THE JOURNAL OF COMPARATIVE NEUROLOGY 305164-176 (1991)

Distribution of Cholecystokinin-Like Immunoreactivity Within the Stomatogastric Nervous Systems of Four Species of Decapod Crustacea G.G. TURRIGIANO AND A.I. SELVERSTON Department of Biology, Brandeis University, Waltham, Massachusetts 02254 (G.G.T.) and Department of Biology, University of California at San Diego, La Jolla, California 92093 (A.L.S.)

ABSTRACT The distribution of cholecystokinin-like immunoreactivity was studied in the stomatogastric nervous systems, pericardial organs, and haemolymph of four species of decapod crustacea, by using immunocytochemical and radioimmunoassay techniques. Whereas cholecystokininlike immunoreactivity was found within the stomatogastric nervous systems of all four species, its distribution in each is unique. Two species (Panulirus interruptus and Homarus americanus) have cholecystokinin-like immunoreactivity within fibers and neuropil of the stomatogastric ganglion (STG); two other species (Cancer antenarius and Procambarus clarkii) do not. Further, the cholecystokinin-like immunoreactivity within the STGs of Panulirus and Homarus arise from distinct structures; from a projection of anterior ganglia in Panulirus, and from somata within the posterior motor nerves in Homarus. The staining in the other ganglia of the stomatogastric nervous system also shows some interspecies variability, although it appears to be more highly conserved than staining within the STG. These differences in staining were confirmed by measuring the amount of CCK-like peptide present in tissue extracts of ganglia by radioimmunoassay . In contrast to the variable staining within the STG, all four species have cholecystokininlike immunoreactivity within the neurosecretory pericardial organs and thoracic segmental nerves. This cholecystokinin-like immunoreactivity is contained within fibers and within varicosities that coat the surface of these structures. The location of this staining and the presence of detectible levels of CCK-like peptide in the haemolymph suggests that CCK-like peptides in decapod crustacea may be utilized as neurohormones. Key words: hormone, peptide, neuromodulator, immunocytochemistry

A large body of work attests to the important role of peptides in shaping nervous system activity (Jan et al., '83; O'Shea and SchaEer '85). An important step in determining the functional role of a peptide is to examine its anatomical and phylogenetic distribution. Immunocytochemical techniques have been used extensively to localize peptides to specific neurons and neural circuits and to identify structurally related peptides in a number of different species. In this report we describe the distributionof cholecystokinin (CCK)like immunoreactivity within the stomatogastric nervous systems of four species of decapod crustacea. The stomatogastric nervous system of decapod crustacea is a model system that has been used extensively to explore the roles of monoamines and neuropeptides in modulating the activity of neural circuits (Marder, '87; Harris-Warrick, '88). The stomatogastric ganglion (STG) contains two O

1991 WILEY-LISS, INC.

central pattern generators (CPGs) that control rhythmic movements of the gut, the gastric mill CPG, and the pyloric CPG (Selverston and Moulins, '87). The output of these simple circuits is not "hard wired," but is highly dependent upon the chemical milieu. A number of peptides and monoamines can profoundly alter cellular properties of these circuits, thereby changing the rhythmic motor program they produce (Flamm and Harris-Warrick, '86; Hooper and Marder, '87; Heinzel and Selverston, '88; Turrigiano and Selverston, '89a). Whereas some of these substances are contained within modulatory neurons that project directly to the STG (Katz and Harris-Warrick, '86, '89; Accepted October 25, 1990. Address reprint requests to Dr. G.G. Turrigiano, c/o Dr. E. Marder, Dept. of Biology, Brandeis University, Waltham, MA 02254.

CCK-LIKE IMMUNOREACTIVITY IN CRUSTACEA Dickinson et al., '88; Nusbaum and Marder, '89a,b), others may be released into the general circulation, where the neurons of the STG are one of several potential targets (Beltz and Kravitz, '83; Schwartz et al., '84; Kobierski et al., '87; Turrigiano and Selverston, '90). Cholecystokinin is one of a family of peptides, including the gastrins and caerulein peptide, that is broadly distributed both anatomically and phylogenetically. Originally isolated from mammalian gut, these peptides function as hormones that control such processes as gut motility and secretion and are also thought to contribute to certain motivational states such as satiety (Baile and Della-Ferra, '85; Smith and Gibbs, '85). The distribution of CCK-like immunoreactivity (CCKLI) throughout the mammalian cortex and the ability of CCK to alter the activity of some CNS neurons suggest that CCK serves an important transmitter or neuromodulatory function in addition to its hormonal roles (Dockray, '76,'78; Brooks and Kelly, '85; Kritzer et al., '87; Skirboll et al., '81). Immunocytochemistry has shown that CCK-like peptides have a broad phylogenetic distribution, having been localized within gut tissue and/or nervous systems of most vertebrates and invertebrates examined (Dockray, '76,'78; Dockray et d . , '81; Osborne et al., '81; Larson and Vigna, '83a,b; Scalise et al., '84; Vigna, '85; Favrel et al., '87). Several invertebrate CCK-like peptides have now been isolated and sequenced, including leucosulfakinin (LSK) (Nachman et al., '86) and drosulfakinin (DSK) (Nichols et al., '88). In previous work we have shown that CCK-like peptides play an important role in the modulation of the STG of the spiny lobster, Panulirus interruptus. In this species a CCK-like peptide is contained within input fibers to the STG and exogenously applied CCK can modulate the output of the gastric mill (Turrigiano and Selverston, '89a). A CCK-like peptide also plays a hormonal role in the control of the gastric mill; this peptide is released into the haemolymph following feeding, where it appears to be necessary for gastric mill activation (Turrigiano and Selverston, '90). The goal of the present study was to determine whether this peptide has a conserved distribution in the stomatogastric nervous systems, neuroendocrine organs, and haemolymph of several species of decapod crustacea. To

