Immunocytochemical Localization Of Multiple Cholecystokinin-Like Peptides In The Stomatogastric Nervous System Of The Crab Cancer Borealis

Over the past three decades, the stomatogastric nervous system (STNS) of decapod crustaceans has emerged as one of the leading systems for studying neuromodulation of small, rhythmically active neural circuits. This region of the crustacean nervous system consists of four ganglia and controls the rhythmic movement of food through the foregut. One of these ganglia, the stomatogastric ganglion (STG), contains the neural circuits responsible for controlling the movements of both the gastric mill and the pyloric valve. Each of these circuits is composed of about half of the 30 or so neurons contained within the STG. Numerous studies have shown that the motor patterns produced by the gastric mill and pyloric circuits are not hard-wired, but are capable of being modulated by a variety of neuroactive substances (Harris-Warrick and Marder, 1991; Harris-Warrick et al. 1992; Marder and Nusbaum, 1989; Marder and Weimann, 1992). These molecules are delivered to the ganglion by the hemolymph and local release from modulatory neurons. Biochemical and immunocytochemical studies have shown that small molecule transmitters and neuropeptides are present in the input fibers that project to the STG. Over 15 distinct neuroactive substances have been localized to these axons, including serotonin (Beltz et al. 1984; Katz and Harris-Warrick, 1989), octopamine (Barker et al. 1979), dopamine (Marder, 1987), histamine (Claiborne and Selverston, 1984) and GABA (Nusbaum et al. 1989), as well as proctolin (Marder et al. 1986), red pigment concentrating hormone (RPCH)-like (Nusbaum and Marder, 1988), FMRFamide-like (Marder et al. 1987; Callaway et al. 1987; Weimann et al. 1993), tachykinin-like (Goldberg et al. 1988; Christie et al. 1993; Blitz et al. 1994), β-pigment dispersing hormone-like (Mortin and Marder, 1991), buccalin-like (Christie et al. 1994), myomodulin-like (Christie et al. 1994) and allatostatin-like (Skiebe and Schneider, 1994) peptides.

Numerous studies have shown that the cholecystokinins (CCKs) are a diverse and widely distributed family of substances. CCK-like molecules have been identified in both vertebrate and invertebrate neural tissue (Dockary, 1976; Dockary et al. 1981, 1985; Favrel et al. 1987; Grimmelikhuijzen et al. 1980; Johnsen and Rehfeld, 1990; Larson and Vigna, 1983; Nachman et al. 1986; Nichols, 1992; Nichols et al. 1988; Vigna, 1985). Moreover, there is an extensive literature demonstrating that these molecules serve important roles as neurotransmitters and/or neuromodulators (Brooks and Kelley, 1985). In a recent series of reports, CCK-like peptides were shown to be present in, and powerful modulators of, the spiny lobster Panulirus interruptus STG (Turrigiano and Selverston, 1989, 1990, 1991; Turrigiano et al. 1994). In this paper, we show that CCK-like immunoreactivity is also present in the STNS of the crab Cancer borealis. Using laser scanning confocal microscopic techniques, we have examined the staining patterns produced by three different antibodies to CCK8, two monoclonal antibodies (P. Sithigorngul, C. Cowden and A. O. W. Stretton, in preparation) and one polyclonal antibody. While the vast majority of profiles label only with a single antibody, some appear to be recognized by both a monoclonal and the polyclonal antibody. These results indicate that a family of CCK-like peptides is distributed in a differential fashion throughout the STNS of C. borealis. Some of this work has appeared previously in abstract form (Christie et al. 1991, 1992).

Rock crabs, Cancer borealis Stimpson (N=91), were obtained from Neptune Lobster and Seafood Company, Boston, MA, USA, and maintained without food in aquaria containing artificial sea water at 10–12 °C.

CCK8SO₄ and proctolin were purchased from Sigma. RPCH was obtained from Peninsula Laboratories. TNRNFLRFamide was synthesized by James Weimann, Brandeis University.

