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THE JOURNAL OF COMPARATIVE NEUROLOGY 395:466–480 (1998) Serotonin Immunoreactivity in the Central Nervous System of the Marine Molluscs Pleurobranchaea californica and Tritonia diomedea LELAND C. SUDLOW, JIAN JING, LEONID L. MOROZ, AND RHANOR GILLETTE* Department of Molecular and Integrative Physiology and the Neuroscience Program, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801 ABSTRACT The central nervous systems of the marine molluscs Pleurobranchaea californica (Opisthobranchia: Notaspidea) and Tritonia diomedea (Opisthobranchia: Nudibranchia) were examined for serotonin-immunoreactive (5-HT-IR) neurons and processes. Bilaterally paired clusters of 5-HT-IR neuron somata were distributed similarly in ganglia of the two species. In the cerebropleural ganglion complex, these were the metacerebral giant neurons (both species), a dorsal anterior cluster (Pleurobranchaea only), a dorsal medial cluster including identified neurons of the escape swimming network (both species), and a dorsal lateral cluster in the cerebropleural ganglion (Pleurobranchaea only). A ventral anterior cluster (both species) adjoined the metacerebral giant somata at the anterior ganglion edge. Pedal ganglia had the greatest number of 5-HT-IR somata, the majority located near the roots of the pedal commissure in both species. Most 5-HT-IR neurons were on the dorsal surface of the pedal ganglia in Pleurobranchaea and were ventral in Tritonia. Neither the buccal ganglion of both species nor the visceral ganglion of Pleurobranchaea had 5-HT-IR somata. A few asymmetrical 5-HT-IR somata were found in cerebropleural and pedal ganglia in both species, always on the left side. The clustering of 5-HT-IR neurons, their diverse axon pathways, and the known physiologic properties of their identified members are consistent with a loosely organized arousal system of serotonergic neurons whose components can be generally or differentially active in expression of diverse behaviors. J. Comp. Neurol. 395:466–480, 1998. r 1998 Wiley-Liss, Inc. Indexing terms: serotonin; immunohistochemistry; Mollusca; Aplysia; Lymnaea Serotonin (5-HT) plays multiple roles in modulating the behavioral state in molluscs, arthropods, annelids, and vertebrates (Jacobs and Fornal, 1993; Weiger, 1997). Its roles range across regulation of affective state in behavior, setting arousal levels in neural networks, and fine-tuning sensory pathways. In molluscs, 5-HT innervation within the central nervous system (CNS) is prominent and so influential in network up-regulation that it has been proposed to act as a general arousal factor (Kupfermann and Weiss, 1981; Sakharov, 1990). Thus, 5-HT acts in arousal mechanisms for defensive behavior, as in gill/ siphon withdrawal in Aplysia (Kandel and Schwartz, 1982; Glanzman et al., 1989), and analogous behaviors in the pteropod Clione (Satterlie and Norekian, 1996) and the pulmonate snail Helix (Zakharov et al., 1995). Serotonin acts as a facilitatory modulator in feeding networks of Aplysia (Kupfermann and Weiss, 1981) and Lymnaea (Kyriakides and McCrohan, 1989), and in the respiratory network of Lymnaea (Moroz, 1991) as well. Serotonin is r 1998 WILEY-LISS, INC. also an intrinsic neuromodulator of motor network and muscle activity in the escape swim of Tritonia (Katz et al., 1994; McClellan et al., 1994; Katz and Frost, 1995a, 1995b) and in parapodial swimming in both Aplysia (McPherson and Blankenship, 1991c) and Clione (Panchin et al., 1996; Satterlie and Norekian, 1996). In Tritonia and Lymnaea, 5-HT may regulate ciliary locomotion by means of serotonergic neurons in the pedal ganglia (Audesirk et al., 1979; Syed et al., 1988). The bilateral pair of serotonergic giant cerebral ganglion cells, the metacerebral giants Grant sponsor: National Institutes of Health; Grant numbers: RO1 NS26838 and PHS 5 T32 MN18412. *Correspondence to: Dr. R. Gillette, Department of Molecular and Integrative Physiology, University of Illinois, Urbana-Champaign, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801. E-mail: [email protected] Received 2 September 1997; Revised 3 February 1998; Accepted 6 February 1998 SEROTONIN IN MOLLUSCS (MCGs) conserved in many opisthobranch and pulmonate species (Weinreich et al., 1973; Senseman and Gelperin, 1974; Sakharov, 1976; Weiss and Kupfermann, 1976; Gillette and Davis, 1977; Granzow and Rowell, 1981; Croll, 1987), exert varying degrees of influence in feeding behavior (Gillette and Davis, 1977; Weiss et al., 1978; Yeoman et al., 1996). In the predatory opisthobranch Pleurobranchaea, 5-HT may play roles in locomotion and in the escape swimming network similar to those of Tritonia, because the animals share apparently homologous networks for these behaviors (Jing and Gillette, 1995; Jing et al., 1997; Jing and Gillette, 1998). In this animal, 5-HT stimulates multiple aspects of behavioral activity, including feeding behavior (Palovick et al., 1982; Gillette et al., 1997). Thus, in Pleurobranchaea as in other molluscs, 5-HT may play an important role as a general arousal factor. Previously, we described the serotonergic innervation of the periphery in both Pleurobranchaea and Tritonia (Moroz et al., 1997). We found it to be extensive, and entirely central in origin. In particular, greatest densities of 5-HT-immunoreactive (5HT-IR) elements were found in the foot and reproductive system, and in multiple feeding structures. Dense 5-HT-IR innervation of chemosensory epithelia suggested neuromodulatory regulation of chemosensory pathways by central elements. In the present work, we describe 5-HT immunoreactivity