Abbreviations

CCK CCKE CCK8S0, CCKLl CG COC DSK FMRFamide

LVN ION IVN NGS n2 OG

PBS RIA

RPCH sn SOG SON STG STN T TX

cholecystokinin cholecystokininmolar equivalents cholecystokinin-8-sulfate chokystokinin-like immunoreactivity commissural ganglion circumesophageal connective drosulfakinin phe-met-arg-phe-amide lateral ventricular nerve inferior esophageal nerve inferior ventricular nerve normal goat serum second root of thoracic ganglion esophageal ganglion phosphate-buffered saline radioimmunoassay red pigment concentrating hormone sensory nerve subesophageal ganglion superior esophageal nerve stomatogastric ganglion stomatogastric nerve thoracic ganglion triton X 100

165 this end, we have used immunocytochemical and radioimmunoassay (RIA) techniques to compare the distribution of CCKLI in the nervous systems of the lobsters Panulirus interruptus and Homarus americanus, the crab Cancer antennarius, and the crayfish Procambarus clarkii. These species were chosen because of the wealth of information on the distribution and activity of several other peptides within their stomatogastric nervous systems. The results presented here demonstrate that CCK-like peptides do not have a phylogenetically conserved distribution in the decapod crustacean STG. Rather, of the four species examined, only two have CCKLI within the neuropil and input fibers of the STG, and of these two, the origins of the staining are different. In contrast, all four species have CCKLJ within neurohaemal organs and detectable levels of CCKLI in the haemolymph, suggesting that one of the functions of CCK-like peptides in decapod crustacea is as a circulating hormone.

MATERIALS AND METHODS Animals Specimens of the California spiny lobster, Panulirus interruptus, were obtained from a local fisherman and kept without food in a running seawater aquarium at 13-15°C until use. Adult Homarus americanus were purchased from local markets and were used immediately; juvenile Homarus were a gift from Ernest Chang at U.C. Davis Bodega Marine Labs. Cancer antennarius were obtained from Marinus and kept without food in a running seawater aquarium. Procambarus clarkii were obtained from College Biological and maintained in freshwater aquaria with free access to food for up to several months. For all species, specimens of both sexes were used.

Peptides and reagents Reagents were obtained from the following sources. CCK8S04,.proctolin, FMRFamide, and activated charcoal were obtsuned from Sigma. RPCH was obtained from Peninsula. Crustacean FMRFamide-like peptide was a kind gift from E. Marder and J. Weimann. DSK was a kind gift from R. Nichols. CCK8S0,1'25, labelled on the n-terminal aspartate by the Bolton-Hunter technique and purified to > 97% by HPLC, was obtained from New England Nuclear Corp.

Antisera Rabbit CCWGastrin antiserum raised against CCK8 conjugated to keyhole limpet hemocyanin (antiserum A) was obtained from Incstar. Rabbit CCWgastrin antiserum C was raised against CCK8 conjugated to bovine thyroglobulin (Turrigiano and Selverston, '89a). An earlier bleed of this antiserum (243-2) was used for immunocytochemistry, and a later bleed (243-4) with the same specificity but a higher titre was used for radioimmunoassay (see below). The cross-reactivities of these antisera with a number of peptides have been determined (Turrigiano and Selverston, '89a): these antisera are specific for peptides that share a terminal sequence with CCK. The secondary antiserum (goat anti-rabbit IgG conjugated to rhodamine) was obtained from Jackson, Inc.

Saline compositions Panulirus and Cancer saline (saline composition in mM): NaCl, 479.0; KC1, 12.7; CaCl,, 13.7; MgSO,, 10.0; N&SO,,

G.G. TURRIGIANO AND A.L. SELVERSTON

166 3.9; glucose, 2.0; Hepes, 5.0; Tes, 5.0; pH, 7.4. Homarus saline: NaC1, 462.0; KC1, 16.0; CaC12, 26.0; MgC12, 8.0; glucose, 11.1; Tris base, 10.0; malaic acid, 5.0; pH 7.4. Procambarus saline: NaCl, 210.1; KC1, 5.3; MgCl2, 2.5; CaC12, 14.0; Tris base, 10.0; Malaic acid, 3.9.

