Cell Tissue Res (2003) 314:437–448DOI 10.1007/s00441-003-0778-1

R E G U L A R A R T I C L E

Roger P. Croll · Dmitri Y. Boudko · Anthony Pires ·Michael G. Hadfield

Transmitter contents of cells and fibers in the cephalic sensory organsof the gastropod mollusc Phestilla sibogae

Received: 14 April 2003 / Accepted: 15 July 2003 / Published online: 4 November 2003� Springer-Verlag 2003

Abstract While the central ganglia of gastropod mol-luscs have been studied extensively, relatively little isknown about the organization and functions of theperipheral nervous system in these animals. In the presentstudy, we used immunohistochemical procedures toexamine the innervation of the rhinophores, oral tentaclesand region around the mouth of the aeolid nudibranch,Phestilla sibogae. Serotonin-like immunoreactivity wasfound in an extensive network of efferent projectionsapparently originating from central neurons, but was notdetected within any peripheral cell bodies. In contrast,large numbers of peripheral, and presumably sensory,somata exhibited reactivity to an antibody raised againsttyrosine hydroxylase (the enzyme catalyzing the initialstep in the conversion of tyrosine into the cate-cholamines). Additional tyrosine hydroxylase-like immu-noreactivity was detected in afferent fibers of theperipheral cells and in several cells within the rhinophoralganglia. The presence of serotonin, dopamine andnorepinephrine in the rhinophores, tentacles and central

ganglia was confirmed using high-performance liquidchromatography. Finally, FMRFamide-like immunoreac-tivity was detected in cells and tangles of fibers foundwithin the rhinophore, possibly revealing glomerulus-likestructures along olfactory pathways. FMRFamide-likeimmunoreactivity was also found in somata of therhinophoral ganglia, in a small number of cells locatedin the body wall lateral to the tentacles and in whatappeared to be varicose terminals of efferent projectionsto the periphery. Together, these results indicate severalnew features of the gastropod peripheral nervous systemand suggest future experiments that will elucidate thefunction of the novel cells and innervation patternsdescribed here.

Keywords Serotonin · Catecholamine · Dopamine ·FMRFamide · Immunocytochemistry · Phestilla sibogal(Mollusea)

Introduction

The cephalic sensory organs (CSOs: e.g., rhinophores,oral tentacles, lips) of gastropod molluscs mediatenumerous modalities. For example, chemoreceptors inthe CSOs of marine opisthobranchs are known to besensitive to odors emanating from food items, conspecif-ics and predators (Jahan-Parwar 1972; Field and Macmil-lan 1973; Audesirk 1975, 1977; Bicker et al. 1982a; Levyet al. 1997). These organs are also sensitive to mechanicalstimuli associated with water currents eliciting rheotaxis(Murray and Willows 1996), light touch eliciting feedingand searching movements (Field and Macmillan 1973;Kupfermann 1974; Lee and Liegeois 1974; Susswein etal. 1976; Teyke et al. 1990) and stronger stimuli elicitingwithdrawal (Kovac and Davis 1980). Dermal photosen-sitivity (Chase 1979b) has also been described.

The innervation of the CSOs and the ultrastructure oftheir resident cell types have been studied and reviewedextensively (Emery and Audesirk 1978; Davis and Matera1982; Matera and Davis 1982; Croll 1983; Emery 1992),

This research was supported by Natural Sciences and ResearchCouncil of Canada Grant #OPG38863 to R.P.C. and Office ofNaval Research Grant #N00014-94-1-0524 to M.G.H.

R. P. Croll ())Department of Physiology and Biophysics,Dalhousie University,Halifax, NS, B3H 4H7, Canadae-mail: roger.croll@dal.ca

D. Y. Boudko · M. G. HadfieldKewalo Marine Laboratory,University of Hawaii,Honolulu, HI 96813, USA

A. PiresDepartment of Biology,Dickinson College,Carlisle, PA 17013, USA

Present address:D. Y. Boudko, Whitney Laboratory,University of Florida,St. Augustine, FL 32080, USA

but many fundamental questions remain unanswered. Forexample, it remains uncertain which cell types mediatewhich sensory modalities. While correlations between theabundance of different cell types and stimulus sensitiv-ities are suggestive of some functions, firmer evidenceremains elusive. In addition, most morphological studieshave focused upon concentrated cell populations found inrestricted body regions, potentially leaving less abundant,and yet still important, cell types unexamined. Finally,while several studies have focused on sensory cells andafferent pathways, very little attention has been paid topossible efferent control or peripheral processing of thesensory inputs.

