Allatostatin-like immunoreactivity in the stomatogastric nervous system and the pericardial organs of the crabCancer pagurus the lobster Homarus americanus and the crayfishCherax destructor andProcambarus clarkiiкод для вставкиСкачать
THE JOURNAL OF COMPARATIVE NEUROLOGY 403:85–105 (1999) Allatostatin-Like Immunoreactivity in the Stomatogastric Nervous System and the Pericardial Organs of the Crab Cancer pagurus, the Lobster Homarus americanus, and the Crayfish Cherax destructor and Procambarus clarkii PETRA SKIEBE* Institut für Neurobiologie, Freie Universität Berlin, Berlin, Germany ABSTRACT The distribution of allatostatin (AST)-like immunoreactivity was studied in the stomatogastric nervous system (STNS) and the neurosecretory pericardial organs (PO) of four decapod crustacean species by using wholemount immunocytochemical techniques and confocal microscopy. AST-like immunoreactivity was found within the STNS of all four species; its distribution in each was unique. In all four species, AST-like immunoreactivity was present in the paired commissural ganglia (CoG), in the esophageal ganglion (OG), in the stomatogastric ganglion (STG), and in their connecting nerves. Within the CoGs, numerous cell bodies and neuropil were stained. In the OG, two cell bodies were immunoreactive, although their branching pattern varies between species. In the STG of C. pagurus and H. americanus, neuropil was stained extensively, but no labeled cell bodies were found. Surprisingly, in C. destructor and P. clarkii, cell bodies were stained in the STG, one brightly stained cell body in both species and an additional two to five weakly stained cell bodies in P. clarkii. In all four species, stained gastropyloric receptor cells were present. In contrast to the variable staining within the STNS, all four species have a similar pattern of AST-like immunoreactivity within the PO. Only in C. destructor, AST-immunoreactive varicosities occur on the surface of the circumesophageal connectives and on the postesophageal commissure and suggest another neurohaemal source for AST-like peptides in this species. The pattern of this staining suggests that AST-like peptides are likely utilized as both neurohormones and as neuromodulators in the STNS of decapod crustacea. J. Comp. Neurol. 403:85–105, 1999. r 1999 Wiley-Liss, Inc. Indexing terms: neuropeptides; neuromodulators; hormones; confocal microscopy; decapod crustaceans; immunocytochemistry The allatostatins (AST) are a family of peptides which were isolated in the process of finding substances which inhibit juvenile hormone synthesis by the corpora allata, a major endocrine organ of insects (Stay et al., 1994). The first AST to be identified were isolated in the cockroach Diploptera punctata (Woodhead et al., 1989; Pratt et al., 1989). Since then, AST have been isolated in a number of other insects including other cockroaches, moths, flies, crickets (reviewed in: Stay et al., 1994; Bendena et al., 1997), and locusts (Veelaert et al., 1996a,b). The majority of these AST peptides share a conserved C-terminal sequence (-Y-X-F-G-L-NH2). Taking advantage of this conserved structure in insects, Duve et al. (1997b) were recently able to isolate a family of AST from a crustacean, r 1999 WILEY-LISS, INC. the crab Carcinus maenas. Twenty different peptides were isolated in this species including the shortest and the longest AST isolated so far, making it the largest AST family. Anatomical studies performed in a variety of insect species (Stay et al., 1992; Duve et al., 1993, 1997a; Bellés et al., 1994; Duve and Thorpe, 1994; Petri et al., 1995; Ude Grant sponsor: DFG; Grant number: SFB 515, C1. *Correspondence to: Dr. Petra Skiebe, Institut für Neurobiologie, Freie Universität Berlin, Königin-Luise-Str. 28–30, D-14195 Berlin, Germany. E-mail: email@example.com Received 19 May 1998; Revised 10 August 1998; Accepted 19 August 1998 86 P. SKIEBE and Agricola, 1995; Veelaert et al., 1995; Würden and Homberg, 1995; Yoon and Stay, 1995; Vitzthum et al., 1996) show that AST-like immunoreactivity is present in many neurons throughout the nervous system, indicating that AST might have other functions in addition to its inhibitory effect on juvenile hormone synthesis. Additional anatomical studies include crustaceans (Abel et al., 1994; Skiebe and Schneider, 1994; Christie et al., 1995a), spiders (Agricola et al., 1995), mollusks (Rudolph and Stay, 1997), and several non-arthropod invertebrates (Smart et al., 1994) and demonstrate that AST-like peptides are common in invertebrates. In insects, an additional function of the AST has been shown for two muscle systems, the hindgut and the antennal pulsatile organ muscle. D. punctata AST is able to inhibit the spontaneous contractions of the hindgut in a dose-dependent manner (Lange et al., 1993, 1995; Duve et al., 1995, 1996, 1997a; Veelaert et al., 1996a). In contrast, application of D. punctata AST to the antennal heart muscle in vitro did not alter the contractions either in P. americana (Hertel and Penzlin, 1992) or in D. punctata. But when D. punctata AST was applied before the excitatory peptide proctolin, the normal enhancement of the contractions produced by proctolin in P. americana (Hertel and Penzlin, 1992) could be reduced. The effect of D. punctata AST on muscles has been studied in detail in the crustacean, C. borealis (SkiebeCorrette et al., 1993; Jorge-Rivera and Marder, 1997) by using muscles innervated by the stomatogastric nervous system (STNS), which was known to contain AST-like peptides (Skiebe and Schneider, 1994). These stomach muscles receive excitatory glutamatergic and cholinergic innervations from the motoneurons of the STNS. JorgeRivera and Marder (1997) could show that D. punctata AST-1 to 4, but in particular D. punctata AST-3, were able to reduce the amplitude of nerve-evoked contractions, excitatory junctional potentials, and excitatory junctional currents at both cholinergic and glutamatergic neuromus- Abbreviations AGR AST ASTir CoG coc d-pon dvn GPR ion ivn lgn ln lvn mgn NGS OG on PB PB-X pdn PO poc pyn son SOG STG stn STNS anterior gastric receptor neuron allatostatin allatostatin-like immunoreactive commissural ganglion/circumesophageal ganglion circumesophageal connective dorsal posterior esophageal nerve (anterior esophageal nerve in crayfish) dorsal ventricular nerve gastropyloric receptor neuron inferior esophageal nerve inferior ventricular nerve lateral gastric nerve labial nerve lateral ventricular nerve median gastric nerve normal goat serum esophageal ganglion esophageal nerve sodium phosphate buffer PB containing 0.5% Triton X-100 pyloric dilator nerve pericardial organs postesophageal commissure pyloric nerve superior esophageal nerve subesophageal ganglion stomatogastric ganglion stomatogastric nerve stomatogastric nervous system cular junctions. Their data suggest that D. punctata AST-3 decreases the postsynaptic action of both neurally released acetylcholine and glutamate. Beside the reduction of muscle contractions in C. borealis, AST has also been shown to play an important modulatory role within the STNS. D. punctata AST-1 to 4 have been shown to inhibit the pyloric (Skiebe and Schneider, 1994) and the gastric rhythms (Skiebe-Corrette et al., 1993) generated by the STNS in a dose-dependent manner. For the crayfish Cherax destructor and Orconectes limosus, injection of D. punctata AST-3 into the hemolymph