Immunocytochemistry Tissues were processed as wholemounts for immunocytochemistry using indirect immunofluorescence, in a modification of the procedure of Beltz and Kravitz ('83). Dissections were carried out in chilled saline. At various times the complete stomatogastric nervous systems, brains and optic lobes, the subesophageal and thoracic ganglia (complete with associated roots), the abdominal ganglia, and the pericardial organs were removed. All ganglia were desheathed, except for those of juvenile Homarus. Panulirus. The tissue was fixed for 16 to 20 hours in 4% paraformaldehyde. The tissue was washed for 6 hours in 0.1 M phosphate buffer with 0.9%NaCl at pH 7.4 (PBS) with 5% normal goat serum (NGS) and 1% Triton X-100 (TX),and then incubated for 16-20 hours in antiserum A or antiserum C. Antiserum A or C was diluted 1 9 0 0 in PBS; for antiserum A 3.3 mg/ml Bovine serum albumen was added, and for antiserum C 3.3 mg/ml bovine thyroglobulin was added. Both primary antisera produced identical staining patterns. The tissue was washed for 6 hours (PBS, 5% NGS), and then incubated with goat antirabbit IgG conjugated to rhodamine, diluted 1:200 (PBS, 5% NGS) for 16-20 hours. The tissue was washed in PBS, cleared, and mounted in buffered glycerol and visualized with a Zeiss fluorescent microscope using rhodamine filters. Homarus, Cancer, and Procambarus. The staining procedure for the other species was identical to that for Panulirus, except that optimal detergent levels for each species were determined. For Homarus, levels were lowered to 0.5%; for Cancer and Procambarus, levels were lowered to 0.3%.Higher detergent levels in these species produced no difference in staining pattern but resulted in worse tissue preservation. There were no differences in staining between juveniles and adults for any of the species examined, and both antisera used produced identical staining patterns. All structures described as displaying CCKLI were observed to stain in at least 80% of the preparations. Structures described as not staining were never seen to stain in more than 20 preparations. For each species, preabsorption controls were performed in which the primary antisera were incubated with CCK M), or proctolin M), FMRFamide M) prior to incubation with the tissue. Data were obtained from more than 20 members of each species.

Haemolymph and tissue extracts One ml samples of haemolymph were withdrawn from either the ventral artery or the dorsal heart sinus directly into a syringe containing 2 ml ethanol; the samples were vortexed and then microfuged for 10 minutes. The supernatants were drawn off the pellet, dried in a speed vac concentrator, and stored a t - 70"until use. The stomatogastric nervous systems were dissected out in ice cold saline, and the STGs, OGs, and CGs frozen immediately on dry ice and stored at -- 70" until use. Tissue samples were extracted two times in methanol, and once with 0.5 M acetic acid, with sonication to disrupt the tissue. Extracts were microfuged and the supernatants from the three extractions

pooled, died in a speed vac concentrator, and stored at - 70" until use. Just prior to assay, the samples were reconstituted in 500 p1 of RIA assay buffer with sonication, and serial dilutions assayed in RIA as outlined below. Recovery for this procedure was estimated by adding CCKIlZ5to haemolymph and tissue samples and treating as above; recovery was approximately 80%.

Radioimmunoassay Antiserum C (243-4) was diluted 1:62,000 into an RIA buffer of 50 mM sodium acetate, 0.2% gelatin, and 10 mM EDTA. Fifty pl of antiserum was incubate overnight with sample and tracer (3,000-6,000 cpm CCKIlZ5),in a final volume of 400 p1, to give a final antiserum dilution of 1:500,000. Bound tracer was separated from free tracer using dextran-coated charcoal, and the pellet and supernatant fractions were counted separately using a Tracor Analytic gamma counter. Blanks (no peptide and no antibody) ran at about 3%, and zeros (no peptide) ran from 50-60%. For each assay a standard curve was constructed using CCK8, in the range of 0 to 20 fm; all points were run in duplicate. As little as 0.2 fm of CCK could be routinely detected in this assay. The amount of CCK-like material in the tissue extracts was estimated by determining the amount of CCK needed to produce the same inhibition of binding; values are therefore expressed as CCK molar equivalents (CCKE). Antiserum C recognizes peptides that share a terminal amino acid sequence with CCK (gastrin and caerulein peptide) equally well, but several unrelated peptides (FMRFamide, proctolin, met-enkephelin, substance P, and RPCH) produced no inhibition of binding in the range of 1to 3,000 fmoles, and crustacean FMRFamidelike peptides produces no inhibition of binding at up to 1 PM.

RESULTS There are major differences, and some similarities, in the distribution of CCKLI in the stomatogastric nervous systems of the four species of decapods examined. The distribution of staining in each structure in the stomatogastric nervous system is described in turn. The stomatogastric nervous system is composed of four ganglia and their connecting nerves (Fig. 1A). The STG contains about 30 neurons that are organized into the pyloric and gastric mill CPGs. These neurons send axons to the muscles of the foregut by way of the dorsal ventricular nerve (dvn), which branches to produce the two lateral ventricular nerves (Ivns). The STG receives descending input from the paired commissural ganglia (CGs) by way of the superior esophageal nerves (sons) and the inferior esophageal nerves (ions),which converge at the stomatogastric nerve (stn), the sole input nerve to the STG. The CGs contain about 300 neurons each. The esophageal ganglion (OG) contains only about 15 neurons, some of which project to the STG through the esophageal nerve (on) and the stn.

