Distribution of catecholamines in the sea scallop, Placopecten ...

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Distribution of catecholamines in the sea scallop, Placopecten magellanicus Stephanie A. Smith, Janette Nason, and Roger P. Croll

Abstract: Catecholamines have previously been implicated in several important physiological processes in molluscs, including reproduction, respiration, and feeding. Much of the previous research has relied upon high-performance liquid chromatography to identify and quantify the various catecholamines and pharmacological experiments to investigate their actions. In the present report, we expand upon these studies by using histochemical techniques to investigate the distribution of catecholamine-containing cells and fibres in the central nervous system and peripheral tissues of the sea scallop, Placopecten magellanicus. Strong catecholaminergic staining was present in the somata and neuropil of all major central ganglia. Catecholamines were also abundantly stained in peripheral neurones and (or) fibres in several other tissues, including the labial palps, lips, intestine, gill filaments, foot, mantle, tentacles, and gonadal integument. It is concluded that catecholamines are widespread in the tissues of the scallop and could have potential neurotransmission roles in both the central nervous system and peripheral tissues of this species. Résumé : Chez les mollusques, les catécholamines ont été impliquées dans plusieurs processus physiologiques, dont la reproduction, la respiration et la nutrition. Jusqu’à présent, la plupart des études avait utilisé la chromatographie liquide à haute performance pour identifier et mesurer les différentes catécholamines et les analyses pharmacologiques pour examiner leurs actions. Dans cette étude, nous avons employé une technique histochimique pour déterminer la répartition des cellules et des fibres contenant des catécholamines dans les systèmes nerveux central et périphérique chez les pétoncles Placopecten magellanicus. Nos résultats démontrent la prédominance de la coloration catécholaminergique dans les corps cellulaires et les fibres de tous les ganglions centraux. De plus, l’analyse histochimique révèle une présence abondante de catécholamine dans les neurones et (ou) dans les fibres périphériques de plusieurs tissus, notamment les palpes labiaux, les lèvres, les intestins, les filaments des branchies, le pied, le manteau, les tentacules et le tégument gonadique. Nous concluons que les catécholamines sont présentes dans les tissus du pétoncle et servent peut-être de neurotransmetteurs dans le système nerveux central et dans les tissus périphériques chez cette espèce. Smith et al.

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Numerous studies have demonstrated the presence and bioactivity of catecholamines, such as dopamine and norepinephrine, in various tissues of bivalve molluscs. The majority of this work has been accomplished using highperformance liquid chromatography (HPLC) and pharmacological experiments to localize, quantify, and manipulate catecholamines in the central nervous system (CNS) (Hiripi et al. 1982; Smith 1982; Paulet et al. 1993) and peripheral tissues (Smith 1982; Osada et al. 1987). For example, HPLC analyses of tissues in the sea scallop, Placopecten magellanicus, revealed a widespread distribution of catecholamines, including high concentrations of dopamine and norepinephrine in the foot, gills, and heart (Pani and Croll 1995). Furthermore, the quantities of catecholamines in the ganglia, gills, and gonads of many bivalve species have been observed to fluctuate in correlation with seasonality and reReceived July 11, 1997. Accepted February 26, 1998. S.A. Smith. Department of Biology, Life Sciences Center, Dalhousie University, Halifax, NS B4H 4J1, Canada. J. Nason and R.P. Croll.1 Department of Physiology and Biophysics, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, NS B3H 4H7, Canada. 1

Author to whom all correspondence should be addressed (e-mail: [email protected]).