We have been using the aeolid nudibranch, Phestillasibogae, as a model species to examine such fundamentalissues concerning gastropod sensory systems. P. sibogaeoffers many advantages for neurobiological investiga-tions. As with other opisthobranchs, its large centralneurons are amenable to electrophysiological analysis(Willows 1985a). In addition, the species is abundant intropical localities and easily reared through its entire lifecycle within the laboratory. Its metamorphosis is reliablyinduced by chemical cues, thus permitting the study ofneuronal events triggering this developmental and behav-ioral event (Hadfield 1978; Hadfield and Pennington1990). Furthermore, its short generation time (<30 days)suggests that it may provide a feasible model for thegenetic analysis of neuronal function and its ontogeny inmolluscs (Miller and Hadfield 1990; Todd et al. 1997).

Importantly for the present series of investigations, theCSOs of P. sibogae have already been studied in certaindetails. For instance, Murphy and Hadfield (1997) studiedthe innervation of the rhinophores and oral tentacles andused electrophysiological techniques to demonstrate thatonly the former were highly sensitive to free amino acidsand to chemical cues emanating from its exclusive food,the hard coral, Porites compressa. In addition, Boudko etal. (1999) used a variety of staining techniques togetherwith electron microscopy to identify three types ofperipheral sensory neurons in the CSOs. Based upontheir morphologies and distributions, these authors sug-gested that: (1) intraepithelial sensory cells concentratedalong the oral tentacles and other regions of the CSOsmight mediate contact chemoreception, (2) subepithelialsensory cells concentrated along the proximal portions ofthe rhinophores might mediate distance chemoreceptionand (3) tufted stiff-cilia sensory cells located at the tips ofthe rhinophores, along the oral tentacles and between theoral tentacles might mediate mechanoreception.

In the present study we extend these studies byexamining the cell and fiber distributions within the CSOscontaining three types of neurotransmitters: the in-doleamine serotonin (5-hydroxytryptamine, 5-HT), cate-cholamines (dopamine and/or norepinephrine) and Phe-Met-Arg-Phe-NH3 (FMRFamide) and related peptides.These transmitters have all been found to be abundantwithin the nervous systems of various gastropods (Walker1986; Greenberg and Price 1992; Moroz et al. 1997;Sudlow et al. 1998; Croll et al. 1999; Croll 2001),

including P. sibogae (Kempf et al. 1992; Pires et al. 2000;Croll et al. 2001). Here we demonstrate that theseneurotransmitters each mark unique features of the CSOsand thus provide insights into both the efferent control ofand afferent flow from these sensory structures.

Preliminary results from this study have appearedpreviously (Boudko et al. 1998).

Materials and methods

Animals

Phestilla sibogae were collected from the wild and maintained atthe Kewalo Marine Laboratory (University of Hawaii) in outdoor,flow-through, seawater tables under ambient light and temperature.They were fed pieces of live Porites compressa ad lib. Over 75specimens, ranging from 3 to 7 mm in length, were examined forthese studies with a minimum of 15 animals being examined foreach transmitter-specific, primary antibody described below.

Immunocytochemistry

Whole-mount immunohistochemistry employed procedures modi-fied from Croll et al. (2001). Briefly, slugs of various sizes weredecapitated using a razor blade. The entire head region was fixedfor 4–12 h either in 4% paraformaldehyde in 0.1 M phosphatebuffer (pH 7.4) at 4�C for eventual detection of 5-HT orFMRFamide-related peptides or in methanol at �18�C for thedetection of tyrosine hydroxylase (TH), which catalyzes the initialstep in the conversion of tyrosine to the various catecholamines (seeCooper et al. 1996; Pani and Croll 1998). The tissues were thenwashed several times in phosphate-buffered saline (PBS; 50 mMNa2HPO4 and 140 mM NaCl, pH 7.2) and then bathed overnight inblocking solution of 1% Triton X-100 and 1% bovine serumalbumen (BSA) in PBS. The tissues were next incubated for 2–3 days in one of the primary antibodies at 4�C. The anti-5-HT andanti-FMRFamide sera were raised in rabbits and obtained fromDiasorin, Inc. (Stillwater, MN). They were used at 1:500 dilution.The monoclonal anti-TH antibody was developed in mouse andalso obtained from Diasorin, Inc. This latter antibody was used at1:50 dilution. (See Voronezhskaya et al. 1999; Croll et al. 2001 andCroll 2001 for demonstrations of the specificity of this antibody ingastropods.)

Following incubation in primary antibodies, the tissues weregiven another three or four 1-h washes in PBS, and were thenincubated for 12–24 h in goat anti-rabbit or sheep anti-mouseantibodies (1:50 dilution) labeled with either FITC or rhodamine(Jackson Labs., West Grove, PA, USA) at 4�C. After anotherseveral washes in PBS, the CSOs were dissected from thesurrounding tissues and mounted between glass coverslips in asolution of three parts glycerol to one part 0.1 M TRIS buffer(pH 8.0) with the addition of 2% n-propyl gallate (Giloh and Sedat1982). Preparations were viewed and photographed on a ZeissAxiophot microscope equipped with filter blocks with 510–560 nmexcitation and 590-nm longpass barrier filters for viewing rhoda-mine and 450–490 nm excitation and 515- to 565-nm bandpassbarrier filters for viewing FITC.