reduces the number of spikes per cycle of some of the neurons and the frequency of the pyloric rhythm (Heinzel et al., 1997), suggesting that AST released from neurohemal organs into the hemolymph might be able to change the rhythms generated by the STNS, as implied for other peptides (Weimann et al., 1997). These effects of D. punctata AST on the crustacean STNS are of particular interest because the effects attributed to AST are the first inhibitory effects described for peptides in this system, although numerous peptides have been studied physiologically including Phe-Met-Arg-Phe (FMRF)amide-like peptides, proctolin, red pigment-concentrating hormone, cholecystokinin-like peptides, crustacean cardioactive peptide, and tachykinin-like peptides (Weimann et al., 1993, 1997; Blitz et al., 1995; Christie et al., 1997; Tierney et al., 1997; reviews: Harris-Warrick et al., 1992; Marder et al., 1997). The demonstrated physiological action of the AST on the muscles and the motor pattern-generating network and its distribution within the crustacean STNS is only known for one species (C. borealis). Because it is known that distribution of peptides can vary among different species (Mortin and Marder, 1991; Turrigiano and Selverston, 1991) and given the complexity of the AST family of peptides, a comparative anatomical study of the distribution of AST within the STNS of several species of crustaceans would be useful to determine the degree to which the AST peptide family has a conserved distribution in the STNS, and to lay the groundwork for physiological studies on identifiable AST-immunoreactive neurons. I have used immunocytochemical techniques to compare the distribution of AST-like immunoreactivity within the Fig. 1. Schematic drawings showing the stomatogastric nervous system (STNS), stomach, heart, and pericardial organs. A: Location of the STNS, stomach, heart, and a pericardial organ (PO) in C. destructor. C. destructor was chosen as an example, because its body shape is similar to that of H. americanus and P. clarkii. B: Enlarged view of the STNS, stomach, heart, and PO. The STNS consists of four ganglia, the paired CoGs, the OG, and the STG, and their connecting and motor nerves. The STNS is located between the brain and the SOG, which are connected by the coc surrounding the esophagus. The poc links both cocs close to the SOG. The paired CoGs lie next to the esophagus. The STG lies on the stomach within the ophthalmic artery. The PO are major neurohemal organs located in the pericardial chamber. Neurohormones released by the PO are pumped through the opthalmic artery to the STG. C: Schematic of an isolated STNS. To isolate the STNS, the connections with the brain and the SOG via the cocs are cut on either side of the CoGs, the poc is cut in the middle, and the motor nerves are removed from the muscles. The CoGs are connected with the OG by the ions. The STG is coupled to the CoG by the sons and the stn. The dvn is the major motor nerve originating from the STG, which splits into two lvns, from which the lgn and mgn branch. Branching from the tip of the lvn are pdn and pyn. Not drawn to scale. Additional nerves not discussed in the paper are not shown. For definitions of abbreviations in this and subsequent figures, see list. AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS Figure 1 87 88 P. SKIEBE Figure 2 AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS STNS and the pericardial organs (PO) of four decapod crustaceans: the crab Cancer pagurus, the lobster Homarus americanus, and the crayfish Cherax destructor and Procambarus clarkii. The PO were included because the data from Heinzel et al. (1997) also suggested a neurohemal role for AST. Two particular factors influenced the choice of animals for comparison. First, Casasnovas (1995) suggested a role for an inhibitory modulator in the maturation from embryonic to adult rhythms generated by the STNS, and much information is available on the development of the nervous system for H. americanus (Factor, 1981; Charmantier et al., 1991; Helluy and Beltz, 1991; Beltz et al., 1990, 1992) and for C. destructor (Sandeman and Sandeman, 1991; Scholtz, 1992; Helluy et al., 1993). Second, the distribution of several other peptides within the STNS of P. clarkii (Mortin and Marder, 1991; Turrigiano and Selverston, 1991; Tierney et al., 1997) is known, and C. pagurus is closely related to Cancer borealis, for which the immunocytochemical distribution of other peptides has been intensely studied (Marder et al., 1994). The results of the present study indicate an overall pattern of AST-like immunoreactivity within the crustacean STNS which is unique for each species, and suggest that AST-like peptides function as circulating hormones and as modulators released by interneurons, by sensory neurons and, possibly, by motoneurons. Preliminary reports of some of these data have appeared in abstract form (Skiebe and Dietel, 1996; Skiebe, 1997). MATERIALS AND METHODS Animals Four species of adult decapod crustaceans of both sexes were used in this study: the crab Cancer pagurus (n ⫽ 7), the lobster Homarus americanus (n ⫽ 6), and the freshwater crayfish Cherax destructor (n ⫽ 10) and Procambarus clarkii (n ⫽ 10). C. pagurus were purchased from a marine biological station (Biologische Anstalt Helgoland, Germany), H. americanus were purchased from Lindenberg & Fig. 2. Wholemount immunocytochemical staining of the STNS of C. pagurus. A: The highest density of AST-immunoreactive (ASTir) cell bodies and neuropil is seen in the paired CoGs. There are ASTir fibers entering or leaving the CoG through the ion and son. In addition, ASTir fibers are present in the coc, which enter, exit, or bypass the CoG. B: The unpaired OG contains two large labeled cell bodies with processes projecting into the ivn. One pair of neurites enters the OG through each ion (asterisks) and both pairs leave the OG via the ivn. Another pair connects both CoGs (arrows). C: ASTir processes are present in the neuropil of the STG. Cell bodies are not labeled. A pair of descending neurites (asterisk) enters this ganglion via the stn and ramifies close to the ganglion and into the neuropil. Ascending axons (arrows) enter the STG via the dvn and leave via the stn. The area of entry into the STG via the dvn is magnified in the inset. Two normal and two weakly stained axons are marked by the arrows. These weakly stained fibers were only seen in one out of seven preparations. D: ASTir axons at the stn-son junction. A single, brightly stained axon is present in each son (asterisks) which continue into the stn. In addition to this pair of strongly stained axons, two weakly stained axons can be seen bifurcating into each son (arrows). The branching points of these weakly stained fibers are enlarged in the inset. E: ASTir axons at the dvn-lvn junction. A pair of stained axons are present in each lvn (arrows), which enters the dvn. One of the fibers seen in the lvn is weakly stained and was only seen in one out of seven preparations. F: ASTir cell body in the lvn, which due to its location and branching pattern is assumed to be a GPR neuron. Scale bars ⫽ 100 µm in A,C, 50 µm in B,E,F, 25 µm in D. 89 Co. GmbH (Berlin, Germany) and the crayfish C. destructor were purchased from Langbein & Co. GmbH (Hamburg, Germany). The crayfish P. clarkii were a gift from Dr. Barbara Schmitz (Technische Universität