in the CNS of Pleurobranchaea and Tritonia. We undertook this study to identify putative serotonergic neurons in Pleurobranchaea’s neural networks for feeding, locomotion, and escape swimming, and those cells that innervate chemosensory areas and the reproductive system. We included Tritonia for comparison, because it represents a line of the nudibranch evolutionary radiation derived from pleurobranchomorphs that conserves some primitive traits of its pleurobranchomorph ancestors and differs in others central to its ecologic specialization. It is also used as a model system of considerable power for which no published immunohistochemical map for 5-HT exists. We found around 170 5-HT-IR cells in the CNS of Pleurobranchaea. Identified neurons that were 5-HT-IR were the MCGs and cells of the dorsal A-cluster group of the cerebropleural ganglia, and the G neurons of the pedal ganglia. The buccal ganglion is densely 5-HT-IR innervated, but lacks 5-HT-IR somata, and the sole source of 5-HT is probably the MCGs. Tritonia, with about 220 5-HT-IR cells, shares many apparently homologous 5-HT-IR cells with Pleurobranchaea, based on their relative sizes and positions, including the MCGs, G cells, and the A-cluster cells. Consideration of the anatomical and physi- Abbreviations 5-HT 5-HT-IR ABC BSA CNS DAB DSI INa,cAMP MCG PAP PBS PBS-GT PBST serotonin serotonin-immunoreactive avidin-biotin complex bovine serum albumin central nervous system 3,38-diaminobenzidine tetrahydrochloride dorsal swim interneuron cAMP gated sodium current metacerebral giant peroxidase-antiperoxidase phosphate buffered saline phosphate buffered saline with goat serum and Triton X-100 phosphate buffered saline with Triton X-100 467 ologic attributes of the 5-HT-IR neurons points to their possible role as a loosely organized arousal network capable of modulating the expression of diverse behaviors. MATERIALS AND METHODS Specimens of Pleurobranchaea californica and Tritonia diomedea were obtained from Mr. Mike Morris (Sea Life Supply, Sand City, CA) or Dr. Rimmon Fay (Pacific BioMarine, Venice, CA) and maintained in 14°C artificial sea water. Twenty Pleurobranchaea, ranging from 10 to 300 g, and four Tritonia, ranging from 20 to 100 g, supplied ganglia for whole-mounts. Ganglia from an additional eight Pleurobranchaea were used in control experiments. Five Pleurobranchaea, ranging from 15 to 300 g, were studied in cryotome sections. Animals were anesthetized by chilling; ganglia were quickly dissected free and transferred to artificial Pleurobranchaea saline (in mM: 420 NaCl, 10 KCl, 25 MgCl2, 25 MgSO4, 10 CaCl2, 10 MOPS buffered with NaOH, pH 7.5). For whole-mount immunocytochemistry, ganglia were pinned to Sylgard and fixed for 1 hour at 0oC, then 5 hours at 4oC in 4% paraformaldehyde in 450 mM NaCl with 20 mM phosphate buffer, pH 7.5. Tissues were washed 24 hours (4 3 6 hours) in equiosmotic phosphate buffered saline (PBS; 450 mM NaCl, 20 mM phosphate, 0.5% thimerosal, pH 7.5) and dehydrated through an ethanol series to absolute ethanol in 30-minute steps. Ethanol was replaced with absolute methanol and H2O2 was added to a final concentration of 0.5 % (v/v) to inactivate endogenous peroxidases. After incubating for 45 minutes in the methanol-H2O2 solution, the tissues were rehydrated through a decreasing ethanol dilution series (30-minute steps) into PBS (150 mM NaCl, 20 mM phosphate, 0.5 % thimerosal, pH 7.5). All further blocking, incubations, and washings were performed at 4oC with agitation. Tissues were blocked in PBS with 3% (v/v) heat-inactivated goat serum (Cappel, Organaon Teknika Corp., Durham, NC) and 0.4% Triton X-100 (PBS-GT) for 4 hours. Two different lots of rabbit anti-serotonin antisera (IncStar, Stillwater, MN) were used over the course of these experiments. Lot 8843026 was used at a final dilution of 1:1,000 in PBS-GT (PBS with 0.25% Triton X-100). Subsequent lots of antisera from different rabbits were used at final dilutions of 1:10,000 , 1:15,000 in PBS-GT. Primary incubations lasted 72 hours. Tissues were washed 24 to 72 hours (minimum 4 3 6 hour washes) in PBS with 0.25% Triton X-100 (PBST). Staining was accomplished by the avidin-biotin complex (ABC) -horseradish peroxidase technique (Vectastain, Vector Labs, Burlingame, CA) or by the peroxidase-antiperoxidase (PAP) technique. Both ABC and PAP reactions produced similar staining patterns of somata in the cerebropleural and pedal ganglia. For ABC staining protocols, tissues were incubated in biotinylated goat anti-rabbit IgG (Cappel) at a final dilution of 1:200 in PBS-GT for 24 hours, washed for 24 to 72 hours in PBST, and incubated 24 hours in the ABC reagent. For PAP reactions, tissues were incubated in goat anti-rabbit IgG (Cappel) at a final dilution of 1:200 in PBS-GT for 24 hours. Ganglia were washed for 24 to 72 hours (minimum 4 3 6 hour washes) and incubated in rabbit PAP (Jackson ImmunoResearch, West Grove, PA) at a final dilution of 1:100 for 24 hours. Tissues were then washed for at least 48 hours in PBST. Tissues were washed in PBS for 4 hours and transferred to 50 mM Tris-HCl (pH 7.2) for 4 hours. In some experiments, the Tris-HCl 468 L.C. SUDLOW ET AL. concentration was increased to 120 mM (pH 7.2). Tissues were incubated at least 2 hours in 0.05% 3,38-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co., St. Louis, MO) in Tris-HCl buffer at room temperature in the dark. Final peroxidase reactions were performed by adding H2O2 to 0.05% to the DAB reaction solution. Progress of the chromogenic reaction was monitored under subdued lighting. Reaction times were typically 5–15 minutes. Tissues were