Stomatogastric ganglion There are striking differences in the pattern of CCKLI within the STG of the four species of decapods examined. As shown previously, in the lobster Panulirus interruptus there are fibers that descend down the stn from anterior ganglia and send out branches that ramify within the STG,

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B

A

COC

+

STG'

bsn4

-c Fig. 1. Schematic representation of the nervous system of decapod crustacea. A. Stomatogastric nervous system. The motoneurons of the STG send axons to the muscles of the digestive system by way of the paired LVNs. The STG receives input from the OG and the CGs by way of the IONS and the SONS, which converge at the STN, the sole input nerve to the STG. The IVN projects from the OG to the brain. The CGs are attached to the rest of the nervous system by the COCs that project to the brain in one direction and to the subesophageal ganglion in the

other. B. The stomatogastric nervous system is connected to the SOG by the COC. The proximal regions of the sns of the SOG and the n2 of the T are neurosecretory structures; these nerves project into the dorsal heart sinus, where they thicken into the pericardial organs, also neurosecretory structures. Anatomy differs somewhat from species to species; notably, in Cancer the subesophageal and thoracic ganglia are fused into the thoracic mass. Not drawn to scale.

where they terminate (Turrigiano and Selverston, '89a). In contrast, the crab Cancer antennarius has no detectable CCKLJ within the STG. Occasionally, very faint staining could be achieved (Fig. 2A), but this may represent weak cross reactivity of the primary antiserum with some other peptide. Like Cancer, the STG of the crayfish Procambarus clarkii displays only occasional, very faint staining with these antisera. There are fibers immunopositive for CCK within the stn, but the neuropil within the ganglion does not stain. The STG of Homarus americanus exhibits yet a third staining pattern. Like Panulirus, the STG of Homarus contains a dense plexus of immunopositive fibers and neuropil (Fig. 2B), but unlike Panulirus, the somata responsible for this projection reside not in anterior ganglia,but in the motor nerves. There are four bipolar somata in each branch of the lvn (Fig. 3A,B), each of which sends an axon anteriorly, where they pass through the STG and continue up the stn. At the stnlson junction a bundle of axons can be seen traveling in each son; as eight fibers can be counted in

the stn and in each son, it is likely that each fiber bifurcates at the stnlson junction and sends one process in each son (Fig. 3C). These axons then ramify within the CGs, where they terminate (Fig. 3D). All CCKLI in the Homarus STG appears to arise from these cells, as only eight immunoreactive fibers were observed in the stn.

Esophageal ganglion The OGs of Cancer, Homarus, and Procambarus all contain two somata that are immunopositive for CCK (Fig. 4A,B).In all three species, these somata send processes out the inferior ventricular nerve (ivn), that travels to the brain. Lucifer yellow backfills of the OG from the ivn confirm that only two somata in the OG have processes in the ivn, and double-labels of these fills show that these are the same cells that are immunoreactive to CCK (data not shown). These cells also have processes in the ions. In Panulirus, there are no somata in the OG that display CCKLI. In Procambarus, there are fibers that travel be-

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Fig. 2. CCKLI in the STGs of Cancer and Homarus. A. Wholemount preparation of the STG from Cancer antennarius. The STG of Cancer does not display CCKLI. Occasionally very faint staining was achieved, as shown here; this staining may reflect very low levels of

CCKLI, or weak cross reactivity with some other peptide. B. Wholemount preparation of the STG from Homarus americanus. The STG contains numerous immunoreactive fibers, terminals, and neuropil; none ofthe somata within the ganglion stain. Scale bar = 100 pm.

Fig. 3. Origin of the CCKLI in the STG of Homarus. A and B. There are four bipolar somata that lie within each LVN, and send one process each to the STG, where they branch profusely (see Fig. 2B). C. At the STNiSONjunction the bundle of axons in the STN appears to bifurcate (arrow).D. The fibers terminate within the CGs. Scale bar = 50 pm.

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Fig. 4. CCKLI within the OG. A. Two immunoreactive somata within the OG ofl'rocambarua B. Two immunoreactivesomata within the OG of Cancer. Scale bar = 50 cLm.

tween the CGs and the OG in the ions and the sons, and two fibers can be seen traveling out a motor branch of the ions toward esophageal muscles. The origin and termination of these fibers was not determined.