München, Germany). C. pagurus were maintained in aerated seawater, and C. destructor and P. clarkii in fresh water, at temperatures between 8 and 14°C. H. americanus were used on the day of purchase. All animals were anesthetized by packing in ice for 20–80 minutes, depending on size, prior to dissection. The stomach, including the STNS, was then removed from the animal. The rest of the dissection was performed in chilled physiological saline. Physiological salines Cancer saline (mM): NaCl, 440; MgCl2, 26; CaCl2, 13; KCl, 11; Trizma base 10; maleic acid, 5; pH 7.4–7.5 (Heinzel et al., 1993); Homarus saline (mM): NaCl, 462; MgCl2, 8; CaCl2, 26; KCl,16; Trizma base 10; maleic acid, 5; pH 7.4 (Marder et al., 1986); crayfish saline (mM): NaCl, 205; MgCl2, 2.6; CaCl2, 13.6; KCl, 0.54; HEPES 7.6; pH 7.4–7.5. (Van Harreveld, 1936). Wholemount immunocytochemistry Wholemount immunocytochemistry was performed by using standard techniques for this system (Beltz and Kravitz, 1983; Marder et al., 1986; Skiebe and Schneider, 1994). STNSs were fixed overnight in 4% paraformaldehyde in sodium phosphate buffer (PB, 0.1 M, pH 7.3–7.4). In the case of C. pagurus and H. americanus, 15% sucrose was added. Following fixation, the preparations were rinsed six times at 1-hour intervals in 0.1 M PB and then were incubated in PB containing 0.5% Triton X-100 (PB-X) and 10% normal goat serum (NGS) for 1 hour. Antisera against each of two principal ASTs, the tridecapeptide AST-1 (provided by Dr. Hans Agricola, Friedrich-SchillerUniversität, Jena, Germany), and the octadecapeptide AST-5 (provided by Dr. René Feyereisen, University of Arizona) were used. It has been shown that the antibody against AST-1 recognizes Diploptera AST-1 through 5, but it is almost two orders of magnitude more sensitive to AST-1 (Vitzthum et al., 1996), indicating that it recognizes the common C-terminus. The antibody against AST-1 was applied in a final dilution of 1:10,000 and the antibody against AST-5 in a final dilution of 1:500 in PB-X containing 10% NGS for 2 days. The AST-5 antibody served as a control and was only applied to one preparation per species, because no differences in the intensity and pattern of staining relative to the AST-1 antibody were able to be detected. Secondary anti-rabbit antibodies labeled with Cy3 (1:400, Dianova, Hamburg, Germany) were applied for 1 day in PB-X containing 10% NGS. After each incubation with antibody, preparations were rinsed six times at 1-hour intervals and finally were mounted in glycerol. The preparations were viewed by using a Leica microscope equipped for confocal microscopy (Leica TCS-4D with a krypton/argon laser). Computer images of the wholemounts were generated from optical sections (2 µm) by using software included with the microscope. The final figures of the micrographs were asssembled with Photoshop (version 2.5) and FreeHand (version 5.0.1) on a Macintosh computer and printed with an Epson Stylus Photo color inkjet printer. The staining of the preparations showed a wide range of intensities, and some lightly stained cell bodies and axons marked by arrows in the figures may not be well reproduced. 90 P. SKIEBE Preadsorption controls were performed by preincubating the primary antibody with 10⫺4 M AST-3 (Bachem Biochemical GmbH, Heidelberg, Germany) for 1 hour at room temperature before adding the combined solution to the nervous system and processing as described above. Lucifer yellow backfills Lucifer yellow (Sigma, Deisenhofen, Germany) backfills of the stomatogastric nerve (stn) were carried out by placing a vaseline well around it. After replacing the saline in the well with a 15% Lucifer yellow solution (diluted in distilled water), the stn within the well was cut and the preparation incubated at 4°C for 24 hours. Then the tissue was fixed in 4% paraformaldehyde and processed for wholemount immunocytochemistry. After 24 hours, the dye traveled to the junction of the stn and the superior esophageal nerve (son) and also into the STG, but did not reach the commissural ganglia or the esophageal ganglion. Backfills were performed on the stn of C. destructor (n ⫽ 3) and P. clarkii (n ⫽ 3) to verify which neurons send their axons into this nerve. RESULTS Figure 1A shows the location of the stomach with the STNS, the heart and one of the two PO within the thorax of C. destructor. A more detailed view of these organs is shown enlarged in Figure 1B. This schematic is also useful for the other three species used in this study, because the relationships of these structures to one another remains essentially the same. The STNS is composed of four ganglia together with connecting and motor nerves (Fig. 1B,C). The paired commissural ganglia (CoGs) lie between the brain and the subesophageal ganglion (SOG), which are interconnected by the circumesophageal connectives (coc). The two cocs are linked by the postesophageal commissure (poc). The esophageal ganglion (OG) is located between the CoGs and is connected with them by the inferior esophageal nerves (ions, Fig. 1C). The OG is also connected with the brain via the inferior ventricular nerve (ivn). The STG is connected with the CoGs by the superior esophageal nerves (sons) and the stomatogastric nerve (stn, Fig. 1B,C). The major nerve which branches from the son is the dorsal posterior esophageal nerve (d-pon, Fig. 1C) in crabs and lobsters, referred to as the anterior esophageal nerve in crayfish. The STG lies within the ophthalmic artery and substances released by the PO, a pair of neurohaemal structures located in the pericardial chamber, are pumped by the heart through the ophthalmic artery to the STG. Nearly all the motoneurons present in the STG send axons to the muscles of the forgut by way of the dorsal ventricular nerve (dvn), which branches into two lateral ventricular nerves (lvn, Fig. 1B,C), from which the lateral gastric nerve (lgn), the medial gastric nerve (mgn), the pyloric dilator nerve (pdn), and the pyloric nerve originate (pyn). The STG receives descending input from the paired CoGs by way of the ions and the sons, which converge at the stn, the sole input nerve to the STG (Coleman et al., 1992). The CoGs contain a few hundred neurons and are thought to be important centers for coordination of the foregut motor pattern (Selverston et al., 1976). Some of the neurons in the OG project to the STG through the esophageal nerve (on) and some project to the brain through the ivn. Because the AST antibodies stain a large number of axons connecting the ganglia of the STNS, the results will be presented separately for each species to simplify describing these interconnections. Cancer pagurus In the STNS of C. pagurus, staining for AST was found in all ganglia. In the CoGs, numerous cell bodies and densely stained neuropil were found (Fig. 2A). Immunostained fibers enter or leave the CoGs through the ions, the sons and through the coc. Within the coc, fibers can also be seen which bypass the CoG. In the d-pon and son are immunoreactive fibers connecting the d-pon with the CoG (not shown). In two out of seven preparations, immunoreactive cell bodies were stained which lie in the d-pon or in the son between the d-pon and the CoG. Two pairs of fibers were found in each ion (Fig. 2B), the polarity and origin of which could not be determined. One pair passes through the OG and connects the CoGs (Fig. 2B, arrows). The other pair from each ion projects into