washed in Tris buffer (3 3 2 hours) and dehydrated through absolute ethanol. Tissues were cleared and mounted either in methyl salicylate or in Permount (Fisher Scientific, Pittsburgh, PA). Soma diameters reported in this study were from the ,100-g animals. For positive controls, a conjugate of bovine serum albumin (BSA) and 5-HT was synthesized after the method of Steinbusch et al. (1983) for use in preabsorption controls. In brief, 1 ml of 3 M sodium acetate (pH 7.2), 9.2 mg of serotonin creatinine sulfate (Sigma) in 1 ml of distilled water, 28.1 mg of BSA (BSA Fraction V, Sigma) in 1 ml of distilled water, and 1 ml of 7.5% freshly made paraformaldehyde were sequentially added to 10 ml of distilled water and reacted overnight in the dark at room temperature. The 5-HT-BSA conjugation reaction was terminated by dialysis (Spectra/Por 4; molecular weight cut-off, 12,000– 14,000; Spectrum Medical Industries, Los Angeles, CA) against running deionized water for 24 hours. The 5-HTBSA conjugate was then aliquotted and lyophilized for future use. Positive immunocytochemical control incubations used 1 mg of conjugate per 10 ml of antisera overnight at 4oC with agitation. Serotonin immunoreactivity could be eliminated by preincubation of the primary antisera with the 5-HT-BSA conjugate (n 5 3, data not shown). Negative controls omitting the primary antisera exhibited no staining of somata or neuropil (n 5 5, data not shown). For cryotome sectioning, fixed preparations were rinsed in two to three changes of 0.5 M PBS, incubated at 4oC for 4–8 hours in 30% sucrose in 0.4 M Tris-HCl buffer solution (pH 7.4), embedded in Tissue-Tec O.C.T. compound (Miles, Elkhart, IN), and frozen on dry ice. Serial sections of four preparations of the Pleurobranchaea CNS were cut at 30 µm with a cryotome, mounted on charged and precleaned microscope slides (ProbeOn Plus, Fisher), and dried for 10–20 minutes at room temperature. The staining protocols were identical both for whole-mount preparations and cryotome sections. The nomenclature of ganglia and ganglionic lobes is used as described earlier (Moroz and Gillette, 1996). RESULTS The fixation and staining protocols preserved and defined immunoreactive elements in the CNS with a high degree of resolution in both Pleurobranchaea and Tritonia. The staining pattern of somata was generally replicable, with slight differences mentioned further. Stained somata and their unstained glial invaginations were distinct even in the perinuclear region (Fig. 1A). Fine 5-HT-IR neuronal processes could be traced in the neuropil (Fig. 1B) and were often seen investing unstained somata (Fig. 1C,D). 2–6). Stained cell bodies of the dorsal surface of the cerebropleural ganglion were distributed in three major, bilaterally paired clusters (Figs. 2A, 3). A dorsal anterior cluster consisted of the MCG (.240 µm diameter) and three medium-sized neurons (150–190 µm) found in the rhinophore lobe; soma positions were consistent among preparations. In sectioned material, axons from the somata of the medium-sized cells were seen to enter the neuropil and run posteromedially toward the cerebral commissure (Fig. 6). One-to-two small 5-HT-IR somata (70–80 µm) near the medium cells in the rhinophore lobe were seen in sectioned material. The second group was the dorsal medial cluster located near the cerebral commissure (Figs. 2A, 3). This group of 5-HT-IR somata consisted of five small-diameter cells (70–80 µm), identified elsewhere as As1, As2, As3, As4 and As-rh, of which As1-4 are elements of an escape swimming pattern generating network (Jing et al., 1997; Jing and Gillette, 1998). The third group was the dorsal lateral cluster found laterally in each cerebropleural hemiganglion near the roots of the anterior and posterior cerebropedal connectives (Figs. 2A, 3). The dorsal lateral cluster consisted of five to seven small (70–80 µm), medium (160–180 µm), and one giant somata. Last, one asymmetrical giant cell was found on the dorsal posterior surface of the left cerebropleural ganglion near the left small body wall nerve (Figs. 2A, 3). The ventral surface of the cerebropleural ganglia had relatively few 5-HT-IR cells (Fig. 4). The MCGs were visible because they occupy the edge of the anterior pole of the cerebral lobe. Those few 5-HT-IR somata were located in a single, ventral anterior cluster of four small (60–80 µm) and three medium-sized (150–180 µm) somata arrayed bilaterally along the medial border of each cerebropleural hemiganglion on the ventral surface posterior to the MCGs (Fig. 4). Finally, a single 5-HT-IR asymmetrical giant soma was found on the ventral surface of the left cerebropleural hemiganglion near the root of the left body wall nerve (Fig. 4). Neuropilar staining was also observed in the cerebropleural ganglion. Serotonin-immunoreactive fibers and varicosities were clearly visible both in whole-mounts (Fig. 2A) and in sectioned material (Fig. 5). The strongest 5-HT-IR neuropilar staining in the cerebropleural ganglia was found lateral to the MCG somata (Figs. 2A, 5A, 6). Additional dense regions of neuropilar staining were observed posterolaterally in the cerebropleural ganglion (Fig. 2A). Serotonin-immunoreactive fibers crossed in the anterior and posterior portions of the cerebral commissure (Fig. 2A, 6). Numerous 5-HT-IR axons of various diameters exited the ganglion from all major nerve roots with the sole exception of the optic nerve, which had none (Fig. 2A). There was an asymmetrical distribution of large-diameter axons between the left and right cerebropleural hemiganglia: large-diameter axons were found in the left body wall nerve, the