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productive activity (Stefano and Catapane 1977; Osada and Nomura 1989a, 1989b; Martínez and Rivera 1994). Ciliary activity of the gills, which appears to be under nervous control, is inhibited by the application of dopamine and dopaminergic agents, thus reducing water pumping rates (Stefano et al. 1977; Paparo 1988; Jones and Richards 1993). In addition, application of catecholamines caused excitation in isolated hearts of Venus mercenaria (Greenberg 1960). Taken together, this evidence suggests potential roles of catecholamines in several physiological processes in bivalves including feeding, respiration, cardiac regulation, and reproduction. The histological work to date on the distribution of catecholamines in bivalves has generally focused only on specific tissues, e.g., the nervous system (Dahl et al. 1966; Zs.Nagy 1967; Matsutani and Nomura 1984; Sweeney 1968), gills (Stefano and Aiello 1975), or gonads (Matsutani and Nomura 1984; Khotimchenko 1991). Comprehensive studies comparing catecholaminergic innervation in different tissues are lacking. In addition, no maps of the locations of central catecholaminergic neurons in bivalves exist. Such maps might provide a foundation for future studies of central control of bivalve behaviour and physiology. In the present study, we address these deficiencies by investigating the distribution of catecholamines in the commercially valuable bivalve species P. magellanicus using histochemical techniques.

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Fig. 1. Distribution of aldehyde-induced catecholamine fluorescence on the surfaces of the CNS. Cerebral and pedal ganglia: (A) anterior and (B) posterior surfaces; parietovisceral ganglion: (C) anterior and (D) posterior surfaces. p, pedal ganglia; cc, cerebral commissure; apn, anterior pallial nerve; pn, pedal nerve; cvc, cerebral–visceral connective; s, statocysts; dcl, dorsal–central lobes; vcl, ventral–central lobe; lll, left lateral lobe; rll, right lateral lobe; al, accessory lobe; on, osphradial nerve; ppn, posterior pallial nerve; glo, glomerulus. Scale bar = 500 µm.

Materials and methods Animals Placopecten magellanicus were purchased from the Great Maritime Scallop Trading Co. (Chester, N.S.) and maintained in circulating artificial seawater (Instant Ocean) at 4–6°C. Animals were prepared for histology within 2 weeks of arrival.

Histological procedures Cerebral, pedal, and parietovisceral ganglia and selected organs were dissected from both juvenile (35–45 mm shell height, N = 20) and adult (70–80 mm shell height, N = 6) scallops and processed using the FaGlu procedure modified from Furness et al. (1977). Tissues were placed in a solution of 4% paraformaldehyde, 0.55% glutaraldehyde, and 35% sucrose in phosphate-buffered saline (PBS: 50 mM Na2HPO4·7H2O and 140 mM NaCl, pH 7.4) for 24– 48 h. Whole ganglia and cryostat-sectioned ganglia and tissues were placed on glass slides and desiccated at room temperature and in the dark for 24–48 h. Slides were mounted in a 3:1 solution of glycerine in PBS or cleared and mounted in methyl salicylate. Mounted slides were viewed through a Leitz Aristoplan microscope equipped for ultraviolet (UV) epifluorescence (D filter block) and photographed using Kodak T-MAX 100 film. Negative controls were also prepared in which no glutaraldehyde was added to the fixative. Such preparations exhibited none of the blue–green fluorescence reported in this study.

Histological localization of catecholamines in the CNS of 1-year-old juveniles and 2- to 3-year-old adults Catecholamines are indicated by aldehyde-induced blue– green fluorescence when viewed with UV illumination. Little or no yellow fluorescence of indoleamines (Furness et al. 1977) was observed. Also, the pattern of blue–green fluorescence obtained using the FaGlu procedure was quite different from the staining pattern observed previously in P. magellanicus using antibodies raised against serotonin conjugated to bovine serum albumin with paraformaldehyde (Croll et al. 1995). Blue–green fluorescent staining was observed in cells and fibres of the neuropil in all the major ganglia in both cryostat-sectioned and whole-mounted preparations. Staining was similar in both the juvenile and adult CNS. In all ganglia, fibre tracts were strongly stained, with somewhat weaker staining occurring in cell bodies. Schematic representations of this staining for both the anterior and posterior surfaces of the ganglia are given in Fig. 1. Within the cerebral ganglia, brightly stained bundles of fibres extended through the ganglia. In addition, what appeared to be a large glomerulus (-100 µm in diameter) of axon terminals was observed near the base of the anterior pallial nerve in each cerebral ganglion, with fibres originat© 1998 NRC Canada