the ivn (Fig. 2B, asterisks), where they join the neurites of two large, monopolar cell bodies stained in the OG. These six fibers reach the brain via the ivn. No neuropil was labeled in the OG. In the STG, the neuropil was strongly stained, but no cell bodies were labeled (Fig. 2C). The neuropil staining can be attributed to two sources: a set of two fibers which descend from the CoGs through the son (Fig. 2D, asterisks) into the stn and branch at the entrance to the STG (Fig. 2C, asterisk), and two faintly stained fibers which enter the STG by the dvn (Fig. 2C, arrows). In one out of seven preparations, the two labeled fibers in the dvn were accompanied by two additional very faintly stained fibers (arrows in the inset of Fig. 2C). The faintly stained fibers in the dvn can be traced into the lvns (Fig. 2E, arrows). These faintly stained fibers originate from bipolar cell bodies located either in the lvn (n ⫽ 12, Fig. 2F) or in the mgn (n ⫽ 1). Due to both their morphology and their location, these cells are assumed to be the gastropyloric receptor neurons (GPRs, Katz et al., 1989). In a previous study, it was shown that the GPR cells in C. borealis (identified by serotonin immunoreactivity) contain AST-like peptides (Skiebe and Schneider, 1994). In the seven preparations, no more than one pair of GPR cell bodies was found: one pair was found in six preparations, and in one preparation, only a single GPR cell body was present. In one of the seven preparations, an additional axon next to a GPR cell body was stained, but for which no cell body could be found (Fig. 2F, arrows). The fibers of the GPR neurons run through the dvn, the STG, the stn, and branch at the stn-son junction into each son (Fig. 2D, arrows and inset). Figure 3 shows stained fibers connecting the CoGs with the SOG, the poc, and the brain. The fibers entering or leaving the CoG by way of the coc are schematically drawn in Figure 3A. These fibers run between the CoG and the SOG (marked with an arrow), the CoG and the brain (marked with an double arrow) or run between the CoG and the poc (marked with a asterisk). In addition to the fibers drawn in Figure 3A, other allatostatin-like immunoreactive (ASTir) fibers pass the CoG and connect the brain with the SOG (Fig. 3B, marked by two open arrows). Strong AST-like immunostaining is present in each PO. The surface of a PO is covered by strongly stained varicosities (Fig. 4A,C), whereas within a PO, the staining is not as dense and stained fibers are visible (Fig. 4B). AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS 91 Fig. 3. ASTir fibers in one of the circumesophageal connectives (coc). A: Drawing of ASTir fibers that enter or exit the CoGs. Fibers bypassing the CoGs were not drawn. The outline was drawn from a montage of eight confocal micrographs. Fibers connect the CoG with the brain (double-headed arrows), and the CoG with the SOG (arrow). The asterisk marks fibers running from the CoG through the poc. The box marks the area which is shown in the confocal micrograph in B, which also shows numerous ASTir fibers in the coc that bypass the CoG (outlined arrows). Scale bars ⫽ 250 µm in A, 100 µm in B. Fig. 4. AST-like staining in the pericardial organ of C. pagurus. A: A confocal micrograph of a part of a PO showing that its surface is covered by strongly immunoreactive varicosities. This micrograph is a composite of all 71 confocal sections separated by 2 µm. B: Composite micrograph of the same area produced by using only the inner confocal sections (22 to 57). Only stained fibers are seen within the PO, the strongly stained varicosities being confined to the surface. C: Enlargement of the framed area in A. Scale bars ⫽ 100 µm in A,B, 50 µm in C. Homarus americanus Staining was present in all four ganglia of the lobster STNS, but the pattern of staining for AST is much simpler than that found for C. pagurus. In the CoGs, numerous cell bodies and densely stained neuropil were found (Fig. 5A). Immunostained fibers enter or leave the CoGs by the ions, the sons, and the coc. In one out of six preparations, immunoreactive cell bodies were stained which lie in the son and project to the CoG (not shown). Some fibers in the coc connect the CoG with the SOG, the brain or the poc, whereas other coc fibers bypass the CoG and connect the brain with the SOG (not shown). In each ion, two pairs of fibers were found (Fig. 5B). One pair of axons in each ion (Fig. 5B, arrowheads) can be traced to the two large cell bodies stained in the OG, from which only one pair of fibers project into the ivn. The other neurite pair from each ion also joins the ivn (Fig. 5B, asterisks), connecting the brain with the CoGs. The staining of these fibers is much 92 P. SKIEBE Figure 5 AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS 93 Fig. 6. AST-like staining in the pericardial organ of H. americanus. A: A confocal micrograph of a part of a PO showing ASTir fibers and strongly stained varicosities on the surface. Montage of three confocal micrographs. B: Enlarged view of the framed area of A. Scale bars ⫽ 100 µm in A, 25 µm in B. stronger. All six fibers reach the brain via the ivn. In the OG, neuropil was not labeled. In the STG, the neuropil, but no cell bodies, was strongly stained (Fig. 5C). In the lobster, the source of this AST-like peptide within the STG seems to be exclusively the GPR neurons, because no descending fibers were found to be stained in the stn. This particular animal had seven stained GPR cell bodies and axons (see also Fig. 14). In general, the number of stained GPR cell bodies varied between six and eight in a given animal and are all located in the lvn (Fig. 5F, arrows). In Fig. 5. Wholemount immunocytochemical staining of the STNS of H. americanus. A: AST-like immunoreactivity in cell bodies and neuropil within the CoG. Fibers from the coc, the ion, and son enter or exit the ganglion. Montage assembled from four confocal micrographs. B: The unpaired OG contains two large labeled cell bodies with processes projecting into the ivn and both ions (arrowheads). Four additional fibers run in the ivn, two entering or exiting from each ion (asterisks). C: The STG shows densely stained neuropil but no stained cell bodies. A bundle of ASTir axons runs through the STG (arrows). Montage of two confocal micrographs. D: These ASTir axons from the STG bifurcate at the stn-son junction and due to their location and branching pattern are likely to be from the GPR neurons. E: These presumed GPR axons also bifurcate at the lvn-mgn junction. F: The cell bodies of GPR neurons are stained within the lvn. Scale bars ⫽ 200 µm in A, 100 µm in B,C,E, 25 µm in D, 50 µm in F. contrast to the staining in C. pagurus, the cell bodies and axons of the GPR neurons are strongly stained in H. americanus. The axons of the bipolar GPR cells enter posterior to the pyn and run anterior through the lvn. In the lvn, axons branch into the mgn (Fig. 5E). The axon of the GPR neurons from both sides join in the dvn, and run through the STG and stn. At the stn-son junction, the axons bifurcate into each son running further to the COGs (Fig. 5D). In H. americanus, strong ASTir varicosities and fibers are also present in the PO (Fig. 6A,B). Cherax destructor The STNS of C. destructor shows staining for AST in all ganglia. In the CoGs, numerous cell bodies and densely stained neuropil were found (Fig. 7A). Immunostained fibers enter