Use of FITC and rhodamine yielded identical results. Additionalcontrol experiments involved the use of identical procedures asdescribed above except for the replacement of the primaryantiserum with either 1% normal mouse serum or with the serumdilutant alone. No staining was observed in any of these controlpreparations. As positive controls, the central ganglia of thedissected preparations were also examined and the distributions ofthe various immunoreactivities are described elsewhere (Croll et al.2001).

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Photography

Histological preparations were photographed using Kodak TMAX100 film. The developed negatives were digitally scanned and theimages were then assembled into plates and labeled usingPhotoshop 6.0 (Adobe Systems, Inc., San Jose, CA). Contrastand brightness of the images were adjusted to provide consistencywithin plates.

Extraction of monoamines and analysis by chromatography

Catecholamines

Oral tentacles, rhinophores and central ganglia (includingrhinophoral, cerebropleural and pedal ganglia) were dissectedfrom adult P. sibogae. Tissues of each type from two individualswere combined in each of four replicate samples to ensureadequate material for chromatographic analysis of catecholamines.Each sample was homogenized in 600 �l of ice-cold perchloratebuffer (Pires et al. 1997) containing 60 pmol dihydroxybenzy-lamine (DHBA) as an internal standard. Homogenates werecentrifuged (15,000�g, 10 min) and catecholamines were extractedover alumina from supernatants as described previously (Pires etal. 1997, 2000). Catecholamines were separated in duplicate 80-�linjections of each extract on a Bioanalytical Systems MF-6213analytical column (100�3.2 mm, 3 �m C-18 reverse phasepacking). The aqueous portion of the mobile phase contained100 mM monochloroacetic acid, 1.3 mM Na2EDTA and 1.3 mMsodium octyl sulfate. Mobile phase was adjusted to pH 3.1 withNaOH. The organic portion of the mobile phase was 6% (v/v)acetonitrile. Flow rate was set at 1.0 ml/min. Catecholamines weredetected with a Bioanalytical Systems LC-4C amperometricelectrochemical detector with a glassy carbon working electrodeset at an oxidizing potential of 650 mV against a Ag/AgClreference electrode. Detector output was low-pass filtered (1.0 Hzcutoff) and sent to a Hewlett-Packard 3395 integrator that printedchromatograms and calculated peak areas and retention times ofcatecholamines in tissue extracts and standard mixtures. Cate-cholamine contents were expressed as picomoles free base/�gprotein. Protein contents of homogenates were determined by theCoomassie blue dye binding method (Bradford 1976) with a bovineserum albumin standard.

Serotonin

Tissues were dissected and combined from two adult P. sibogae tomake a single sample each of oral tentacles, rhinophores and centralganglia. Tissues were homogenized and centrifuged as above.Supernatants were diluted 1:1 with deionized water and a single 80-�l injection of each was made directly into the chromatographicsystem under the conditions described above. Further injections ofthe ganglionic sample were made at a different pH (5.0) andacetonitrile concentration (9%) to confirm the identity of theputative 5-HT peak by coelution with 5-HT standards.

Results

The cephalic sensory organs (CSOs) are densely inner-vated by several nerves exiting from the cerebropleuralganglia (Fig. 1). The rhinophores are paired dorsalappendages innervated by arborizations of four rhino-phoral nerves which leave each of the paired rhinophoralganglia. These small ganglia are, in turn, attached directlyto the anterior margin of each cerebropleural ganglion.(Details of the ganglia and the origins of the variousperipheral nerves are shown elsewhere; Croll et al. 2001.)

The oral tentacles, which are more ventral appendages,derive their innervation from a branch of the commonlabial and oral tentacle nerve (CLOTN), which alsoinnervates a region of the body wall lateral to the tentacleand anterior and lateral areas around the mouth. Thecephalic shield, which constitutes the anterior body wallin a region between the rhinophores and tentacles, isinnervated by the cephalic shield nerve (CSN). Finally themouth is innervated along its lateral and anterior marginsby a branch of the CLOTN, while its posterior margin isinnervated by the posterior oral nerve (PON).

5-HT-like immunoreactivity

Serotonin (5-HT)-like immunoreactive (lir) fibers wereabundant throughout the CSOs (Fig. 2). A large numberof the fibers appeared to terminate subepithelially, withno fibers penetrating the epithelium itself in therhinophore (Fig. 2A, B), oral tentacle (Fig. 2C), oranterior margin of the cephalic shield (Fig. 2D). Termi-nals of 5-HT-lir fibers were also concentrated around thelips (Fig. 2E).