left cerebrovisceral connective, and the left small body wall nerve; the contralateral nerves lacked the large-diameter axons. Cerebropleural ganglion: Pleurobranchaea Pedal ganglia: Pleurobranchaea Serotonin-immunoreactive somata in the cerebropleural ganglion were limited to four major clusters of cell bodies distributed on both the dorsal and ventral surfaces (Figs. The pedal ganglia of Pleurobranchaea exhibited the most numerous 5-HT-IR somata and processes in the CNS. In both left and right pedal ganglia, 5-HT-IR somata were SEROTONIN IN MOLLUSCS 469 Fig. 1. Patterns of serotonin immunoreactivity in individual neurons of Pleurobranchaea (cryotome sections). Asterisks indicate 5-HTimmunonegative neuronal somata densely surrounded by serotoninimmunoreactive (5-HT-IR) terminals. A: Serotonin immunoreactivity in two giant neurons (open and filled arrows) from the posterior lateral lobe of the left pedal ganglion. Note a specific pattern of the serotonin immunoreactivity in the larger neuron (open arrow). Perinuclear areas (black curved arrow) stain homogeneously, whereas more distant cytoplasmic areas (arrowheads) show non-uniform staining due to the presence of numerous glial processes in the cytoplasm of the giant cell (see also Results section). B: Serotonin-immunoreactive neuron in the anterior medial lobe of the right pedal ganglion. Note the 5-HT-IR soma (arrow) next to the immunonegative soma (asterisk). C,D: Neighboring 40-µm sections from a giant neuron in the lateral cerebropleural ganglion. Note the basket-like pattern of 5-HT-IR neuronal terminals (arrow in C) around the neuronal somata. n, nucleus. Scale bar 5 150 µm (applies to A–D). found predominately at the dorsomedial lobes of the ganglia (Figs. 2B, 3, 5B). This group of somata was termed the ‘‘G’’ cells in earlier studies (Huang and Gillette, 1991, 1993; Sudlow and Gillette, 1995, 1997). When left and right pedal ganglia from the same animal were compared in whole-mount preparations, there were both similarities and differences in the distribution of 5-HT-IR somata (Fig. 3). Although there were similar numbers of largediameter (4) and medium-diameter (12–13) 5-HT-IR somata in the dorsomedial lobe of the pedal hemiganglia, there were fewer small-diameter somata in the right pedal than in the left pedal ganglion (Fig. 3). Also, somata by the roots of the posterior and medial pedal nerves in the right pedal ganglion were both fewer and larger than in the left ganglion (Fig. 3). The dorsal surfaces of the left and right posterior lateral lobes of the pedal ganglia had similar numbers of 5-HT-IR somata. Lightly stained cells were observed in the accessory lobes of the pedal ganglia. When the ventral surfaces of pedal ganglia of the same animal were compared, an asymmetry was observed in the distribution of medium and small-diameter 5-HT-IR somata (Figs. 3, 4). Serotonin-immunoreactive processes were observed exiting the pedal ganglia in all major nerve and connective roots (Fig. 2B). Serotonin-immunoreactive axons ran in the anterior and posterior cerebropedal connectives and in the pedal and parapedal commissures. Tracts of fibers coursed through the ganglia between the anterior and posterior cerebropedal connectives and the posterior pedal nerves (Fig. 2B). Axons arising from the 5-HT-IR somata at the dorsomedial lobe coursed laterally in the pedal ganglion (Fig. 5B). Axons exited the pedal ganglia in all peripheral nerves. Buccal and visceral ganglia: Pleurobranchaea The buccal ganglion of Pleurobranchaea was devoid of 5-HT-IR somata (Figs. 7A, 8). One large-diameter, and occasionally a small-diameter, axon entered the buccal ganglion from each of the bilateral cerebrobuccal connectives. Processes of these fibers ramified in the buccal ganglion and surrounded many of the somata (Figs. 7B, 8). Because no 5-HT-IR somata were visible in the buccal 470 Fig. 2. Serotonin immunoreactivity in the central nervous system of Pleurobranchaea (whole-mount preparation). Arrowheads point to somata of the metacerebral giant neurons, immediately caudally adjacent to which are neurons of the dorsal anterior cluster of serotonin-immunoreactive (5-HT-IR) somata. A: Cerebropleural gan- ganglion and only one large-caliber axon was present in the cerebrobuccal connectives, the ramified 5-HT-IR processes were most likely associated with this large axon. Because the axon of the MCG is the only giant, putatively serotonergic axon identified in the cerebrobuccal connectives (Gillette and Davis, 1977), it is most likely that the MCG is the source of the ramified 5-HT-IR processes. Serotonin-immunoreactive processes could be observed crossing to the contralateral buccal hemiganglion. Axons exited the buccal ganglion through the buccal and stomatogastric roots, similar to the known branching pattern of the MCGs in the buccal ganglion (Gillette and Davis, 1977). No 5-HT-IR somata were observed in the visceral ganglion (Fig. 9); however, fibers were observed both enmeshing somata and coursing through from the left and right cerebrovisceral connectives to exit to the periphery. L.C. SUDLOW ET AL. glion, dorsal surface. Arrows indicate the five 5-HT-IR somata of the A-cluster of the dorsal medial 5-HT-IR cluster (As1-4 and As-rh). B: Pedal ganglion, dorsal surface. The arrow points to the G cells. Scale bar 5 500 µm (applies to A,B). Cerebropleural ganglia: Tritonia Serotonin immunoreactivity in the cerebropleural ganglia of Tritonia is similar in many ways to Pleurobranchaea (Figs. 10–12). The MCGs in Tritonia were most striking for their large size (.280 µm) relative to other ganglion cells (Figs. 10, 12), but the