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ing ipsilaterally from the cerebral–visceral connectives (Figs. 1, 2, and 3). On the lateral edge of each ganglion near the glomerulus was a cluster of -50 somata (6–10 µm in diameter) (Figs. 1A, 1B, 2, and 3). Another cluster of -80 smaller cells (4–7 µm in diameter) was also observed on the medial edge of the anterior surface of each cerebral ganglion (Fig. 1A). Other somata were scattered throughout the center of the ganglia, with more cells observed on the anterior surface than on the posterior surface (Figs. 1A, 1B, and 2). Brightly stained fibre tracts were also observed in the cerebral–pedal connective and the pedal ganglia (Figs. 1A and 1B) as well as in the pedal nerves that innervate the foot (Figs. 1A and 1B). Small cells (4–7 µm) were also observed on the anterior surface of the pedal ganglia near the pedal nerves (Figs. 1A and 4). In the parietovisceral ganglion, -75 small neurones were observed surrounding the two dorsal–central lobes on the anterior surface and in the ventral–central lobe on both surfaces (-50 cells anterior, -25 cells posterior) (Figs. 1C, 1D, and 5). Small fibre tracts in the cerebral–visceral connectives splayed out as they approached the outer margins of the lateral lobes of this ganglion (Figs. 1C, 1D, and 6). In the lateral lobes, these brightly stained fibres created a pattern of small patches that were devoid of staining (Fig. 6). Many small cells (4–7 µm in diameter) were observed in clusters (-15–25 cells per cluster) along the entire length of the outer edge of both lateral lobes (Figs. 1C, 1D, and 6). More cells were observed in the larger left lateral lobe than in the right lobe. Catecholamines in sectioned peripheral tissues of juvenile scallops Catecholamine staining was also observed in neurones and fibres in several peripheral tissues of juvenile scallops, which were used because their smaller organs could be more readily sectioned in their entirety and repeatedly sampled in several individuals. Brightly stained cells (10–20 µm in diameter) were observed at the base of the labial palps near the edge of the digestive gland. Fibres extended from these cells into the palps (Fig. 7). Similar cells and fibres were also observed in clusters in the lips (data not shown). In the foot, smaller cells (-10 µm in diameter) were observed surrounding what appeared to be connective tissue lamellae (Fig. 8). Small fibres protruded from these cells toward the lamellar spaces. In the gills, numerous cells (10–20 µm in diameter) and a network of axons were observed in the epithelium of the distal gill filaments and in the epithelium of filaments near their insertion into the gill axis (Figs. 9 and 10). Catecholaminergic fibres were also observed in the circumpallial nerve running through the mantle and extended from this nerve toward the ocelli (Fig. 11). Cells (10– 20 µm in diameter) and small fibres were also observed throughout the tentacles (Fig. 12). Catecholaminergic cells were observed in the epithelium of the intestine, which passes through the gonad (Fig. 13). The cells in the intestinal epithelium are elongated and bipolar, with processes extending towards the intestinal lumen and axons extending in the opposite direction to form a network along the outer edge of the intestinal wall. In the gonad itself, catecholaminergic staining was observed in cells (10–15 µm in diam-

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eter) and fibres in the gonadal integument only and not in the gonadal tissue (Fig. 14). Fibres in the gonadal integument seem to extend toward the internal edge of the integument, where they branch to form a network. No catecholaminergic staining was found in the adductor muscle, heart, digestive gland, or kidneys of these juvenile animals. Catecholamines in gonads of 2- to 3-year-old scallops Gonads from both male and female adult scallops were also processed to determine whether the distribution of catecholamines changed as scallops became reproductively mature. Results showed similar staining in both male and female scallops as was observed in juveniles. Neurones and fibres were located in the gonadal integument and intestinal epithelium, with no staining in the gonadal tissue.