Commissural ganglia Unlike the staining within the STG, the pattern of CCKLI in the CGs of the four decapods examined show a number of similarities. In Cancer, there is one brightly stained neuron in each CG, of about 30 pm in diameter, that sends an axon out the ganglion and up the circumesophageal connective (coc), as well as several smaller, more weakly stained somata (Fig. 5A). The brightly stained neuron has numerous branches within the CG, that may represent its dendritic arborization (Fig. 5B). The axon of this neuron can be traced all the way to the thoracic mass, close to the first sn, where it produces a number of immunopositive varicosities (Fig. 5C). It was not possible to trace this axon any farther. This cell resembles a neuron described in the Panulirus CG in size and position; the axon of this neuron in Panulirus has an initial trajectory similar to the cell in Cancer, but could not be traced as far (Turrigiano and Selverston, '89a). In Procambarus there is one large somata and several smaller somata in a similar position within the CGs. No such somata were observed in Homarus. In Cancer and Homarus there are two to three immunoreactive somata that lie close to the sons; in Panulirus somata in a similar position were occasionally observed. In Procambarus, there is one large immunoreactive somata that lies close to the ions. A feature of the CCKLJ in the CGs that is similar in the four species of crustacea examined is a projection from the thoracic side of the COC. In Cancer a fiber can be seen entering the CG from the thoracic side of the COC (Fig. 1B) and ramifying into a number of processes. These produce a dense neuropilar plexus that extends throughout much of

the ganglion (Fig. 6A). A similar projection can be detected in Panulirus, Homarus, and Procambarus (Fig. 6B,C). The source of this projection has not been determined, In Cancer, there is a similar projection from the brain side of the COC, that produces a smaller region of immunoreactive neuropil. The other species examined also had an additional neuropilar region within the CGs, but the source was not determined. In all species examined there are several fibers that travel in the COC between the brain and the subesophageal ganglion and thoracic ganglia. In addition, in each species there are numerous immunopositive somata and neuropilar regions within the brain and the optic lobes. There are 6-10 immunoreactive fibers in the optic peduncles that enter the brain, but could not be followed to their point of termination. Homarus, Procambarus, and Panulirus have 30 or more somata in the optic lobes that display CCKLI and several regions of immunoreactive neuropil. The optic lobes of Cancer were not examined. The precise distribution of somata and neuropil displaying CCKLJ in the brain and optic lobes was not determined. The staining pattern within the stomatogastric nervous systems of the four crustacea examined is illustrated in Figure 7.

Segmental nerves and pericardial organs The segmental nerves (sns) of the subesophageal ganglia and the thoracic second roots and the homologous region of the thoracic mass in Cancer project dorsally into the pericardial chamber (Fig. 1B). There they thicken into the pericardial organs, or pericardial plexus. Both the proximal region of the sns and the pericardial organs themselves are well-known neurosecretory structures that are thought to release neurohormones directly into the haemolymph around the heart. From the pericardial chamber hormones would be drawn into the heart during diastole and from there would be pumped throughout the animal. The sto-

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~

Fig. 5. CCKLI within the CG of Cancer. A. There is one strongly immunoreactive cell in each CG of Cancer, that sends a process out the CG and up the COC toward the thoracic mass. A smaller somata next to this cell also stains (small arrow), as do three small cells close to the SON (large arrow). B. The brightly stained CG cell has a number of

~

-

.

.

dendritic branches within the neuropil of the CG. C. The process of'this cell can be traced through the COC to the thoracic mass, where it produces a number of immunopositive varicosities. A, 100 pm; 15, 50 pm; C, 20 pm.

Fig. 6. Neuropil displaying CCKLI within the CGs. A. Cancer. B. Procambarus. C. Homarus. In all three cases an immunoreactive fiber travels down the COC from the direction of the thoracic ganglia, and terminates within the CGs. Scale = 50 wm.

matogastric ganglion sits within an artery just anterior to the heart, where it is in an excellent position to respond to factors released by the pericardial organs. We therefore wished to determine if the segmental nerves and pericardial organs display CCKLI.

In all four species, the sns display intense CCKLI. In Procambarus, several fibers can be seen traveling out the sns, and the perimeter of the roots are covered with dense imrnunoreactive varicosities (Fig. 8A). The somata responsible for this staining have not been localized. There are

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A. Panulirus

B. Homarus

c. Procambarus

D. Cancer

I

Fig. 7. Schematic representation of the distribution of CCKLI in the stomatogastric nervous systems of four species of decapod crustacea. Drawing has been simplified in the interest of clarity; e.g., all eight neurons in the Homarus LVN send axons to the CGs. Not drawn to scale.

bilaterally paired clusters of immunoreactive somata within the subesophageal ganglion (Fig. 8B) and a number of immunoreactive somata in the thoracic ganglia that are possible sources of this staining. In general the background was high enough in these wholemount preparations of thoracic ganglia that it was not possible to trace processes

to their sites of termination. Similar staining was seen in the sns of Cancer, Homarus, and Panulirus. In Panulirus, a number of immunoreactive fibers can be seen in the pericardial plexus (Fig. 9A), and there are densely staining varicosities that surround the roots and each of the many small processes that make up the pericar-

172

G.G. TURRIGIANO AND A.L. SELVERSTON - 10-3M) had no effect on staining of FMRFamide these or any other structure (Fig. lOA,B,C).