dorsal anterior cluster of smaller somata observed in Pleurobranchaea was not present. There were few other stained somata on the dorsal surface. In the dorsal medial cluster, five intensely stained somata of small diameter (50–80 µm) were found medially near the anterior border of the cerebral commissure (Figs. 10A, 12A). Of these, the three most anterolateral are likely to be the serotonergic ‘‘dorsal swim interneurons,’’ or DSIs identified in Tritonia’s swim network (Getting et al., 1980; McClellan et al., 1994; Katz et al., 1994), and SEROTONIN IN MOLLUSCS 471 Fig. 3. Schematic diagram of the distribution of serotoninimmunoreactive (5-HT-IR) neurons on dorsal surfaces of cerebropleural and pedal ganglia of Pleurobranchaea. Cerebropleural ganglion: aCPC, anterior cerebropedal connective; BWN, body wall nerves (left and right); CBC, cerebrobuccal connective; CC, cerebral commissure; CVC, cerebrovisceral connective; MN, mouth nerve; ON, optic nerve; OVN, oral veil nerve; pCPC, posterior cerebropedal connective; RhL, rhinophore lobe; RN, rhinophore nerve; sBWN, left small body wall nerve of cerebropleural ganglion; SCC, subcerebral commissure; TN, tentacular nerve. Pedal ganglion: AccL, accessory lobe; aPN, anterior pedal nerve; aLBWN, anterior lateral body wall nerve of the pedal ganglia; DML, dorsomedial lobe; mPN, medial pedal nerve; PC, pedal commissure; pLBWN, posterior lateral body wall nerve of the left pedal ganglion; PLL, posterior lateral lobe; pPC, parapedal commissure; pPN, posterior pedal nerve. homologs of the As1-3 neurons of Pleurobranchaea (Jing et al., 1997). The remaining two most posteromedial cells may correspond to the As-rh and As4 cells of Pleurobranchaea (Jing and Gillette, 1998). A second small cluster of lightly stained, medium-sized (,120 µm) somata (shaded cells illustrated in Fig. 12A) was located near the posterior margin of the cerebral commissure, sometimes mingled with the putative As-rh and As4 homologs. The ventral surface of the Tritonia cerebropleural lobes, as in Pleurobranchaea, exhibited relatively little serotonin immunoreactivity (Fig. 12). All labeled somata were found in the anterior cerebral lobes. Two medium-sized (,110– 140 µm) and 11–14 small-sized (50–70 µm) somata, the ventral anterior cluster, were observed just caudal to the MCGs. The posterior lobes of the cerebropleural ganglion did not exhibit consistent serotonin immunoreactivity. An occasional 5-HT-IR soma was found in these lobes. How- ever, these oddly located 5-HT-IR somata were inconsistent, suggesting that their occurrence in the posterior lobes of the cerebropleural ganglion was a developmental vagary. The ventral neuropil of the Tritonia cerebropleural ganglion had one area in each hemiganglion showing a higher density in neuropil labeling. This lateral region in the cerebral lobes, near the roots of cerebral nerve 4 (Figs. 10A, 12B), is an area associated with mechanoreceptive inputs to the cerebral ganglion (Audesirk, 1979). A similar area in Pleurobranchaea was located near the roots of the oral veil, tentacular, and mouth nerves (Fig. 2A). Pedal ganglia: Tritonia Serotonin-immunoreactive somata on the dorsal surface of the Tritonia pedal ganglia were clustered as a group posterolaterally (Figs. 11, 12). There was some variation in the sizes and distributions of somata between the left and 472 L.C. SUDLOW ET AL. Fig. 4. Schematic diagram of the distribution of serotonin-immunoreactive neurons on the ventral surface of the cerebropleural and pedal ganglia of Pleurobranchaea. Pedal abbreviation: VML, ventromedial lobe. For other abbreviations, see the legend to Figure 3. right ganglia, as well as between specimens. The dorsal surface of the left pedal ganglion exhibited a characteristic giant soma (250–280 µm) near the root of the cerebropedal connective (Figs. 11, 12), possibly the previously identified ‘‘left pedal 1 neuron’’ (Willows et al., 1973). In some specimens, additional giant somata could be observed posterior and slightly ventral to the medial giant. These may have been ventral somata that migrated to a slightly more medial location, and hence could be readily observed from the dorsal surface at nearly the same plane of focus as the medial giant. In whole-mounts of the left ganglion, 3–5 medium-sized (90–220 µm) and 20–24 small-diameter somata (30–90 µm) were observed (Figs. 11, 12). The dorsal surface of the right pedal ganglion lacked the medial giant found in the contralateral ganglion. Three asymmetrical medium-sized and several small (30–35 µm) somata were slightly posterior in the posterolateral region of the right pedal ganglion. The ventral surfaces of left and right pedal ganglia were the richest in serotonin immunoreactivity of the Tritonia CNS (Fig. 12). The somata were located near the anterior and the posterior margins of each ganglion, leaving a zone between the margins relatively devoid of 5-HT-IR somata (Fig. 12). At least 20 giant somata could be observed in each pedal ganglion, mostly near the posterior margin. Medium-diameter and small-diameter somata were found predominately near the anterior margin of the pedal ganglia. Buccal ganglion: Tritonia As in Pleurobranchaea, the buccal ganglion of Tritonia lacked 5-HT-IR somata (not shown). Stained fibers coursed through the buccal ganglion to branch and exit in nerves, and processes were also observed enmeshing the somata of the buccal ganglion. DISCUSSION Localization of identified 5-HT-IR somata in Pleurobranchaea The present morphologic results can be combined in several cases with physiologic data on identified neurons and with previous data on the peripheral