Histological analyses contained in the present paper demonstrate that catecholamines are abundant in cell bodies and fibres in both the CNS and several peripheral tissues of the sea scallop, P. magellanicus. This finding is in agreement with previous chromatographic evidence (Pani and Croll 1995) that also demonstrated the presence of catecholamines together with their putative precursors and metabolites in various tissues of this bivalve mollusc. The histochemical techniques employed in this study were similar to those used by others to specifically stain catecholamines in a wide variety of animals (Schöler and Armstrong 1982; Hauser and Koopowitz 1987; Molist et al. 1993) including molluscs (Croll and Chiasson 1990; Teyke et al. 1993; Croll et al. 1997; Quinlan et al. 1997). While application of such techniques has largely been superseded in many preparations by immunocytochemical demonstration of the presence of tyrosine hydroxylase (the initial and rate-limiting enzyme for catecholamine synthesis), use of this latter procedure has been problematic in molluscs. Certain antibodies raised against vertebrate tyrosine hydroxylase appear to cross-react with peptide sequences found in noncatecholaminergic cells (Croll and Chiasson 1990). The one such antibody that appears to immunoreact with the enzyme in certain gastropods (Hernadi et al. 1992; Sakharov et al. 1996) failed to label any central or peripheral cells in P. magellanicus or other gastropods such as Aplysia (S.A. Smith, J. Nason, and R.P. Croll, unpublished data). Nonetheless, future refinements in immunocytochemistry could eventually be used to confirm the catecholaminergic nature of the cells described in this study and provide further details of their morphologies. Immunocytochemistry could also be used to help distinguish whether specific neuronal populations contain primarily dopamine or norepinephrine, since both these compounds have been found consistently in molluscs, including P. magellanicus. The finding of large numbers of catecholamine-containing somata and fibres in the various central ganglia is consistent with past chromatographic analyses in this (Pani and Croll 1995) and other bivalve species (Hiripi et al. 1977; Smith 1982). It is also consistent with previous histological procedures used to detect catecholaminergic elements in central ganglia (Dahl et al. 1966; Zs.-Nagy 1967). However, © 1998 NRC Canada



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Figs. 2–6. Catecholamine fluorescence in the CNS. Fig. 2. Whole mount of the anterior surface of a cerebral ganglion showing glomerulus (g) and fluorescent cell bodies (solid arrows). Fig. 3. Sectioned preparation of cerebral ganglion showing cell bodies along the lateral edge (solid arrows). A portion of the glomerulus is also indicated (open arrow). Fig. 4. Section through a pedal ganglion, with arrows indicating fluorescent cell bodies near the pedal nerve (pn). Fig. 5. Section through the parietovisceral ganglion, showing fluorescent cell bodies (solid arrows) near one dorsal–central lobe (dcl). The open arrow indicates fluorescent fibres that run along the cerebral–visceral connective and through the ganglion. Fig. 6. Section through the parietovisceral ganglion, showing cell bodies (open arrows) and fibres (solid arrow) in the lateral lobes. Scale bars: 40 µm in Figs. 2 and 5; 30 µm in Fig. 3; 25 µm in Fig. 4; and 170 µm in Fig. 6.

while previous studies have detected catecholamines, the present study represents the first attempt at the construction of maps showing the distribution of catecholamines within the CNS, where these monoamines are likely to play several important roles. The staining of numerous cells within the various central ganglia is consistent with the possibility that

these neurones may use catecholamines as neurotransmitters/ neuromodulators in interactions with other central neurones. Such central actions for catecholamines are well known in gastropod molluscs, where, for example, the giant dopaminergic neurone, R.Pe.D1, has been shown to be a part of the central pattern generator for respiration in the pond

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Figs. 7–10. Sections of peripheral tissues, showing catecholamine fluorescence in cell bodies and fibres. Fig. 7. Labial palp (lp) with fluorescent cell bodies (solid arrows) at the junction with the digestive gland (dg). Fluorescent fibres (open arrow) also extend into the palp. Fig. 8. Foot with cell bodies (solid arrows) and processes around the connective tissue lamellae (cl). Fig. 9. Fluorescent cell bodies (solid arrows) and fibres (open arrow) in epithelium of distal gill filaments. Fig. 10. Fluorescent cell bodies (solid arrows) and fibres (open arrow) in epithelium of gill filaments near insertion into the gill axis. Scale bars: 285 µm in Fig. 7; 140 µm in Fig. 8; 175 µm in Fig. 9; and 50 µm in Fig. 10.