Many, if not all, of the 5-HT-lir fibers appeared tooriginate centrally; no 5-HT-lir somata were observedperipherally, and the small terminal branches of fibers inthe CSOs often appeared to derive from larger fiberswhich originated in the central nervous system. This was

Fig. 1 Schematic diagram of frontal view of P. sibogae showinginnervation of the cephalic sensory organs. The left side of thediagram shows the innervation patterns of the rhinophoral nerves(RhN), the common labial and oral tentacle nerve (CLOTN), thecephalic shield nerve (CSN) and the posterior oral nerve (PON).Also shown are the locations of the cerebropleural ganglia (CPG),pedal ganglia (PeG) and rhinophoral ganglion (RhG). The rightside of the diagram shows the locations of each of the cephalicsensory organs: rhinophore (Rh), oral tentacle (OT), and cephalicshield (CS). Also shown are the locations of the mouth (M) and theposition of the left side of the internal buccal mass (BM)

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most apparent in the rhinophore (Fig. 2B). Generally fourlarge fibers entered the base of each rhinophore and thenbranched repeatedly. Two large fibers running througheach rhinophoral ganglion appeared to be the source ofthe 5-HT-lir fibers found in the rhinophores. In addition tothese large fibers in passage, the rhinophoral ganglia alsoappeared to receive serotonergic input from smallerfibers. One group of small 5-HT-lir fibers appeared toterminate within the ganglionic neuropil (Fig. 2F), while anetwork of 5-HT-lir fibers surrounded the cortex of theganglion (Fig. 2G).

Tyrosine hydroxylase (TH)-like immunoreactivity

The pattern of TH-like immunoreactivity was strikinglydifferent from that of 5-HT-like immunoreactivity in the

CSOs. A small number of TH-lir somata were scatteredalong the lengths of the rhinophores (Fig. 3A) withincreased concentrations near their tips (not shown). Incontrast, the small, TH-lir somata were much moreabundant over the entire lengths of the tentacles (Fig. 3B).TH-lir somata were also abundant over the surface of thecephalic shield (Fig. 3C) and around the mouth, but theirpresence ended abruptly at the lips (Fig. 3E, F).

In all regions of the CSOs the TH-lir somata weresmall and located subepithelially. They possessed apicalprocesses which penetrated the epithelium to terminate atthe outer surface of the body wall. Because of the tightpacking of the somata, it was difficult to estimate theirdensities. However, over the relatively flat surface of thecephalic shield, it was possible to find focal planescontaining only the apical processes (Fig. 3D) and toaccurately count their numbers. The density of such TH-

Fig. 2A–G Serotonin (5-HT)-like immunoreactivity in the cephalicsensory organs of P. sibogae. A Numerous small fibers (examplesindicated by arrows) running the length of the rhinophore. Fibersdo not invade the epithelium (Epi). B Higher magnification view ofbase of rhinophore showing three large, immunoreactive fibers(arrows). A fourth large fiber is out of focus. C The tip of the oraltentacle showing that numerous immunoreactive fibers (examplesindicated by arrows) are excluded from the epithelium. D Anteriormargin of cephalic shield showing numerous fibers which runbeneath but without entering the epithelium. E Numerous immu-

noreactive fibers (examples indicated by arrows) and theirterminals concentrated around the opening of the mouth. F Largefibers (medium-sized arrows) running though the rhinophoreganglion. Smaller immunoreactive fibers terminate within therhinophoral ganglion neuropil (smaller arrows). Also shown is alarge immunoreactive cell (hollow arrow) lying within the attachedcerebropleural ganglion (CPG). G Small immunoreactive fibers(smaller arrows) on the surface of the same rhinophoral ganglionshown in F. Scale bars approximately 60 mm (A, D, E), 25 mm (B,C), 80 mm (F, G)

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lir processes was 15–20 elements in regions measuring100�100 mm (or about 1,500–2,000 elements/mm2).

Small, TH-lir fibers were often observed projectingcentrally from the subepithelial somata (Fig. 3C). Inaddition, the various nerves innervating the tentacles andthe cephalic and oral shields contained numerous smallTH-lir fibers (data not shown but see Croll et al. 2001). Incontrast, the rhinophore nerves contained relatively fewsuch fibers. However, the rhinophoral ganglia containedseveral small TH-lir somata (Fig. 3G). Two larger TH-lir

somata were reliably found at the base of each ganglion.A single axon projected into the cerebropleural ganglionfrom each of these somata.

FMRFamide-like immunoreactivity

FMRFamide-like immunoreactivity presented a distinctlypatchy pattern within the rhinophores (Fig. 4A). Uponcloser examination, the patches appeared to consist of

Fig. 3A–G Tyrosine hydroxylase (TH)-like immunoreactivity inthe cephalic sensory organs of P. sibogae. A A few smallimmunoreactive somata (examples indicated by medium-sizedarrows) are scattered along the length of the rhinophore. Increasedbackground fluorescence near the tip is due to a larger number ofthese cells which lie out of focus. A process (smaller arrow) fromone of these cells can be seen penetrating the epithelium near thetip. B A much larger number of immunoreactive somata (examplesindicated by medium-sized arrows) are present in the oral tentacles.Many processes (smallest arrows) from these cells can be seenpenetrating the epithelium. Largest arrows indicate nerves con-taining immunoreactive fibers. C Higher magnification view oftightly packed subepithelial cells (examples indicated by medium-sized arrows) along the anterior margin of the cephalic shield.