or leave the CoGs through the ions and the sons. Within a coc, stained fibers connect the CoG with the SOG, the brain and the poc and additional fibers in the coc bypass the CoG (not shown). In each ion, six axons were found, four of them strongly stained and two of them weakly (Fig. 7B). The weakly stained axons belong to two weakly stained cell bodies within the OG and run within the ivn and both ions. The first branching point of these weakly stained axons are marked by arrowheads in Figure 94 P. SKIEBE Figure 7 AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS 95 Fig. 8. Wholemount of the STG and the son-stn junction following double-labeling by backfilling the stn with Lucifer yellow and subsequent AST immunocytochemistry in C. destructor. A: The ASTir cell body in the STG (A1, arrow) also shows Lucifer yellow staining (A2, arrow), suggesting that its axon projects into the stn. In addition, nine other cell bodies were labeled by the backfill including the AGR cell body in the dvn. Each image is a montage from three confocal micrographs. B: Lower magnification of the son-stn junction, which shows that the ASTir cell bodies in this junction (B1, asterisks) are also labeled by Lucifer yellow (B2, asterisks), indicating that their axons project into the stn. C: The double-labeling is more prominent at higher magnification (asterisks). Scale bars ⫽ 100 µm in A,B, 25 µm in C. 7B. The four strongly stained axons also run within the ivn and both ions (Fig. 7B, asterisks), connecting the brain with both CoGs. The polarity and origin of these four axons could not be determined. In the other five preparations, there was sparse neuropil-like staining in the OG. In the STG, the neuropil was strongly stained and one cell body was also labeled (Fig. 7C, thick arrow). This cell body appears to send its axon through the stn, bifurcating at the son-stn junction into both sons (Fig. 7D, thick arrow). In addition, one axon from each son runs in the stn (Fig. 7D, double-headed arrows) connecting the CoGs with the STG. Two pairs of bipolar neurons, assumed to be GPR neurons, were stained in C. destructor. Their cell bodies lie within the lvn (Fig. 7E, arrows) and their axons run through the lvn, the dvn, the STG (Fig. 7C, small arrows) and the stn, where they bifurcate into each ion (Fig. 7D, small arrow). In the stn-son junction, two cell bodies were stained (Fig. 7D, asterisk) that did not stain in either C. pagurus or H. americanus. These cells send their axons into the stn, but because they are very thin and faintly stained, it was not possible to trace them further. In the stn-son junction, neuropil-like structures were also ASTir (Fig. 7D, hollow ellipse). The structures were not only present at the stn-son junction, but also within the stn and sons, themselves. These structures were prominently stained in C. destructor, but were visible in the other three species as well. To confirm whether the ASTir neuron in the STG and the neurons within the stn-son junction send their axons into the stn, Lucifer yellow backfills of the stn were combined with wholemount immunocytochemistry in C. destructor. The ASTir neuron in the STG was double-labeled in all experiments (n ⫽ 3, Fig. 8A1,2 thick arrows), indicating that it has an axon in the stn. Within the STG, 10 (n ⫽ 1) or 11 (n ⫽ 2) cell bodies were labled by Lucifer yellow, although with different intensities. Among them was the anterior gastric receptor neuron (AGR), which was identified by its position and by the fact that it is bipolar (Fig. 8A2). The two cell bodies in the stn-son junction which exhibit AST-like immunoreactivity were also labeled in two of the three Lucifer yellow backfills of the stn (Fig. 8B,C, asterisks), although they have thin axons. Fig. 7. Wholemount immunocytochemical staining of the STNS of C. destructor. A: Within the COG, cell bodies and neuropil were stained. Fibers from both the ion and son enter or exit the ganglion. B: In the OG, two cell bodies are stained, which send their axons through the ivn and both ions. First branching points of the axons of these cells are marked with arrowheads. Four additional stained fibers run in the ivn, which interconnect the CoGs (asterisks). C: One stained cell body (large arrow) and densely stained neuropil are present in the STG. In addition, a fiber bundle presumably from the GPR cells enter the STG via the dvn and exit via the stn (small arrows). Montage of three confocal micrographs. D: Two cell bodies (asterisks) and axons are ASTir at the stn-son junction. One brightly stained axon bifurcates at the stn-son junction (thick arrow), probably originating from the stained cell in the STG. In addition, two other axons enter the stn, one from each son (double-headed arrows). The remaining fibers marked with the arrow are likely from the GPR neurons. In addition to cell bodies and axons, neuropil-like structures stained (hollow ellipses). E: C. destructor has two pairs of GPR-like cell bodies, located in the lvn. Scale bars ⫽ 100 µm in A,C,D, 50 µm in B,E. 96 P. SKIEBE Fig. 9. AST-like staining in the pericardial organ of C. destructor. A: A confocal micrograph of a PO showing ASTir fibers and strongly stained varicosities on the surface. Montage from six confocal micrographs. B: Magnified view of ASTir fibers within a PO within the framed area in the middle of A. C: Magnified view of ASTir varicosities within the area framed at the top of A. Scale bars ⫽ 100 µm in A, 50 µm in B,C. The surface of the PO in C. destructor is also covered with strongly stained varicosities (Fig. 9). In Figure 9B, some of the ASTir fibers are enlarged, and in Figure 9C, the varicosities are enlarged. Surprisingly, strongly stained varicose structures were also present on the surface of the coc and the poc in C. destructor (Fig. 10A) which were not seen in the other three species. The intensity of this staining was extremely strong, compared with the staining in other parts of the STNS such as the CoG seen in Figure 10C. Figure 10A shows a drawing of these structures on the surface of the coc and the poc, excluding stained axons, neuropil, and cell bodies to better show the web-like appearance of this structure. Especially on the surface of the poc, these structures look like a tightly woven web with swollen varicosites (Fig. 10B), sending out branches away from the middle of the poc. Within the poc, fibers were stained, some of them connecting the poc with both CoGs. This web-like structure extends over the surface of the coc close to the CoGs (Fig. 10C). The superficial location and the intensity and varicosal nature of the staining of these structures suggests that they are neurohemal. The fibers from the CoGs did not seem to be the source of the neurohemal-like structure because the fibers did not show any branching points. No cell bodies were found to be connected to this neurohemal-like structure. fibers enter or leave the CoGs through the ions and the sons. Within the coc, stained fibers connect the CoG with the SOG, the brain and the poc, and additional fibers bypass the CoG (not shown). In each ion, six axons were found (Fig. 11B). Two of the axons belong to two stained cell bodies within the OG and run within both ions and the ivn. The first branching point of these weakly stained axons are marked by arrowheads. The other four stained axons also run within the ivn and both ions, connecting the brain with