snail, Lymnaea stagnalis (Winlow et al. 1981; Syed et al. 1990). In addition, fluorescent fibres and potential catecholaminergic actions within the central ganglia could also de-

rive from the many peripheral cells identified in this study (see below). Unfortunately, the relative dearth of information on the physiology and functional anatomy of the © 1998 NRC Canada



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Figs. 11–14. Sections of peripheral tissues, showing catecholamine staining. Fig. 11. Mantle showing fluorescent fibres in the circumpallial nerve (cn) and fluorescent fibres (solid arrow) in the tentacles (t). Catecholamine staining between tentacles (open arrow) occurs where ocelli protrude (ocelli are not shown in this section). Fig. 12. Closer view of a tentacle, showing fluorescent neurones (solid arrows) and fibres (open arrow). Fig. 13. Arrows indicate bipolar fluorescent cells in intestinal epithelium that passes through the gonad. The autofluorescent crystalline style (cs) is visible within the lumen. Fig. 14. Fluorescent cell bodies (solid arrows) and fibres (open arrow) in the gonadal integument. The outer wall of the intestine (i) is visible in the lower right-hand corner of the photograph. Scale bars: 200 µm in Fig. 11; 100 µm in Figs. 12 and 14; and 130 µm in Fig. 13.

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bivalve nervous system hinders discussion of such potential roles for catecholamines in the CNS. The lateral lobes of the parietovisceral ganglion, however, might provide one promising focus for future studies along this vein. These lobes are known to receive sensory input from the numerous ocelli along the edge of the mantle of the scallop (Wilkens 1991). We interpret the patchy staining pattern observed in this study to indicate that catecholaminergic somata and fibres surround the glomeruli formed by the sensory afferent fibres (Wilkens 1991). Central responses to visual inputs are therefore likely to be influenced by catecholamines, as well as by other putative transmitters such as serotonin (Croll et al. 1995) and FMRFamide-related peptides (Too and Croll 1995). Catecholaminergic fibres in the neuropilar regions of central ganglia have received past commentary regarding their abundance and intensity of staining (Dahl et al. 1966; Zs.Nagy 1967). These fibres could derive from central neurones (e.g., motoneurones) with fibres projecting to the periphery, where catecholamines are also known to have actions (Muneoka et al. 1991). Such peripheral actions must, however, be interpreted in light of the large number of catecholamine-containing cells observed in a variety of tissues in the present study. Catecholaminergic neurones have previously been identified in peripheral tissues of both larval (Croll et al. 1997) and adult (Sweeney 1968) bivalves, and at least some of these neurones have been suggested to play sensory roles. Motor roles cannot be ruled out, however, in light of the fact that the peripheral nervous systems of molluscs can mediate both sensory and motor functions autonomous of the CNS (Bullock 1965). Among the peripheral tissues, the gills appear to contain the largest number of catecholaminergic cells. The observation is consistent with the previous finding of relatively abundant amounts of catecholamines and related metabolites in the gills of Placopecten. In addition, application of dopamine to isolated gill preparations and live animals of other bivalve species causes inhibition of lateral ciliary activity and thus reduces water pumping rates (Stefano et al. 1977; Jones and Richards 1993). Application of the dopamine antagonist ergometrine blocks the inhibition of the lateral cilia by both dopamine and the “brown tide” alga Aureococcus anophagefferens, which may be releasing a water-soluble dopamine-mimetic compound (Gainey and Shumway 1991). Peripheral catecholaminergic cells may therefore be involved in the ciliary control of respiratory and (or) filtering functions of the gills. Control of gill cilia may also be exerted from the CNS via innervation by the branchial nerve (Sweeney 1968; Stefano and Aiello 1975; Paparo 1988). Both the detection of catecholaminergic neurons in this study and the previous use of chromatography (Pani and Croll 1995) to demonstrate the presence of catecholamines in the lips and labial palps further indicate a potential role for catecholamines in the feeding processes of these animals. The labial palps are also ciliated structures and are believed to be involved in the sorting of food particles from the gills (Beninger and Le Pennec 1991). Thus, catecholamines may be involved in the ciliary function of these organs as well as in the gills (Smith 1982). This hypothesis is strengthened by preliminary studies (S.A. Smith, A.K. Pani, and R.P. Croll, unpublished data) showing that administration of α-methyl-