Smallest arrows indicate examples of processes from these cellswhich penetrate the overlying epithelium (Epi). Largest arrowsindicate examples of centrally projecting axons from the cells. DEpithelial terminals (examples indicated by arrows) of immunore-active processes over the surface of the cephalic shield. E Tightlypacked immunoreactive somata (examples indicated by arrows) inthe region surrounding the mouth. F Higher magnification ofsomata (arrows) near the mouth. G Small immunoreactive somata(examples indicated by smallest arrows) scattered within therhinophoral ganglia. Medium-sized arrows show two larger immu-noreactive cells which were reliably located near the base of theganglion and which possessed centrally projecting axons (largestarrows). Scale bars approximately 100 mm (A, B), 40 mm C, F),25 mm (D), 80 mm (E), 45 mm (G)

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Fig. 4A–I FMRFamide-like immunoreactivity in the cephalicsensory organs of P. sibogae. A Patches (examples indicated byarrows) of immunoreactivity within the rhinophore. B Highermagnification view of patches (examples indicated by largerarrows) and interconnecting fibers (smaller arrows) in therhinophore. C Another high-magnification view of the rhinophoreshowing isolated small somata (arrows) lying deep within thetissue. D Immunoreactivity within dissected rhinophoral nervesshowing entangled fibers and terminals (largest arrows) and somata(medium-sized arrows) with interconnecting fibers (smallest ar-rows). E Patches (larger arrows) of entangled fibers and numerouslongitudinal fibers (examples indicated by smaller arrows) in theoral tentacle. F Numerous immunoreactive terminals speckle the

body wall surrounding the mouth. The fibers from which theterminals originate are generally out of focus. G Immunoreactivesomata (larger arrows) and their axons (smaller arrows) lyingwithin the body wall lateral to the oral tentacle. H Smallimmunoreactive somata (examples indicated by smaller arrows)scattered within the rhinophoral ganglion. A larger soma (largerarrow) with a centrally projecting axon is reliably located near thebase of the ganglion. I Lower magnification view of rhinophoralganglion showing both the smaller somata (examples indicated bysmaller arrows) and a large region of immunoreactivity locateddeep within the base of the ganglion (large arrow). Scale barsapproximately 75 mm (A), 40 mm (B, C), 30 mm (D, I), 100 mm (E),60 mm (F), 45 mm (G), 35 mm (H)

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concentrations of tightly knotted fibers and terminals(Fig. 4B). Often one or two small fibers were foundlinking adjacent patches. Small somata were also en-countered deep within the rhinophores (Fig. 4C). Upondissection of the rhinophores, it became apparent thatboth the patches of entangled fibers and the somata werelocated in the major nerve branches running through therhinophore (Fig. 4D).

In contrast to the rhinophores, each oral tentaclecontained only two such patches of entangled fibers(Fig. 4E). These patches were somewhat larger than thosefound in the rhinophores and were located near the lateralmargin of the tentacle at its base. The rest of the tentaclepossessed more continuous fibers which appeared to runthe length of the structure (Fig. 4E). These fibers, likethose found within the patches of entangled fibers in therhinophores, generally possessed numerous large vari-cosities along their lengths.

The cephalic shield and regions around the mouth alsocontained FMRFamide-lir fibers and varicosities whichwere particularly concentrated near the lips (Fig. 4F). Inaddition, six to eight somata were routinely observed inthe body wall lateral to the base of the tentacles (Fig. 4G).These cells were distinctly different from those observed

in the rhinophores. The cells near the tentacle were larger,were located subepithelially within the body wall, andappeared to be monopolar with a single, prominent,centrally projecting axon.

In addition to the FMRFamide-lir elements locateddistally in the CSOs, immunoreactive cells and fiberswere also observed within the rhinophoral ganglia(Fig. 4H, I). Each ganglion contained numerous smallimmunoreactive somata which were particularly concen-trated in a region midway along the lateral margin. Eachrhinophoral ganglion also contained a distinctly largersoma with a centrally projecting axon near its connectionto the cerebropleural ganglion. Finally each rhinophoralganglion also contained a region of immunoreactivitywhich was generally less intensely labeled than theimmunoreactivity in the above-mentioned somata andfibers. This sharply defined region of immunoreactivitywas located in the center of the rhinophoral ganglion.While the region may have been composed of a singlecell, no associated axon was ever observed.