both CoGs. The polarity and origin of these four axons were not determined. No neuropil was stained in the OG. In the STG, the neuropil and one cell body (Fig. 11C, thick arrow) were strongly stained. In addition to the one strongly stained cell body, two to five additional weakly stained cell bodies were present. In four out of eight STGs, a total of six cell bodies were stained (Fig. 11C); in one STG, five cell bodies were stained, and in the other three STGs, only three cells were marked. The axon of the strongly stained cell body appears to project through the stn, bifurcating at the son-stn junction into both sons. Two additional axons connect the CoGs with the STG: one axon in each ion runs in the stn (Fig. 11D, double-headed arrows). In only three of 10 preparations, weakly ASTir cell bodies were able to be detected within the son-stn junction (Fig. 11D, asterisks; this example represents the strongest staining obtained). The weakly stained cell bodies in the STG likely send their axons through the dvn, in which weakly stained axons are visible (Fig. 11E, arrows). These axons bifurcate into both lvns. In two out of ten preparations, four axons were weakly stained in the dvn; in four preparations, three Procambarus clarkii Staining for AST was found in all ganglia of the STNS of P. clarkii. In the CoGs, numerous cell bodies and densely stained neuropil were found (Fig. 11A). Immunostained AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS 97 Fig. 10. AST-like staining suggests that the surface of the connectives and of the postesophageal commissure are neurohemal in C. destructor. A: Drawing of strongly stained structures on the surface of the cocs and the poc. Drawn from a montage of 20 confocal micrographs. Other stained structures, including axons in the coc and cell bodies and neuropil in the CoG, were not drawn. B: AST-like staining on the surface of the poc. The picture is a composite of four confocal micrographs. C: Confocal micrograph showing the detailed structure on the surfaces of the connectives. Composite of four confocal micrographs. Scale bars ⫽ 500 µm in A, 200 µm in B,C. axons; in two preparations, two axons; in one preparation, one axon; and in another preparation, none. Six additional strongly stained axons were always present in the dvn, three of which running in each lvn (Fig. 11E) and later sending branches into the mgn. Based on this branching pattern, these axons likely belong to the GPR neurons. Although there were always three pairs of strongly labeled axons, there were never more than two pairs of stained bipolar cell bodies in the lvn (Fig. 11F), suggesting that one of the three pairs did not belong to GPR neurons, or that one pair of cell bodies was hard to locate, or that one pair of GPR cell bodies did not stain. These presumed axons of the GPR neurons run through the lvn, the dvn, the STG and the stn, where they bifurcate into each ion (Fig. 11D, small arrow). Additional axons were also labeled in the lvn, perhaps those of the weakly stained cell bodies in the STG. As in C. destructor, Lucifer yellow backfills of the stn were combined with wholemount immunocytochemistry to further examine the stained cells with axons in the stn. The strongly stained ASTir neuron in the STG was doublelabeled in all experiments (n ⫽ 3, Fig. 12A, thick arrow), strongly suggesting that it sends an axon into the stn. In addition, the AGR neuron was also double-labeled (doubleheaded arrow), identified by its position and by the fact that it is bipolar. In all backfill experiments, nine (n ⫽ 3) cell bodies were labeled by Lucifer yellow in the STG, although with different intensities. The two cell bodies in the stn-son junction which exhibit AST-like immunoreactivity were also labeled in two of the three Lucifer yellow backfills of the stn (Fig. 12B, asterisks), although they have thin axons. Similar to the other three species, immunostained fibers and strongly stained varicosities were present in the PO of P. clarkii (Fig. 13). DISCUSSION The results presented here demonstrate that AST-like peptides are present in both the PO and the STNS of the four different species of decapod crustaceans examined. Both AST-1 and AST-5 antisera used for these studies (antibodies from Agricola and Feyereisen) were found to stain the same structures with the same relative intensity in each species. This similarity of staining and the fact that the antibody against AST-1 recognizes Diploptera AST-1 to 5 (Vitzthum et al., 1996) indicate that both antibodies recognize the common C-teminal pentapeptide sequence -Y-X-F-G-L-NH2. Because the AST found in both in the crab C. maenas (Duve et al., 1997b) and in the crayfish O. limosus (Dircksen et al., 1998) also share this C-terminal sequence, it is likely that these antibodies bind 98 P. SKIEBE Figure 11 AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS to many if not all members of the crustacean AST peptide family. The different intensities of immunoreactivity found in this study could result both from differences in the amounts of peptide present in particular cells and/or in the different binding affinities of members of the peptide family with the antibody. That the distribution of AST could be complex is suggested by the recent work of Duve et al. (1997b) in the crab, C. maenas, where a family of at least 20 different AST peptides were isolated. Pericardial organs AST-like peptides are present in the PO of all four species of decapod crustacea investigated. This is not surprising, because many peptides have been localized in the PO (Dircksen et al., 1987; Dircksen, 1990; Christie et al., 1995a) and the rhythms of the STG can be influenced by injections of AST into the heart chamber (Heinzel et al., 1997). The presence of AST-like immunoreactivity within the PO suggests that AST also functions as a neurohormone in decapod crustacea. Putative neurohaemal organs on the surface of the connectives and the post oesophageal commissure of C. destructor A surprising finding was the presence of ASTir structures on the surfaces of coc and poc of C. destructor, because similar staining was not found in the other species investigated (Figs. 10, 14C, black). These web-like structures on the coc (Fig. 10C) and on the poc (Fig. 10B) of C. destructor are superficial and have varicosity-like swellings. Due to their superficial location and varicosal staining, these structures appear to be neurohemal, and electron microscopic experiments support this hypothesis (Skiebe et al., 1998). ASTir fibers running in the middle of the poc connecting the CoGs are present in all investigated species and might not therefore be the origin of the web-like structure. Fibers interconecting the CoGs via the poc have been earlier demonstrated by methylene blue staining for Penaeus braziliensis (Knowles, 1953) and by gamma aminobutyric acid (GABA) immunocytochemistry for the crayfish Pacifastacus leniusculus (Mulloney and Hall, 1990). Fig. 11. Wholemount immunocytochemical staining of the STNS of P. clarkii. A: Within the CoG, cell bodies and neuropil were stained. Fibers from the coc, the ion, and son enter or exit the ganglion. There are also ASTir fibers bypassing the CoG. B: In the OG, two cell bodies are stained, which send their axons through the ivn and both ions. The first branching point of the axons of these two cells is marked (arrowheads). Four additional fibers run in