Tissue and haemolymph extracts The ganglia of the stomatogastric nervous system of each species were extracted and measured in RIA to determine the levels of CCK-like peptide present. Most structures that displayed CCKLI contained measurable amounts of CCKlike peptide (Table 1).The STGs ofPanulirus and Homarus, the CGs of all four species, and the OGs of Homarus and Cancer, all of which display CCKLI, contained measurable amounts of CCK-like peptide. The STGs of Cancer and Procambarus and the OG of Panulirus, which do not display CCKLI, did not contained measurable amounts of CCK-like peptide, even when up to five ganglia were pooled; in these pooled extracts the limit of dectectibility was 0.04 fm/ganglia, so the level of CCKE in these ganglia was below this value. The OG of Procambarus does display CCKLI, but did not contain measurable amounts of CCKLI in RlA, even when five or six ganglia were pooled; levels were Fig. 8. CCKLI within the segmental nerves and thoracic ganglia of Procambarus. A. Immunoreactive fibers and varicosities within the sn therefore below 0.04 fm/ganglia. Serial dilutions of extracts 2 of Procambarus. B. Two bilaterally paired clusters of immunoreactive of ganglia and haemolymph produced inhibition of binding somata within the subesophageal ganglion ofProcambarus. Scale bar = curves that were nearly parallel to that produced by CCK 50 Wm. (Fig. 111, so this assay can be used to measure CCKE in these tissue and haemolymph samples. The levels of CCKE in ganglia of the stomatogastric dial plexus (Fig. 9B,C). No somata were seen within the nervous system varied from species to species, with Panupericardial plexus, and it was not possible to follow the luris and Homarus having the highest levels and Cancer immunoreactive fibers to their somata of origin. Similar and Procambarus having the lowest. These values were not staining was seen in the pericardial organs of the other corrected for wet weight of tissue and may in part reflect three species. differences in the size of ganglia in the different species, Panulirus and Homarus having the largest and Cancer Specificity of staining antennarius and Procambarus the smallest ganglia. The Preabsorption controls were performed, in which the levels of CCKE measured in single ganglia by RIA, on the primary antisera were incubated with peptides prior to order of 0.2 to 20 fm, are low compared to values obtained incubation with the tissue. Preincubation with CCK (10-5M) for the amount of FMRFamike-like peptide or proctolin in completely abolished staining of even the most brightly these ganglia, by one to two orders of magnitude (Marder et stained structures, such as the Cancer CG cell and the al., '86, '87). This may reflect poor cross-reactivity of this with the endogenous peptide. This antisepericardial plexus of Panulirus, whereas preabsorption antiserum (243-4) with the same or higher concentrations of proctolin or rum has only about a 1 : l O O cross-reactivity with the

Fig. 9. CCKLI within the pericardial plexus of Panulirus interruptus. A. Several fibers run within the main branch of the pericardial plexus, and branch as the plexus does. B. The fine branches o f the pericardial plexus close to where they terminate around the heart; the

plexus displays intense CCKLI. C. At higher magnification the CCKLI can be resolved into densely packed immunoreactive varicosities. Scale bars = A, 50 p n ; B, 100 wm; C, 20 wm.

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Fig. 10. Specificity of staining of antiserum C. A. Preabsorption of the primary antiserum with lO-'M CCKSSO, completely abolishes staining in the pericardial plexus (compare with Fig. 9). B. Preabsorption with CCKSSO, abolishes staining in the CG of Cancer. C.

Preabsorption of the primary antiserum with M FMRFamide leaves staining unaffected; the neuropil and axon of the brightly stained CG cell are not in the plane of focus. Scale bar = 100 km.

TABLE 1. CCKE in the Stomatogastric Nervous Systems and Haemolymph of Decapod Crustacea'

rior ganglia, and ramify within the STG to produce a densely immunoreactive neuropil (Turrigiano and Selverston, '89a). In contrast, the crayfish Procambarus clarkii and the crab Cancer antennarius have little or no CCKLI within the STG, and the CCKLI within the STG of the lobster Homarus americanus arises from somata in the motor nerves. In number, position, and projection pattern, these cells in Homarus are identical to the previously identified serotonergic GPR cells (Beltz et al., '84; Katz and Harris-Warrick, '86); as no other somata have been described within these motor nerves (Dando and Maynard, '74), it is likely that the CCK-like immunoreactive cells in Homarus are indeed the GPR cells. These cells are proprioceptors that are activated by stretch of gastric muscles and may contribute to the ability of the pyloric and gastric CPGs to produce rhythmic activity in the intact animal (Katz and Harris-Warrick, '89; Katz et al., '89). A CCK-like peptide thus may be acting synergistically with serotonin, and possibly other substances, to contribute to the generation of rhythmic activity within the STG of Homarus. It is difficult to interpret negative immunocytochemical results, as it is always possible that a lack of staining is due not to absence of antigen, but instead to factors such as antiserum penetrability. In the case of Cancer and Procambarus numerous protocols were tried in an attempt to improve staining in the STG, with no success. Antiserum penetrability in the STG is generally better than in thick structures such as the CGs for which staining in Cancer and Procambarus was robust and could be achieved using a variety of protocols. Differences in tissue levels of CCKE were also demonstrated in RIA, and with one exception these differences confirmed the differences observed by immunocytochemistry (Table 1).It therefore seems unlikely that lack of staining in the STGs of Procambarus and Cancer is due to factors such as antiserum penetrability. It is possible that in Cancer and Procambarus our inability to detect CCK-like peptide in the STG by immunocytochemistry and RIA is because the peptide in these species has