distribution of SEROTONIN IN MOLLUSCS 473 Fig. 5. Serotonin immunoreactivity in the somata and neuropil in the central nervous system of Pleurobranchaea. A: Giant metacerebral serotonin (5-HT) -containing cells (long black arrows) and serotoninimmunoreactive (5-HT-IR) somata of the dorsal anterior cluster (short black arrows) in the anterior lobe of cerebropleural ganglion (horizontal cryotome sections). Curved open arrows indicate areas of intense neuropilar 5-HT-IR staining (asterisks) in the anterior cerebral and rhinophore lobes of the cerebropleural ganglion; small open arrow indicates 5-HT-IR neurons of the dorsal lateral cluster of the cerebro- pleural ganglion; arrowhead indicates a couple of small 5-HT-IR neurons in the medial part of the ganglion. B: Identified pedal 5-HT-IR neurons. Medial G-cluster (black arrows) in the right pedal ganglion with associated group of smaller moderately 5-HT-IR neurons in the posterior part of the same lobe (small open arrow). Asterisk indicates 5-HT-IR in the neuropil area; large open arrow indicates the cluster of 5-HT-IR neurons in the posterior lateral lobe of the ganglion. Scale bars 5 500 µm in A, 400 µm in B. 5-HT to yield a fuller picture of serotonergic function in Pleurobranchaea. In particular, we found three identified groups of 5-HT-IR cells whose members already have been identified and characterized to varying degrees: (1) the G neurons of the dorsomedial lobes of the pedal ganglia; (2) the As1-4 and As-rh neurons of the dorsal medial cluster in the cerebropleural ganglion; and (3) the MCG and some members of the dorsal anterior cluster of the cerebropleu- ral ganglion. Each of these clusters may compose a functional group of cells with specific roles in different behaviors. The G neurons of the pedal ganglion are already known to form a functional group whose members are extensively electrically coupled (J. Jing and R. Gillette, unpublished observations) and send peripheral axons to the foot through the pedal nerves (Jing et al., 1993). By analogy to the 474 L.C. SUDLOW ET AL. Fig. 6. Organization of serotonin-immunoreactive (5-HT-IR) neuropil in the cerebropleural ganglion of Pleurobranchaea (cryotome sections). Nomarksi interference contrast image of 5-HT-IR somata in the anterior cerebral lobe. An open arrow indicates the cluster of 5-HT-IR neurons sending processes to the central neuropil areas. Small black arrow indicates 5-HT-IR somata of the dorsal lateral cluster. Asterisk indicates 5-HT-IR in the neuropil area. cc, cerebral commissure. Scale bar 5 80 µm. apparently homologous neurons of Tritonia (Audesirk et al., 1979) and Lymnaea (Syed et al., 1988), they may act as effectors of ciliary locomotion. Additionally, the present demonstration that the G neurons are 5-HT-IR suggests that this group may also compose an intrinsically modulatory motor network; it has been shown that they themselves respond sensitively to 5-HT with depolarization (Sudlow and Gillette, 1995, 1997). Serotonergic activation of the G neurons is mediated by receptors coupled to adenylyl cyclase, whose cyclic AMP product activates a cyclic AMPgated cation current, INa,cAMP (Sudlow and Gillette, 1995, 1997). The elements of the As1-4 group also form a functional group. These neurons are members of the escape swimming pattern generating network in which they provide neuromodulatory effects that promote the swim episode (Jing et al., 1997; Jing and Gillette, 1998). They are both electrically and chemically coupled, and fire synchronously; their outputs descend to the pedal ganglion. These neurons express INa,cAMP (Jing et al., 1997), as do many feeding cells (Green and Gillette, 1983), the G neurons (Sudlow and Gillette, 1995, 1997), and the MCG (K. Huang and R. Gillette, unpublished observations). Whether INa,cAMP in As1-4 responds to 5-HT stimulation is not yet known, but their reciprocal long-lasting EPSPs are consistent with such neuromodulation. Some evidence suggests that the dorsal anterior cluster of 5-HT-IR cells is also a functional group of coupled, coactive elements. The MCG neurons were previously found to be electrically coupled to multiple adjacent neurons with which they fire synchronously during generation of the feeding rhythm (Gillette and Davis, 1977); it is likely Fig. 7. Serotonin immunoreactivity in the buccal ganglion of Pleurobranchaea (whole-mounts). A: Serotonin-immunoreactive (5-HTIR) process can be seen coursing through the buccal ganglion. B: Nomarksi interference contrast image of somata of buccal ganglion exhibit basketlike enmeshing by 5-HT-IR termini. Scale bar 5 500 µm in A, 200 µm in B. SEROTONIN IN MOLLUSCS 475 Fig. 8. Serotonin immunoreactivity in the buccal ganglion of Pleurobranchaea (cryotome sections). A: General dorsal view. Open arrows indicate intensely stained neuropil areas in the medial parts of the ganglion lobes. B: Central neuropil area. Asterisks indicate the serotonin (5-HT) -immunonegative neuronal somata surrounded by 5-HTimmunoreactive terminals. Scale bars 5 500 µm in A, 80 µm in B. that the other serotonergic neurons of this cluster are those coupled cells, by analogy with the electrically coupled networks of the G neurons and the As1-4 group (Jing et al., 1997; J. Jing and R. Gillette, unpublished observations). The MCG itself is activated by touch, chemosensory stimuli and activity in the feeding oscillator motor network, of which it is an element. This neuron provides most or all of the serotonergic innervation of both the feeding network in the buccal ganglion and the feeding musculature of the buccal mass and mouth area; its coupled partners may serve analogous