p-tyrosine, which blocks catecholamine synthesis, potently inhibits feeding in otherwise healthy scallops. In addition, the localization of catecholamines in cells within the intestine suggests a role for catecholamines in digestive processes in this animal. The finding by Giard et al. (1995) that dopamine evokes the release of α-amylase from the stomach – digestive gland complex of the scallop Pecten maximus further indicates that catecholamines may several roles in the feeding and (or) digestive processes of bivalves. Catecholaminergic cells were also observed in the foot of the scallop, a finding consistent with previous chromatographic evidence (Pani and Croll 1995). The location of these cells around connective tissue lamellae, with projections extending toward the lamellar spaces, suggests a role for catecholamines in byssal thread production (Beninger and Le Pennec 1991). Catecholamines may also be involved in muscular contractions of the foot, as dopamine is known to have modulatory effects on the anterior byssus retractor muscle, which is closely associated with the foot in the mussel Mytilus edulis (Muneoka et al. 1991). While the presence of catecholamines in the gonadal integument of P. magellanicus may indicate some involvement of catecholamines in bivalve reproduction, our findings contrast with those of Matsutani and Nomura (1984) who reported more extensive catecholaminergic innervation of the gonad from both the parietovisceral ganglion and the cerebral–visceral connectives in the Japanese scallop, Patinopecten yessoensis. This inconsistency must be further investigated. Our findings also contrast with previous work showing extensive serotonergic innervation of gonadal tissue and collecting ducts of P. magellanicus (Croll et al. 1995) and other bivalve species (Matsutani and Nomura 1984; Ram et al. 1992; Smith and Croll 1997). A direct major role for catecholaminergic innervation in the reproduction of P. magellanicus is therefore not supported by our findings, although hormonal actions cannot be overlooked. In addition, catecholamines may affect feeding and digestion and thus indirectly affect gamete production and maturation (Sastry 1975). Hormonal and (or) other indirect actions may thus explain previous evidence for involvement of catecholamines in bivalve reproduction (Osada et al. 1987; Osada and Nomura 1989a, 1989b; Smith and Croll 1997). No catecholamine staining was found in the heart, digestive gland, kidneys, or adductor muscle of P. magellanicus. In reference to the heart, this result is in agreement with previous studies on heart tissues of the gastropod molluscs Aplysia californica (Carpenter et al. 1971) and Helix aspersa (Sloley et al. 1990). In A. californica, however, the heart was found to be capable of concentrating dopamine several times over the bath concentration (Carpenter et al. 1971). Diffusely located catecholamines might not be detected by our histological procedures but could nonetheless explain previous reports of significant levels of catecholamines in the bivalve heart (Pani and Croll 1995), where they have long been known to be bioactive (Greenberg 1960). By identifying specific cell types that might synthesize or otherwise accumulate catecholamines, this study complements previous chromatographic analyses (Pani and Croll 1995) indicating the presence, synthesis, and catabolism of these monoamines in a variety of tissues. The locations and innervation patterns of these cells are suggestive of numer© 1998 NRC Canada



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ous potential roles for catecholamines in both the CNS and periphery. Previous physiological and pharmacological experiments support several hypothesized roles, notably those involved with feeding and respiration. Further work, however, is obviously needed to fully explain the abundance and widespread distribution of catecholamine-containing cells identified in this study.

This work was supported by a strategic grant from the Natural Sciences and Engineering Research Council of Canada to R.P.C.

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