Species Panulirus interruptus STG OG CG HAEMOLYMPH Homarus americanus STG OG CG HAEMOLYMPH Cancer antenarrius STG OG CG HAEMOLYMPH Procambarus clarkii STG OG CG HAEMOLYMPH

CCKFJorgan (fm)

# ganglia assayed

17.1 ? 4.9 O?O 5.2 ? 0.5 6.2 ? 3.5

20

4c 1.3 ? 5.1 c 31.5 t

0.8 0.6

1.8 1.1

0.2 c 0.1 0.5 ? 0.1 11.3 ? 4.8

15 15 24 15

O?O O?O 0.41 c 0.1 22.0 c 1.5

10 11 15 3

O Z O

'Tissue extracts were assayed in RIA using antiserum C (243-4);the limit of detection of this assay is 0.2 fin. For organs with the smallest amounts of CCKE, 5 to 10 ganglia were pooled and extracted; limits of detection for these pooled samples was approximately 0.04 fdganglion. Each determination was made a t least twice. Values are mean t SEM. Haemolymph values are expressed as fm/ml.

invertebrate CCK-like peptide DSK (data not shown), and probably has a similarly low cross-reactivity with the endogenous crustacean CCK-likepeptide. If so these values are underestimated by as much as a factor of 100. All four species had detectable levels of CCKE in the haemolymph (Table 1).

DISCUSSION Of the four species of crustacea examined in this study, all have CCKLI within the stomatogastric nervous system. The pattern of staining for each species, however, is unique (see Fig. 7). In the Pacific spiny lobster Panulirus interruptus, immunoreactive fibers travel down the stn from ante-

G.G. TURRIGIANO AND A.L. SELVERSTON

174

0.5

1

8F -

0.45

-+-CCK ---t Cancer STG Homarus STG

0.4

B/T

+

+Cancer Haemolymph

0.35

+Panulirus CG +Panulirus OG

0.3

0.25

0.2 0.15

0.1

I 0.1

I

I

I I I

'''I

1/12 116 113 I

I 1 1 1 1 1 1 ~

1

10

1 I

I I Ill

100

CCK (fm) Fig. 11. Dilution curves of CCK and of tissue extracts of Cancer, Homarus, and Panulirus in RIA using antiserum C (243-4). BIT = counts bound by the antiserum divided by the total counts; X axis inset represents dilution factor of extracts. Homarus STG, Cancer haemolymph, and Panulirus CG extracts produce inhibition of binding curves that are roughly parallel to that produces by CCK (slopes:

CCK = -0.15, Homarus STG = -0.14,Panulirus CG = -0.16,and Cancer haemolymph = -0.14).Panulirus OG and Cancer STG, which do not display CCKLI, produce no inhibition of binding in this assay. Five ganglia of each type were extracted and serial dilutions assayed as described in the methods; the limit of detection in this assay was 0.06 fmiganglion.

undergone a structural change and is no longer recognized by the antisera used here. The CCK-like innervation of the STG described in Panulirus interruptus appears not to be a general feature of the decapod STG. Only two out of four species examined have such an innervation, and of those two, the projections are likely to serve distinct functions. There are cells in Cancer and Panulirus argus (and most likely in Panulirus interruptus) that correspond to the GPR cells in Homarus (Dando and Maynard, '74; Katz et al., '89), yet in Cancer and Panulirus interruptus these cells do not display CCKLI. Thus whatever function is subserved by CCK-like peptides in the GPR cells of Homarus is either absent or performed by some other substance in the GPR cells of Cancer and Panulirus. Similarly, the projection from anterior ganglia in Panulirus is absent in the other species examined. These data suggest that there is no conserved neuromodulatory function for CCK-like peptides in the control of the STG. In contrast to the interspecies distribution of CCKLI, the distribution of several other peptides in the STGs of Cancer, Panulirus, and Homarus is more highly conserved. Proctolin immunoreactivity and substance P-like immunoreactivity are found in the STGs of all three species (Marder et al., '86; Goldberg et al., '881, and FMRFamide-like immunoreactivity is found in the STGs of all species examined to date (Marder et al.,'87). The distribution of the monoamine serotonin resembles that of the CCK-like peptide more closely; serotonin-like immunoreactivity is lacking in the STG of Panulirus, but is present in Cancer and Homarus, notably in the GPR cells (Beltz et al., '84; Katz and Harris-Warrick, '86). The pattern of CCKLI in other structures of the stomatogastric nervous system is more highly conserved. In the esophageal ganglia of Procambarus, Homarus, and Cancer, two cells with axons within the ivn display CCKLI. In Cancer, these cells have been described previously and are known to contain FMRFamide-like immunoreactivity (Marder et al., '87), and red pigment concentratinghormonelike immunoreactivity (Nusbaum and Marder, '88). These