Mouse anti-CCK monoclonal antibodies C36-9H and C37-4E were gifts from Dr Anthony Stretton (University of Wisconsin, Madison, USA). Both antibodies were generated from clones produced by immunizing mice with a mixture of CCK8/bovine serum albumin conjugate and CCK8/keyhole limpet hemocyanin conjugate. For each immunogen, two coupling agents, glutaraldehyde and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC), were used to cross-link the peptide to the carrier protein. The specificity of both antibodies for CCK-like molecules has been determined (P. Sithigorngul, C. Cowden and A. O. W. Stretton, in preparation). Antibody C36-9H shows a strong recognition of CCK8 but relatively low recognition of CCK4. Antibody C37-4E recognizes CCK8 and CCK4 equally. Both C36-9H and C37-4E were used at a final dilution of 1:300.

Antibody 243-4, a rabbit polyclonal antibody, was raised against a glutaraldehyde-cross-linked CCK8/bovine thyroglobulin conjugate (Turrigiano and Selverston, 1991). This serum shows strong recognition of a variety of CCK-like peptides (CCK8, caerulian peptide and gastrin). As with the two monoclonal antibodies, 243-4 was also used at a final dilution of 1:300.

Since the use of these three anti-CCK antibodies is novel in either decapod crustaceans (C36-9H and C37-4E) or C. borealis (243-4), preabsorption controls were conducted. In these experiments, each antibody was incubated with CCK8SO₄, proctolin, RPCH or TNRNFLRFamide for 2 h at room temperature prior to application to tissue. Incubation of each of the CCK antibodies with 10⁻⁷ mol l⁻¹ CCK8SO₄ completely abolished staining (N=4 preparations for each antibody), whereas preincubation with the same or higher concentrations of proctolin, RPCH or TNRNFLRFamide (10⁻⁷ to 10⁻⁴ mol l⁻¹) had no effect [N=4 for each antibody at each peptide concentration (10⁻⁷ mol l⁻¹, 10⁻⁶ mol l⁻¹, 10⁻⁵ mol l⁻¹ and 10⁻⁴ mol l⁻¹)].

Secondary antibodies were goat anti-mouse and goat anti-rabbit affinity-purified IgGs labeled either with FITC or with Rhodamine (Boehringer-Mannheim). All secondary antibodies were used at a final dilution of 1:25.

Prior to fixation and staining, the entire STNS was dissected from each animal (Selverston and Moulins, 1987). The STNS (Fig. 1) consists of the STG, the oesophageal ganglion (OG) and the paired commissural ganglia (CGs). There are 28–32 neurons in the STG, approximately 18 in the OG and approximately 400 in each CG (V. Kilman, unpublished results). A network of nerves connects the four ganglia to the musculature of the foregut.