HPLC

Dopamine (DA) and norepinephrine (NE) were quantifiedin homogenates of rhinophores, oral tentacles and centralganglia of P. sibogae. A representative chromatogram ofan extract of rhinophores is shown in Fig. 5. Amounts ofboth DA and NE per unit soluble protein were roughly anorder of magnitude greater, and more variable, in centralganglia than in rhinophores and oral tentacles. In all threelocations DA was the predominant catecholamine, ex-ceeding NE by a factor of 12 in central ganglia and byhigher ratios in peripheral tissues (Fig. 6).

Fig. 5 Chromatograms of A catecholamine standards, and Balumina extract of rhinophores pooled from two adult P. sibogae.Standards contained 0.8 pmol each of l-DOPA, norepinephrine(NE) and dihydroxybenzylamine (DHBA), and 4 pmol of dopamine(DA). Vertical arrows mark changes in detector current scale asindicated

Fig. 6 Amounts of A dopamine and B norepinephrine per micro-gram soluble protein, in homogenates of rhinophores (Rh), oraltentacles (OT), and central ganglia (CNS) of P. sibogae. Valuesindicate means +1 SEM based on four replicate homogenates

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Serotonin contents were determined by HPLC in oneof the samples that had also been analyzed for cate-cholamines. Values for serotonin in the three tissue typeswere: 11.0 pM/mg protein in the rhinophores, 3.3 pM/mgprotein in the oral tentacles, and 45.5 pM/mg protein in thecentral ganglia.

Discussion

The three primary antibodies employed in this studydisclosed distinctly different, but complementary, picturesof innervation within the cephalic sensory organs (CSOs)of P. sibogae. Immunoreactivity for 5-HT revealedexclusively efferent innervation, found within fiberswhich appeared to terminate primarily in subepithelialregions, although innervation of deeper structures (e.g.,muscles) is also likely. In contrast, TH-like immunoreac-tivity revealed a large class(es) of peripheral, possiblysensory neurons and their afferent projections. FMR-Famide-like immunoreactivity revealed the most complexpattern, which consisted of at least two types of peripheralneurons, what may be numerous, distributed sites fordistal processing of sensory information, and probably

also some efferent innervation. The distribution patternsfor 5-HT-, TH- and FMRFamide-like immunoreactivitiesare summarized in Fig. 7.

In addition, the different antibodies also providedsome of the first views of the organization of therhinophoral ganglia in opisthobranch gastropods. Ele-ments revealed in the present study included: (1) efferentfibers in passage; (2) smaller efferent fibers whichterminated in the ganglionic neuropil; (3) numerous,small, intrinsic cells; (4) a smaller number of slightlylarger cells with projections into the adjacent cerebro-pleural ganglion; and (5) what may be a single large cellin the center of the ganglion.

Finally, the present study also offered chromatographicevidence confirming the presence of 5-HT and alsoindicating that dopamine is the predominant catechol-amine in the CSOs, although norepinephrine was alsodetected in smaller amounts.

5-HT immunoreactivity

Although the distribution of 5-HT-containing cells hasbeen well studied in the central ganglia of various

Fig. 7A–C Summary of the distributions of immunoreactivity inthe cephalic sensory organs of P. sibogae. The top portion of eachpart of the figure shows the innervation of the right cephalicsensory organs in frontal view comparable to that seen in Fig. 1.The distribution of immunoreactive somata in the rhinophoralganglion (RhG) is also shown. Other abbreviations are indicated inFig. 1. The bottom of each part of the figure is a schematicrepresentation of higher magnification views of innervation withinand beneath the epithelium of the body wall of the cephalic sensoryorgans. The locations of the epithelial cells (EC), sensory cells(SC), glomerulus-like structures (G) and basement membrane (BM)are indicated. A 5-HT-like immunoreactivity, found exclusively in

fibers which terminate beneath the epithelium. B TH-like immu-noreactivity, expressed in numerous small cells found throughoutthe cephalic sensory organs. These subepithelial somata areparticularly concentrated in the tentacles, around the mouth andat the tips of the rhinophores. C FMRFamide-like immunoreactivityis found in numerous fibers which terminate either in glomerulus-like structures in the rhinophores and at the bases of the tentacles orsubepithelially throughout the cephalic sensory organs. Immuno-reactivity is also found in somata located in rhinophore nerves andin a small population of larger, monopolar cells located lateral tobases of the tentacles

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molluscs (Croll and Lo 1986; Longley and Longley 1986;Croll 1987, 1988; Croll and Chiasson 1989; Sudlow et al.1998; Katz et al. 2001) including P. sibogae (Croll et al.2001), the distribution of 5-HT-containing elements inperipheral tissues has received much less attention.However, both histological and chromatographic evi-dence presented in this study suggests that 5-HT isabundant and probably plays important roles in thevarious CSOs.