areas in cerebropleural ganglion neuropil and in the musculature of the head region. The MCG also 476 L.C. SUDLOW ET AL. expresses INa,cAMP in its soma (K. Huang and R. Gillette, unpublished observations); the autosensitivity of this cell and its coupled partners to 5-HT is not yet tested. Origin of peripheral innervation The peripheral 5-HT-IR staining in the ciliary layer of the foot noted previously (Moroz et al., 1997) must originate in the G cells of the medial lobe of the pedal ganglia. Similarly, axons of the MCG must provide the extensive serotonin immunoreactivity of the buccal mass and esophagus through its branches known to innervate these areas (Gillette and Davis, 1977). For the most part, the central neurons providing the extensive 5-HT-IR innervation of the reproductive system and chemosensory areas remain to be identified through correlation of the present maps with nerve backfills and double-labeling experiments. However, one notable exception is the finding of serotonin immunoreactivity in As-rh and swim interneuron As4 (J. Jing and R. Gillette, unpublished observations): these cells provide the 5-HT-IR innervation of the rhinophore and tentacle and are thus likely to be the source of serotonergic innervation of the chemosensory epithelium (Moroz et al., 1997). Identification of these cells may enable rigorous testing of the role of 5-HT in modulating chemosensory pathways at the level of receptors, primary afferents, or both. Comparative analysis of serotonin immunoreactivity in gastropods Fig. 9. Serotonin-immunoreactive (5-HT-IR) processes in the visceral ganglion of Pleurobranchaea (whole-mount preparation). Scale bar 5 200 µm. Fig. 10. Serotonin immunoreactivity in the central nervous system of Tritonia (whole-mounts). The large-diameter stained somata in both A and B are the metacerebral giant somata. A: Dorsal view. The long white arrows point to the three dorsal swim interneurons (DSIs) and two additional serotonin-immunoreactive somata, putatively As-rh and As4 homologs of the dorsal medial cluster. The small arrowheads Cerebropleural 5-HT-IR somata in Pleurobranchaea were confined to four primary clusters. Similar clustering has been noted in cerebral ganglia of other gastropod molluscs (Kistler et al., 1985; Land and Crow, 1985; Longley and Longley, 1986; Kemenes et al., 1989; Diefenbach and Goldberg, 1990; Soinila and Mpitsos, 1991; Satterlie et al., 1995). The dorsal and ventral anterior clusters including the symmetrical 5-HT-IR MCG somata, perhaps the most well studied pair of homologous molluscan serotonergic neurons (Weinreich et al., 1973; Weiss and Kupfermann, 1976; Sakharov, 1976; Gillette and Davis, 1977; Granzow and Rowell, 1981; Croll, 1987), and several adjacent smallto medium-sized 5-HT-IR somata near the MCG are clearly recognizable here in Pleurobranchaea and in other species, whereas Tritonia lacks the smaller diameter dorsal anterior cluster cells. Similar symmetrical groups of 5-HTcontaining medium-sized cells and MCGs were also described earlier in Tritonia sp. by Manokhina and Kuz’mina point to lightly staining somata (shaded cells in Fig. 12A) near putative As-rh and As4 homologs. B: Ventral view. The open arrow points to the densely staining neuropil enmeshing the somata in the lateral region of the cerebral ganglion. The small arrowheads point to the ventral anterior cluster. Scale bar 5 200 µm (applies to A,B). SEROTONIN IN MOLLUSCS Fig. 11. Serotonin immunoreactivity in the pedal ganglia of Tritonia (whole-mount preparation). The view is of the dorsal surface of the left pedal ganglion. The large-diameter soma is located near the root of the cerebropedal connective. Scale bar 5 200 µm. (1971) with the formaldehyde-induced fluorescence technique. The locations of the homologous clusters, as identified by the MCG, vary somewhat across species. The dorsal anterior cluster was found at the anterior pole of the cerebropleural ganglia in Pleurobranchaea (Figs. 2, 3, 10, 12) and in the cerebral ganglion of the pteropod Clione (Satterlie et al., 1995). In contrast, in Aplysia (Ono and McCaman, 1984; Longley and Longley, 1986; Soinila and Mpitsos, 1991), Lymnaea (Kemenes et al., 1989), and Helisoma (Diefenbach and Goldberg, 1990), the likely homologous cluster is found somewhat caudally on the dorsal ganglion surface. A likely homologous dorsal medial 5-HT-IR cerebral cluster was found near the cerebral commissures in all the above species (Longley and Longley, 1986; Kemenes et al., 1989; Diefenbach and Goldberg, 1990; Nolen and Carew, 1994; Satterlie et al., 1995), but this cluster was not described in earlier formaldehyde-induced fluorescence studies on Tritonia (Manokhina and Kuz’mina, 1971). In Aplysia, the left and right CB1 5-HT-IR interneurons involved in the modulation of the gill-siphon withdrawal circuit in Aplysia belong to this dorsal medial cerebral cluster (Mackey et al., 1989). These cells in Pleurobranchaea and Tritonia (named the As1-3 and the DSI-A-C cells, respectively) are components of a homologous escape swimming network and potentiate its output (Getting et al., 1980; Lennard et al., 1980; McClellan et al., 1994; Katz et al., 1994; Jing et al., 1997; Jing and Gillette, 1998). 