Tissues were processed for immunocytochemistry as whole mounts using indirect immunofluorescence methods modified from Beltz and Kravitz (1983). The entire STNS was dissected in chilled (approximately 4 °C) physiological saline (440 mmol l⁻¹ NaCl; 11 mmol l⁻¹ KCl; 26 mmol l⁻¹ MgCl_2; 13 mmol l⁻¹ CaCl₂; 11 mmol l⁻¹ Trizma base; 5 mmol l⁻¹ maleic acid; pH 7.4–7.6), fixed overnight with 4 % paraformaldehyde in 0.1 mol l⁻¹ sodium phosphate buffer (pH 7.3–7.4) and subsequently rinsed six times over approximately 6 h in a solution of 0.1 mol l⁻¹ sodium phosphate (pH 7.2) containing 0.3 % Triton X-100 (P-Triton). Fixation, and all subsequent processing, was carried out at approximately 4 °C. The incubation in primary antibody (or antibodies in the case of double labels) was performed in P-Triton for 18–48 h (10 % goat normal serum was added to all reactions to reduce nonspecific binding). Tissues were again rinsed six times in P-Triton over approximately 6 h. Secondary antibody incubation was also carried out in P-Triton. For double-label experiments, a cocktail of goat anti-mouse and goat anti-rabbit IgG was employed. After incubation with secondary antibody (12–24 h), each preparation was rinsed six times at 1 h intervals in 0.1 mol l⁻¹ sodium phosphate buffer (pH 7.2). Following the final rinse, ganglia were mounted on glass coverslips using a solution of 80 % glycerine and 20 % 20 mmol l⁻¹ sodium carbonate, pH 9.5. Fragments of glass coverslips were placed near each preparation before covering to minimize compression of the tissue. All preparations were viewed with a Biorad MRC 600 laser scanning confocal microscope equipped with a krypton/argon mixed gas laser and the standard YHS [for Rhodamine (excitor filter, 568 nm DF10; dichroic reflector, 585 nm DRLP; emission filter, 585 nm EFLP)] or BHS [for FITC (excitor filter, 488 nm DF10; dichroic reflector, 510 nm LP; emission filter, 515 nm LP)] filter blocks provided by the manufacturer. For double-label preparations, the K1/K2 filter set (K1: 488 and 568 nm dual excitation, dual dichroic reflector; K2: dichroic, DR 560 nm LP; green emission filter, 522 nm DF35; red emission filter, 585 nm EFLP) supplied by the manufacturer was employed.

Comos (Biorad) and NIH Image (software for the Macintosh available via anonymous ftp from: zippy.nimh.nih.gov) were used for all measurements of cell number and diameter. Kaleidagraph software (Synergy Software) was used for all statistical computations. All measurements are given as the mean value ± one standard deviation. As all measurements are taken from fixed fluorescently labeled tissue, sizes are given only to permit comparison between the antibodies used and do not necessarily reflect the true size of the structures described.

Although none of the CCK antibodies used in this study labels any of the 28–32 neuronal somata found in the STG, each gives rise to intense labeling within the neuropil of the ganglion (Fig. 2). With each antibody (243-4, N=8 ganglia; C36-9H, N=6 ganglia; C37-4E, N=7 ganglia), immunoreactive fibers can be seen to descend through the stomatogastric nerve (stn) from the anteriorly located CGs and/or OG and to ramify within the STG, where they terminate. Since the fibers labeled by each of the antibodies generally travel as one or more tightly associated fascicles, it is difficult to determine accurately the number of fibers stained by each of the antibodies. However, in several preparations, antibodies 243-4 (N=4 of eight ganglia) and C36-9H (N=4 of six ganglia) can be seen to label at least four fibers, while only two fibers are routinely seen in C37-4E-labeled ganglia (N=5 of seven ganglia).

Within the STG neuropil, each of the patterns of labeling is composed of immunopositive neurites and varicosities. Striking differences are seen in the size of the neuropilar varicosities labeled by each of the antibodies. With C37-4E, measurements of the major cross-sectional diameter of sample populations of labeled profiles show that essentially all of the varicosities are less than 10 μm in diameter (hereafter referred to as ‘small’ profiles). Similar measurements taken from ganglia labeled with 243-4 and C36-4E show that, in addition to these small profiles, a population of varicosities greater than 10 μm in diameter (hereafter referred to as ‘large’ profiles), including a significant number of profiles greater than 15 μm, is also present. While the small profiles stained by all three antibodies are distributed throughout the entire peripheral neuropil, the large profiles are concentrated at the anterior end of this structure. This differential distribution is particularly evident in the C36-9H-labeled ganglion shown in Fig. 2B.

Approximately 40 of the 50 or so axons that project to the STG in C. borealis originate in the paired CGs (Coleman et al. 1992). Antibodies 243-4, C36-9H and C37-4E all label complements of neuronal somata and neuropil within the CGs (Fig. 3). 243-4-labeled ganglia (N=16) show 5–10 immunopositive somata (mean 7±1 somata) as well as an intensely stained neuropilar plexus (Fig. 3A,B). The labeled somata ranged in size from 12 to 76 μm in diameter (mean diameter 36±15 μm, N=115 somata). The staining pattern seen with C36-9H (N=12 ganglia) contains 2–6 labeled somata (mean 4±1 somata), which ranged in size from 14 to 59 μm (mean diameter 32±11 μm, N=45 somata) as well as an intensely labeled neuropil (Fig. 3C,D). 5–12 immunopositive somata, ranging in size from 13 to 52 μm (mean diameter 36±9 μm, N=103 somata) and a large neuropilar plexus are seen in C37-4E-labeled ganglia (N=14 ganglia; Fig. 3E,F).