the ivn and also connect both COGs. C: The STG shows densely stained neuropil and one strongly stained cell body (thick arrow, see also Fig. 12A1) plus five additional faintly stained cell bodies (thin arrows). Composite of two confocal micrographs. D: In the son-stn junction are ASTir cell bodies stained in three out of 10 preparations. One fiber runs in each son between each of the CoGs and the STG (double-headed arrows). Putative GPR axons also bifurcate at the son-stn junction (small arrow). Two cell bodies are also stained (asterisks). E: Nine stained fibers are present in the dvn. Six of them are strongly stained and separate at dvn-lvn junction, three of them running in each lvn. These fibers presumably belong to the GPR cells, although only a maximum of two pairs of GPR cell bodies were found in a single preparation. The other three faintly stained fibers in the dvn (arrows) bifurcate at the dvn-lvn junction, all of them sending an axon into each lvn. F: A GPR cell body next to a bundle of axons within the lvn. Scale bars ⫽ 100 µm in A,C,D, 50 µm in B,E,F. 99 Neurohemal structures associated with the poc have been described by Knowles (1951) for the prawn P. braziliensis and named postcommissure organ. Postcommissure organs also have been described for the prawn Leander serratus (Knowles, 1953), the stomatopod Squilla mantis (Carlisle and Knowles, 1959), and in 12 brachyuran species including Cancer borealis (Maynard, 1961; Fingerman et al., 1965). In P. braziliensis, L. serratus, S. mantis, and the two fiddler crab species investigated by Fingerman et al. (1965), the postcommissure organs are located close to the poc and are connected to the poc by the a short postcommissure nerve (Knowles, 1953; Carlisle and Knowles, 1959). In the 10 brachyuran species investigated by Maynard (1961) though, they are located further apart, connected both to the poc by a longer postcommissure nerve and to the CoG by a nerve which also innervates esophageal muscles and joins the postcommissure nerve. Is the ASTir web-like structure a postcommissure organ? From its web-like structure it resembles very much the ‘‘meshwork of finely branched fibers many of which have a beaded appearance after methylene blue staining,’’ reported by Carlisle and Knowles (1959, p. 30 and plate V[b] p. 80). A postcommissure nerve or clear lamellae-like structure has not been found in C. destructor, although there was a flap connected with the poc into which some of the ASTir fibers could be seen to extend. I have examined only the STNS in this study and have not tried to locate the postcommissural organs in C. destructor or in the other investigated species. It is interesting to find a neurohemal structure so close to the STNS, especially because Carlisle and Knowles (1959) suggested that its innervation comes from the brain. The AST-like immunoreactivity within these structures further supports a neurohormonal role for AST, but more detailed studies are necessary. STNS In the STNS, a detailed comparison of the distribution of AST-like peptides reveals a number of distinct differences from species to species, as shown in the colored schema of Figure 14. In all four species, both neuropil and numerous cell bodies are stained in the CoGs (Fig. 14, black). Due to the density of staining in the CoGs, the branching patterns of single neurons were not able to be traced. It was therefore not possible to identify the cell bodies of CoG neurons which project to the STG in the crab and both crayfish. Even homologous neurons like the GPR cells and the pair of ASTir neurons in the OG show strong differences in the intensity of their immunoreactivity between species. Variations in staining intensity between different preparations of a species also occur, and make it difficult to obtain an exact count of stained cell bodies. Therefore, no attempt was made to count the stained cells in the CoGs or to homologize these neurons. All investigated species also have immunostained fibers which enter or leave the CoGs through the ions, the sons, and the coc (Fig. 14, black). All species have two cell bodies in the OG, although the exact branching pattern differs (Fig. 14, orange). In C. pagurus, the axons run exclusively in the ivn (Fig. 14A, orange), whereas in the other three species the cells have collaterals that run through both ions. 100 P. SKIEBE Fig. 12. Double-labeled whole mount of the STG and the son-stn junction following a Lucifer yellow backfill of the stn and subsequent AST-like immunocytochemistry in P. clarkii. A: The strong ASTir cell body in the STG (A1, arrow) as well as the ASTir AGR neuron (double-headed arrow) also show Lucifer yellow staining (A2), suggesting that both these neurons send their axons into the stn. In addition, eight other cell bodies were labeled by the backfill. Each image is a montage from three confocal micrographs. B: Higher magnification of the son-stn junction, showing that the ASTir cell bodies in this junction (B1, asterisks) are also labeled by Lucifer yellow (B2, asterisks), indicating that their axons project into the stn. Scale bars ⫽ 100 µm in A, 25 µm in B. GPR cells putative third pair is marked in light blue. Katz and Tazaki (1992), though, describe only two pairs of GPR cells in P. clarkii. This means that either the third pair of GPR cell bodies is hard to find or that the third pair of axons does not stem from GPR cells, but additional experiments are necessary to clarify this point. All GPR axons run through the lvn, dvn, STG into the stn, where they bifurcate into the sons and are strongly stained, with the exception of those of C. pagurus, where the axons are weakly stained. A relatively weak staining of these axons was also found for C. borealis (Skiebe and Schneider, 1994), suggesting a possible difference in the type or amount of AST-like peptides in the GPR cells of these related crabs compared to lobster and crayfish. All four species have ASTir GPR cells, although the number of stained cells and their intensity varies between species. A similar variation in number of stained cells and in cotransmitter content is known for other GPR cotransmitters (Katz, 1991; Katz and Tazaki, 1992). In C. pagurus, one pair of weakly ASTir GPR cells is definitely present (Fig. 14A, dark blue), whereas in C. borealis, two pairs of weakly staining GPR cell bodies were able to be found (Skiebe and Schneider, 1994). In C. pagurus, one of seven preparations had an additional pair of weakly stained axons, indicating that there might be an additional pair (Fig. 14A, light blue). In H. americanus, six to eight GPR cells are ASTir (Fig. 14B, dark blue), and in C. destructor, two pairs of GPR cells are clearly stained; three pairs of strongly stained axons are present in the dvn and lvn, but only two pairs of cell bodies (Fig. 14D, dark blue), suggesting two or three pairs of GPR cells. The ASTir neurons in the stn In both crayfish, two ASTir cell bodies were stained in the stn-son junction, which send an axon into the stn. In AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS 101 Fig. 13.AST-like staining in the pericardial organ of P. clarkii. A: A confocal microgragh of a PO showing ASTir fibers and strongly stained varicosities on the surface. The picture is a composite of six confocal micrographs. B: Magnification showing details of ASTir varicosities within the area framed in A. Scale bars ⫽ 100 µm in A, 50 