477 Furthermore, we surmise that the dorsal lateral cerebropleural cluster found in Pleurobranchaea is homologous to the lightly stained cluster found at the posterior margin of the Tritonia cerebral commissure. The presence and distribution of giant 5-HT-IR somata in the posterior lobes of Pleurobranchaea’s cerebropleural ganglion is an unusual finding. These lobes were presumably pleural lobes in origin, based on their position relative to the cerebral commissure. No such staining has been reported for the pleural ganglia or pleural lobes of any of the gastropod or pteropod species studied thus far (Goldstein et al., 1984; Ono and McCaman, 1984; Longley and Longley, 1986; Jahan-Parwar et al., 1987; Kemenes et al., 1989; Nolen and Carew, 1994). In contrast, putative serotonergic neurons have been identified in the parietal (intestinal or abdominal) ganglia of Lymnaea (Kemenes et al., 1989) and Aplysia (Ono and McCaman, 1984; Longley and Longley, 1986; Jahan-Parwar et al., 1987; Soinila and Mpitsos, 1991). Because Pleurobranchaea lacks distinct or separate parietal ganglia, it is likely that these 5-HT-IR somata in the posterior cerebropleural lobes represent the remnants of the parietal (intestinal) ganglia found in other molluscs (cf. Bullock and Horridge, 1965). Because Tritonia does not have routinely staining 5-HT-IR somata in its pleural lobes, the remnants of Tritonia’s parietal lobes may have been fused into the pedal ganglia. The buccal ganglia in Pleurobranchaea and Tritonia both lack 5-HT-IR somata even though varicosities and fibers in their buccal ganglia were stained. This finding is consistent with other species (Ono and McCaman, 1984; Longley and Longley, 1986; Jahan-Parwar et al., 1987; Kemenes et al., 1989; Nolen and Carew, 1994; Satterlie et al., 1995) and suggests that the MCGs are the major and perhaps sole source of 5-HT innervation. The investment of the buccal somata by numerous 5-HT-IR varicosities suggests axosomatic synapses similar to MCG innervation in Aplysia (Goldstein et al., 1984). An interesting twist in the centralization of the CNS has left the majority of giant 5-HT-IR somata (the G cells) on the ventral surface of Tritonia’s pedal ganglia, whereas they reside dorsally in Pleurobranchaea (Figs. 3, 12). Presumably, the half-twist in the cerebropedal connectives of the notaspids (the likely ancestral condition) is straightened out by a rotation of the ganglia in the nudibranch as they fuse to the cerebropleural complex, leaving the cell clusters in the ventral position. Pedal 5-HT-IR somata, similar in location to the Pleurobranchaea G cells, are found in a wide variety of opisthobranch, anaspidean, and pteropod molluscs (Ono and McCaman, 1984; Land and Crow, 1985; Longley and Longley, 1986; Kemenes et al., 1989; Diefenbach and Goldberg, 1990; Satterlie et al., 1995). Outside of Tritonia and Pleurobranchaea in which they may effect ciliary locomotion, they act in Aplysia brasiliana and Aplysia californica as modulators of the parapodial muscles (McPherson and Blankenship, 1991a, 1991b, 1991c, 1992) and similarly in Clione limacina (Satterlie, 1995). CONCLUSIONS AND FUTURE DIRECTIONS Serotonin has been proposed to modulate the general arousal state associated with feeding, locomotor, defensive, and other behaviors in gastropods and diverse invertebrates (Sakharov, 1990; Weiger, 1997) based on its abilities to up-regulate spontaneous activity and reactivity in both neural networks and muscle. In general, our 478 L.C. SUDLOW ET AL. Fig. 12. Schematic diagram of the distribution of serotoninimmunoreactive neurons in the central nervous system of Tritonia diomedea. A: Dorsal view. B: Ventral view. Empty circles represent somata that stained in a small number of preparations. Shaded circles represent lightly stained somata. Dark circles represent heavily stained somata. Shaded areas on the ventral lateral region of the cerebral ganglion represent the intensely stained neuropil in Fig. 10B. Nerve notations are based on the anatomical scheme of Willows et al. (1973): CBC, cerebrobuccal connective; CC, cerebral commissure; CeN, cerebral nerve; CPC, cerebropedal connective; PdN, pedal nerve; PlN, pleural nerve. emerging picture of a serotonergic arousal network in Pleurobranchaea bears similarities to the compartmental system of serotonergic cells proposed to modulate swimming in Clione (Satterlie and Norekian, 1996) in which the component clusters can act either in concert and perhaps differentially as well. For Pleurobranchaea, the different serotonergic clusters are associated with aspects of feeding behavior (the cerebral anterior cluster, including the MCG), SEROTONIN IN MOLLUSCS escape swimming (dorsal medial cluster), and creeping locomotion (pedal medial lobe cluster, or G neurons). Our observations indicate that neurons within clusters are coupled electrically, chemically, or both, and are coactive, and that clusters may interact with each other (Jing et al., 1997; J. Jing and R. Gillette, unpublished observations). Clusters are also known to act in different states of coordination: during the escape swim the As1-4 fire in intense bursts locked to the swim rhythm, and during feeding the MCG firing pattern is itself locked to the feeding rhythm, whereas the As1-4 group is silent (J. Jing and R. Gillette, unpublished observations). Thus, the putatively serotonergic clusters have distinct target neural networks, and they potentially coordinate behavior through actions either in concert or semi-independently. It is also notable that some of the putatively serotonergic neurons identified here, including the MCGs and the G cells of the pedal ganglia, may produce another potent neuromodulator, nitric oxide. These neurons coexpress NADPH-diaphorase staining, a marker for nitric oxide synthase (Moroz and Gillette, 1996). The colocalization of serotonin immunoreactivity and NADPH-diaphorase staining in these neurons suggests possible interactions of the two signaling molecules on target cells. 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