With all anti-CCK labels, fibers can be seen to exit the CG by way of the superior oesophageal nerves (son), to pass through these nerves and to enter the STG via the stn. All anti-CCK labels also show immunopositive fibers in the inferior oesophageal nerves (ion). In preparations labeled with 243-4 and C37-4E, these fibers can be seen to project into the oesophageal nerve (on). No labeled fibers are seen in the on with C36-9H. All three of the CCK antibodies stain fibers in the circumoesophageal connectives (coc) that connect each CG with both the supraoesophageal ganglion (the ‘brain’) and the thoracic nervous system. A feature seen in 243-4- and C37-4E-labeled preparations, but absent in those stained with C36-9H, is fibers in the inferior ventricular nerve (ivn), a second connection between the STNS and the brain.

In addition to the STG inputs originating in the CGs, several OG neurons have been shown to project to the STG (Nusbaum and Marder, 1989; Coleman et al. 1992). While C36-9H labels no structures in the OG (N=6 ganglia), 243-4 (N=8 ganglia) and C37-4E (N=7 ganglia) each stain a pair of somata which range in diameter from 43 to 66 μm (mean diameter 56±6 μm, N=16 somata) and from 42 to 67 μm (mean diameter 56±8 μm, N=14 somata), respectively (Fig. 4). These somata send projections into the ivn and do not project to the STG.

The pattern of immunoreactivity seen with each of the three CCK antibodies in the C. borealis STNS is summarized schematically in Fig. 5.

To ascertain the extent to which the structures that are labeled by either of the monoclonal antibodies are contained within the set of structures stained by the polyclonal serum, double-label experiments were conducted. In these experiments, tissue was incubated with a primary antibody cocktail containing either 243-4 and C36-9H or 243-4 and C37-4E. These studies reveal that the majority of structures labeled by each monoclonal antobody are stained uniquely by that antibody. Likewise, most of the profiles labeled by 243-4 show no labeling with either of the monoclonal antibodies. As can be seen in Fig. 6A,B, several CG neurons do show co-localization of 243-4 and C36-9H immunoreactivities. In ganglia stained with 243-4/C36-9H cocktails (N=6), co-localization was present in 1–3 somata (mean 2±1 somata), which ranged in diameter from 21 to 66 μm (mean diameter 40±16 μm, N=12 somata) and were most commonly seen immediately adjacent to the son. Similarly, some of the CG neuropil and 1–2 fibers in each coc can be seen to contain both 243-4 and C36-9H immunoreactivities.

An identical situation exists in CGs stained using 243-4/C37-4E cocktails (Fig. 6C,D). In these ganglia (N=6), 2–4 somata (mean 3±1 somata), a subset of the neuropil and several coc fibers can be seen to exhibit both 243-4 and C36-9H immunoreactivities. Somata exhibiting this pattern of colocalization, as with those immunopositive for both 243-4 and C36-9H, range in diameter from 17 to 64 μm (mean diameter 39±11 μm) and are also found immediately adjacent to the son. In the OG (N=5 ganglia), 243-4 and C37-4E label the same pair of neurons (Fig. 6E,F).

Within the STG neuropil (N=5 ganglia), all of the stained varicosities greater than 10 μm in major cross-sectional diameter exhibit both 243-4 and C36-9H immunolabeling (Fig. 7A), as do some of the smaller profiles (Fig. 7B). All of the large varicosities can be seen to arise from two double-labeled input fibers that travel to the STG from the CGs via the son. No profiles within the STG neuropil (N=5 ganglia) show a colocalization of 243-4 and C37-4E immunoreactivities (Fig. 7C,D).