µm in B. C. destructor, these cells were clearly and strongly stained; in P. clarkii the staining was weak. These cells also stain in P. clarkii (Tierney et al., 1997) and in C. destructor (Skiebe et al., 1998) with an antibody against FMRFamide. Cell bodies in the STG Surprisingly, ASTir cell bodies were also found in the STG of C. destructor and P. clarkii. To date, neuropeptideimmunoreactive cell bodies have only been found within the STG of the shrimp Palaemon serratus (Meyrand and Marder, 1991). Using two different antibodies against FMRFamide, Meyrand and Marder (1991) found one large and two small immunoreactive cell bodies in the STG. In P. serratus, two immunoreactive axons leave the STG through the dvn, one of which can be traced into each lvn and to the nerves innervating the cardiopyloric valve muscle 1, the dilator muscle of the pyloric chamber. This muscle is innervated by the pyloric dilator neurons, but their cell bodies do not show FMRFamide immunoreactivity, leaving the identity of those cell bodies undetermined. The presence of these immunoreactive cells, though, could relate to the simplicity of the stomach in P. serratus compared with the other decapod crustaceans. In P. serratus, for example, both the gastric teeth and their extrinsic muscles and the muscles and the neurons of the cardiopyloric valve are missing (Meyrand and Moulins, 1986, 1988). The STG, though, has nearly as many neurons (25) as C. borealis (25–26, Kilman and Marder, 1996), H. americanus (about 30, Maynard, 1971), Panulirus interrup- tus (27–32, King, 1976), and O. limosus (25–26, Eitner, 1997). Other studies investigating FMRFamide immunoreactivity in the STNS including C. borealis (Marder et al., 1987), P. interruptus (Marder et al., 1987), and the crayfish P. clarkii (Tierney et al., 1997) and C. destructor (Skiebe et al., 1998), do not find FMRFamide-immunoreactive cell bodies in the STG. The study investigating the distribution of AST within C. borealis does not describe ASTir cell bodies in the STG (Skiebe and Schneider, 1994). All the other immunocytochemical studies done on the STNS of decapods with various antibodies against peptides also found no evidence for peptidergic cell bodies within the STG (buccalin: Christie et al., 1994; cholecystokinin: Turrigiano and Selverston, 1991; Christie et al., 1995b; myomodulin: Christie et al., 1994; proctolin: Marder et al., 1994; red pigment-concentrating hormone: Nusbaum and Marder, 1988; tachykinin: Blitz et al., 1995) including crayfish (␤-pigment-dispersing hormone: Mortin and Marder, 1991). Backfills of the stn labeled nine to eleven cell bodies in the STG of crayfish, a number which is similar to the number of neurons backfilled in C. borealis (10, Goldberg et al., 1988; Coleman et al., 1992). Among these cells with axons in the stn are six identified neurons including interneurons (anterior burster neuron, interneuron 1), motoneurons (anterior median neuron, and in crayfish also the inferior cardiac neuron) and a sensory neuron (AGR; Claiborne and Ayers, 1987; Katz and Tazaki, 1992; Eitner, 102 Fig. 14. A–D: Schematic showing the distribution of AST-like immunoreactivity in the STNS of four different decapod crustaceans. Color code: black marks cell bodies, axons, and neuropil that is stained, but unidentified. Red marks axons projecting from the CoG to the STG. Orange marks cell bodies in the OG and their axons. Green P. SKIEBE marks strongly stained cell bodies in the STG and their axons. Light green marks weakly stained cell bodies in the STG and their axons. Blue marks GPR cell bodies and their axons. Light blue marks putative GPR cell bodies and their axons. Purple marks stained cell bodies in the stn and their axons. AST-LIKE IMMUNOREACTIVITY WITHIN THE STNS 1997; Weimann, 1992), leaving three to five unidentified neurons. Can the strongly ASTir cell bodies in the STG of C. destructor and P. clarkii be one of the identified neurons? The AGR neuron can be excluded, because its distinct bipolar cell body is located in the dvn close to the STG and it shows weak ASTir in P. clarkii. The anterior median neuron can also be excluded because its axons leaves the stn before the stn-son junction into the anterior median nerve, and no ASTir axons are present in this nerve. In crayfish, the cardiac dilator 2 and inferior cardiac neurons should have their axons in the posterior esopageal nerve, which does not show ASTir, leaving the anterior burster neuron and interneuron 1 as the most likely candidates. The size of this cell body would be consistent with either of these identified interneurons, but the other unidentified neurons cannot be excluded. These could include nonspiking STG neurons which are known to exist in C. borealis (Weimann, personal communication). To positively identify the strongly stained ASTir neuron, intracellular recording in combination with immunocytochemistry has to be done. In summary, H. americanus has the simplest staining pattern; the source of all AST-like immunoreactivity within the STG is the GPR cells (Fig. 14B, dark blue). The next simplest pattern was found in C. pagurus, in which the AST-like immunoreactivity within the STG comes from the GPR cells and two descending axons (Fig. 14A, dark blue and red). This pattern of staining in C. pagurus is identical to that previously reported for C. borealis (Skiebe and Schneider, 1994), with the exception of the GPR cells. In C. destructor and P. clarkii, the AST-like immunoreactivity within the STG comes from the GPR cells, two descending axons, and the ASTir cell or cells in the STG itself plus the two cells stained in the stn-son junction (Fig. 14C,D, dark blue, red, green and purple). This comparative study has revealed a number of important differences in the pattern of distribution of AST-like peptides in the STNS of different decapod crustaceans and should provide a basis for several useful preparations for further physiological experiments. The strongly stained GPR cells in H. americanus are the only source of AST-like immunoreactivity in the STG in this species and provide an opportunity to study AST in sensory neurons in the absence of other sources of AST. The role of AST in network activity can be examined by using the single ASTir cell in the STG of C. destructor. This neuron also has an ascending axon and could be involved in providing feedback to the CoGs. The two ASTir neurons in stn-son junction of the crayfish are also easily accessible, and it would also be interesting to study their function. ACKNOWLEGDMENTS Thanks to Dr. Hans Agricola (Jena, Germany) and Dr. René Feyereisen (Tucson, AZ) for providing the antibodies and Dr. Barbara Schmitz for supplying Procambarus clarkii. I also thank Dr. Brian J. Corrette and Heike Wolfenberg for technical support and Dr. Brian J. Corrette for reading the manuscript. My gratitude also to Dr. Hans-Joachim Pflüger and Dr. Randolf Menzel for their support. 103 LITERATURE CITED Abel B, Dircksen H, Agricola H. 1994. Allatostatin-immunreaktive Neuronensysteme im zentralen und periferen Nervensystem von Crustaceen. Verh Dtsch Zool Ges 87:3. Agricola H, Quillfeld P, Seyfarth E-A. 1995. Immunoreactivity to the neuropeptide allatostatin in the spider CNS. Proc 23rd Göttingen Neurobiology Conference. Stuttgart: Thieme, p. 617. Bellés X, Maestro J-L, Piulachs M-D, Johnson AH, Duve H. 1994. 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