTRP Ia from the ACO into the circulatory system. Non-endocrine neurons within the CoG [specifically CoG projection neurons (CPN) that innervate and modulate the stomatogastric neural circuit] may also be targets of paracrine signals from the ACO. If these neurons are influenced by CabTRP Ia released from the ACO, then a profound reorganization of the motor patterns produced by the motor neurons (MN) of the circuits contained within the stomatogastric ganglion could occur. Abbreviations not defined in this legend are as per Fig. 1.