Here we demonstrated that 5-HT-like immunoreactiv-ity is found in large numbers of what appear to be efferentfibers originating from the central ganglia; we found noimmunoreactive peripheral somata, although we previ-ously demonstrated numerous central neurons which were5-HT-lir (Croll et al. 2001). The absence of 5-HT-lirperipheral somata is consistent with reports of onlyefferent fibers in selected peripheral tissues, such as theheart and buccal mass, of other gastropods (Weiss et al.1978; Buckett et al. 1990; Skelton et al. 1992). Morerecently, Moroz et al. (1997) conducted a more compre-hensive study of various peripheral tissues in two otheropisthobranchs, Pleurobranchaea and Tritonia, and theyalso reported what appeared to be exclusively efferentfibers with no peripheral somata. Similar results havebeen obtained in other molluscs; Pani and Croll (1995)presented chromatographic evidence for the presence of5-HT in various tissues of a bivalve, while histologicalstudies indicated that the 5-HT originated from theefferent projections of central neurons (Croll et al. 1995).

The primary goal of the present study was to screen forgeneral patterns of immunoreactivity in the CSOs, andthus identify potential topics for future detailed investi-gation. Without higher resolution microscopy and cor-roborating electrophysiological evidence, we are unableto determine the roles that 5-HT may play in the CSOs ofP. sibogae. In other gastropod and bivalve molluscs,however, 5-HT has been shown to be a potent modulatorof muscular activity and contractility (Twarog 1954;Weiss et al. 1978; Ram et al. 1981, 1999). The variousmuscles which move the rhinophores, tentacles and lipsand the network of subepithelial muscle fibers responsiblefor local contractions are, therefore, likely recipients ofserotonergic input.

Our results, showing 5-HT-lir fibers terminating bothimmediately beneath the sensory epithelia of the CSOsand in the neuropil of the rhinophoral ganglia, are alsoconsistent with a suggestion by Moroz et al. (1997) that 5-HT may play a role in the peripheral modulation ofsensory input to the CNS. Preliminary electrophysiolog-ical studies on the CSOs of P. sibogae give support to thishypothesis (Boudko and Hadfield 1995). The most likelysource of serotonergic innervation of the rhinophores andpossibly the tentacles derives from a set of approximatelyfive dorsal cells found in the cerebropleural (or simplycerebral) ganglia of most gastropods (Longley andLongley 1986; Croll 1987, 1988; Katz et al. 2001),including P. sibogae (Croll et al. 2001). Sudlow et al.(1998) specifically mentioned that one or two of thesecells innervate the CSOs of closely related nudibranch

and cephalaspidean opisthobranchs. Croll and Lo (1986)and Croll and Chiasson (1989) had previously suggestedthat similarly positioned 5HT-lir cells innervated theCSOs of prosobranch and pulmonate snails.

Tyrosine hydroxylase (TH)-like immunoreactivity

A growing body of evidence shows that catecholaminer-gic neurons are widespread in the peripheral tissues ofboth gastropod (Salimova et al. 1987; Kabotyanskii andSakharov 1990; Croll et al. 1999; Croll 2001) and bivalve(Smith et al. 1998) molluscs. In several instances, thecells possess what appear to be sensory dendrites whichpenetrate the overlying epithelium and also axons whichproject centrally and/or form subepithelial plexuses.Furthermore, these cells first appear in embryonic orlarval stages of development (Croll et al. 1997; Dickinsonet al. 1999; Voronezhskaya et al. 1999; Dickinson et al.2000; Pires et al. 2000). The present study now extendsthese observations to postlarval P. sibogae and providesthe first description of regional differences in theabundance of these cells over the CSOs of an opistho-branch.

The different CSOs are well known to possess varyingsensitivities to environmental stimuli. For instance, therhinophores are generally thought to be involved in thedetection of dilute substances used in the behavioralorientation to distant food items or to conspecifics (Leeand Liegeois 1974; Audesirk 1975; Chase 1979a; Croll1983; Murphy and Hadfield 1997). The relative paucity ofTH-lir cells along the lengths of the rhinophores, there-fore, suggests that these cells do not mediate distancechemoreception. Instead, concentrations of these cells onthe oral tentacles, cephalic shield and around the mouthmay be more consistent with roles in contact chemore-ception or mechanoreception. However, Boudko et al.(1999), consistent with earlier authors, ascribed the role ofcontact chemoreception to a morphologically distinctpopulation of intraepithelial neurons, thus suggesting thatmechanoreception is the most likely modality for the TH-lir cells. Finally, it should be noted that the TH-lir cellsare morphologically similar to a population of subepithe-lial tufted, stiff cilia (TSC) cells previously suggested tomediate mechanoreception in Phestilla (Boudko et al.1999). While we report relatively higher concentrations ofTH-lir cells in the tentacles, cephalic shield and aroundthe mouth than previously reported for the TSC cells,such differences might arise from sampling procedures orfrom the labeling of overlapping, but not identical, subsetsof cells (for instance, TH-like immunoreactivity might beexpressed in two cell types, only one of which possessestufted, stiff cilia).