cells have not been described functionally, and it is not known why they contain so complex a mixture of peptides. The homologous cells in Homarus and Procambarus have not previously been determined to contain peptides, and in Panulirus these cells display FMRFamide-like immunoreactivity (Marder et al., '871, but not CCKLI. No other OG cells in these four species display CCKLI. The distribution of CCKLI in the CGs of all four species is similar. The number and position of immunoreactive somata are roughly comparable, and all species have a neuropilar projection from the thoracic ganglia, and a number of immunoreactive fibers coursing through the coc. In Cancer, the axon of the largest immunoreactive neuron can be traced all the way to the thoracic mass, where it produces a number of immunoreactive varicosities reminiscent of those in the sns. It was not possible to trace its axon any farther, but it is possible that this cell is contributing to the CCKLI observed in the sns and pericardial organs.

Specificity of staining The specificities of the antisera used in this study have been extensively characterized (Turrigiano and Selverston, '89a). Both antisera recognize peptides that have a terminal amino acid sequence homologous to the terminal sequence of CCK (gastrin, caerulein peptide, and DSK), but do not recognize a number of unrelated peptides known to be contained in the crustacean pericardial organs and stomatogastric nervous system, such as crustacean FMRFamides, proctolin, and red pigment concentrating hormone. Ad&tionally, preabsorbtion of the primary antisera with CCK completely abolishes staining in all four species, whereas staining is unaffected by preabsorbtion with FMRFamide or proctolin. We therefore believe that these antisera are specific for peptides that are structurally related to CCK. It is possible that the staining described here represents several different CCK-like peptides. There are several mammalian forms of CCK that are localized to different regions of the nervous system and gut (Dockray et al., '85; Rehfeld

CCK-LIKE IMMUNOREACTIVITY IN CRUSTACEA et al., '85). The immunological techniques used in this study can not distinguish between different molecular forms of CCK-like peptides, but will recognize anything that shares a terminal amino acid sequence with CCK. Characterization of FMRFamide-like peptides from crustacea has revealed several forms of the peptide in the pericardial organs, none of which appear to be authentic FMRFamide (Marder et al., '87; Trimmer et al., '87); it is not yet clear whether there are species differences in peptide sequence. In contrast, all proctolin-like immunoreactivity in the pericardial organs (Schwartz et al., '84) and in the STG (Marder et al., '86) appears to be authentic proctolin. It will be interesting to ascertain whether the CCKLI in the stomatogastric nervous system is identical to that in the pericardial organs and to determine the extent of any interspecies differences in sequence.

A hormonal role for CCK-like peptides in crustacea Like homologous peptides in vertebrates, CCK-like peptides in crustacea may serve a dual function as both neurotransmitters or neuromodulators, and as hormones. In all four species of crustacea examined, the sns and the pericardial organs contain fibers and numerous varicosities that display intense CCKLI. The presence of detectable levels of CCKLI in the haemolymph, at levels of about lo-" M, suggests that the pericardial organs or perhaps other neurohaemal structures are releasing CCK-like peptide into the haemolymph, where it would have access to a variety of targets. Favrel('88) has reported the presence of CCKLI in the haemolymph of prawn, in concentrations of lo-" to lo-'' M. Following ingestion, Favrel noted an approximately twofold increase in CCKLI in the haemolymph, and we have noted a two- to sixfold increase in levels after feeding in Panulirus (Turrigiano and Selverston, 'go), suggesting that CCK-like peptide levels are regulated by feedindfasting cycles. The specimens used in this study had not been uniformly fasted, so some of the variability in haemolymph levels can be attributed to this factor. Other important factors might be light-dark cycles and moult cycles. These possibilities have not been investigated. One potential target of CCK-like peptides in the haemolymph is the STG, which sits within an artery directly anterior to the heart. We have previously shown in Panulirus that CCK8S0, can modulate the output of the STG in vitro (Turrigiano and Selverston, '89a). In addition, in unrestrained lobsters the time-course of gastric mill activation following feeding is correlated with the time-course of the rise and fall in CCKLP levels in haemolymph. Injections of CCK into haemolymph can activate the gastric mill, and injections of the CCK antagonist proglumide can inhibit feeding-induced gastric mill activity (Turrigiano and Selverston, '90). These data strongly suggest that a CCK-like hormone has a role in the in vivo activation of the gastric mill following feeding. The presence of CCKLI in the haemolymph and pericardial organs of all species of crustacea examined suggests that, unlike the neuromodulatory role, the hormonal role of CCKLPs in the control of the STG is phylogenetically conserved. The only neurophysiological role for CCK-like peptides in crustacea that has been established as yet is in the modulation of the STG. Given the presence of CCK-like peptides in a variety of crustacea, and in a number of neural structures including the brain, CCK-like peptides in crustacea are

175

likely to serve a number of important functions, both as neuromodulators and as neurohormones.

ACKNOWLEDGMENTS We are grateful to Lynn Ogden for her expert technical assistance. This work was supported by NIH and ONR grants to A.I. S. and an NIH predoctoral training grant to G.G. T.

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