Fig. 8 summarizes in schematic form the distribution of structures that exhibit 243-4/C36-9H (Fig. 8A) and 243-4/C37-4E (Fig. 8B) colocalization.

Recent reports have shown that peptides similar to CCK are found in the STG and neurohemal organs of several crustacean species, including the spiny lobster P. interruptus and the American lobster H. americanus (Turrigiano and Selverston, 1989, 1990, 1991; Turrigiano et al. 1994). In P. interruptus, five distinct CCK-like moieties (peaks A–E) were isolated from the pericardial organs, with two additional species (peaks F–G) being found only in the STG. In the one species of crab investigated, Cancer antenarius, no CCK-like immunoreactivity was found in the STG; however, immunoreactivity was present in neurohemal structures (Turrigiano and Selverston, 1991). On the basis of these findings, it was postulated that, in the STG of lobsters, CCK-like peptides function as both neurohormones and local modulators, while in the crab, they serve only a neurohormonal role. Our findings suggest that, in at least C. borealis, a local modulatory function is also likely.

In studying the distribution of CCK-like peptides in the C. borealis STNS, we have employed three antibodies, each generated against mammalian CCK8. While all show strong and specific recognition for CCK-like peptides, each stains a unique set of structures within the STNS. The most likely explanation for these different patterns of distribution is that each antibody is recognizing a different CCK-like peptide(s) and that these distinct peptides are differentially distributed within the STNS. Using the same monoclonal antibodies employed in our study, P. Sithigorngul, C. Cowden and A. O. W. Stretton (in preparation) obtained similar results in the nematode Ascaris suum. As the presence of multiple CCK-like peptides has been documented in a number of organisms, it will be interesting to see whether the antibodies employed in our study can be used to elucidate differential distributions of CCK-like peptides in these other systems.

Families of related peptides have been described in many systems. Two recently documented examples are the buccalin and myomodulin families of peptides. Both the buccalins and the myomodulins were originally isolated from the accessory radula closer nerve–muscle system of the marine mollusc Aplysia californica (Cropper et al. 1987a,b, 1988; Miller et al. 1993a,b). Nineteen distinct buccalin-like peptides (buccalin A–S) and nine distinct myomodulin-like peptides (myomodulins A–I) are present in A. californica (Miller et al. 1993a,b). In the C. borealis STNS, two FMRFamide-like peptides, TNRNFLRFamide and SDRNFLRFamide, have recently been biochemically isolated (Weimann et al. 1993).

The detection of a family of differentially distributed CCK-like peptides raises the question of the individual function of each of the related molecules. While the differential distribution of the CCK-like peptides points towards the possibility that each peptide elicits a distinct repertoire of behavioral outputs, it is possible that some or most of the molecules share a common function. All physiological studies of CCK-like peptide modulation of the STG have been carried out on the spiny lobster P. interruptus. Physiological application of one of the partially purified P. interruptus peptides, peak E, to the STG of this animal has shown that this native crustacean peptide is a more powerful modulator of the STG than is CCK8SO₄ itself (Turrigiano et al. 1994). The physiological actions of the other partially purified P. interruptus peaks have yet to be determined. A series of experiments designed to determine the actions of CCK-like molecules on the STG motor patterns of C. borealis is planned, as are the isolation and characterization of the native CCK-like peptides from this animal.

We thank Dr A. Stretton (University of Wisconsin, Madison) for the anti-CCK monoclonal antibodies C36-9H and C37-4E and Ms Simona Retter for her participation in some preliminary experiments using the monoclonal anti-CCK antibodies. Research supported by NS 17813 (E.M.), NIH Shared Instrument Grant RR05615 (Brandeis University), NS15697 (K.G.) and a grant from the Human Frontiers Science Program Organization. E.M. gratefully acknowledges the support of the W. M. Keck Foundation.