Clearly more studies are needed to identify the roles ofboth the subepithelial TH-lir cells and the TH-lir cellslocated in the rhinophoral ganglion, a structure whichprobably plays a major role in the processing of sensoryinput to the central nervous system (Bicker et al. 1982b).Such studies will be aided by our chromatographic

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evidence demonstrating both that catecholamines aremore plentiful in the tentacles than in the rhinophores andthat dopamine is the most abundant catecholamine in P.sibogae as in most other molluscs studied to date(McCaman et al. 1973; McCaman 1984; Pani and Croll1995). Norepinephrine, the only other catecholaminedetected here, is found in much lower concentrations.With the identification of candidate neurotransmitters, thefunctions of the peripheral catecholaminergic neurons canbe tested pharmacologically by using specific agonists,antagonists and transmitter depletors.

FMRFamide-like immunoreactivity

As with TH-like immunoreactivity, the FMRFamide-likeimmunoreactivity demonstrated that the innervation pat-terns of the rhinophores and tentacles are very differentfrom each other. The dominant feature of FMRFamide-like immunoreactivity in the rhinophores was that ofpatches of entangled fibers and isolated somata along thelengths of the major nerves. These entanglements maycorrespond to “glomerulus-like” structures noted inprevious studies of the CSOs of P. sibogae (Boudko etal. 1999). Glomeruli have also previously been noted inassociation with the sensory epithelium of the dorsaltentacle of land snails (Chase and Tolloczko 1986).Unfortunately, we cannot assess the possible relationshipbetween the entangled FMRFamide-lir fibers and thesensory afferent fibers in the rhinophore of Phestilla sincethe major class of these latter neurons was not identifiedin the present study. Presumably, however, these receptorcells constitute the class of subepithelial cells whichBoudko et al. (1999) described as residing along portionsof each rhinophore. Nonetheless, it is tempting tospeculate that the glomerulus-like structures are involvedin the local processing of distant chemoreceptive stimuli,as has been demonstrated for olfactory processing in awide variety of other animals (Shepherd 1972; Korsching2002; Lei et al. 2002). The tentacles, in contrast to therhinophores, possess only two such patches of tangledFMRFamide-lir fibers, lying laterally near the base of theappendage, and have no specialized ganglia associatedwith them. These glomerulus-like structures correspond tothe positions of secondary populations of putativedistance chemoreceptor, subepithelial sensory neuronsdescribed by Boudko et al. (1999) as being locatedlaterally near the base of each tentacle.

In addition to the numerous small cells located alongthe branches of the rhinophoral nerves, the rhinophoralganglia also possessed several similar neurons and a largeimmunoreactive structure lying within its core. The natureof this latter structure could not be determined from thepresent study but would likely be resolved in futurestudies of histological sections. The role of the rhino-phoral ganglion itself is also unclear since it might beinvolved in processing of afferent information, efferentcontrol over sensory receptors or even specialized motorcontrol for the rhinophores.

The majority of FMRFamide-like immunoreactivity inthe tentacles, over the surfaces of the cephalic shield andaround the mouth is more likely derived from efferentprojections originating with the many FMRFamide-lirsomata found in the central nervous system (Croll et al.2001). As with the 5-HT-lir terminals in these regions, theFMRFamide-lir terminals, characterized by their largevaricosities, may be involved either in the modulation ofmuscular activity and/or contractility (Cawthorpe andLukowiak 1990; Evans et al. 1999) or in the modulationof sensory cell activity. This issue can only be resolvedwith further research.

A final source of FMRFamide-like immunoreactivityin the periphery originated from a small set of neuronslocated lateral to the bases of the tentacles. These cellsappeared to be monopolar with no sensory dendrites andtheir axons could not be traced to their terminations.While putative sensory neurons exhibiting FMRFamide-lir have been described in the optic tentacles of stylom-matophoran pulmonate gastropods (Suzuki et al. 1997)and within the osphradia of basommatophoran pulmonategastropods (Nezlin and Voronezhskaya 1997), theyappear to be distinctly different in morphology fromthose observed in P. sibogae, and therefore presumablyhave different functions.

Conclusions

The gastropod central nervous system has been thesubject of close scrutiny for many years (Kandel 1979;Willows 1985b, 1986; Kits et al. 1991; Chase 2002).However, the peripheral nervous system, while longknown to be extensive and complex (Bullock andHorridge 1965), has remained relatively unexplored. Inthe present study we employed immunohistochemicaldetection of three neurotransmitter types to examineinnervation patterns in the CSOs of P. sibogae. 5-HT-likeimmunoreactivity revealed what appeared to be peripheralprojections of central neurons, and suggested numerouspossibilities for efferent control of sensory processingboth in the epithelium and in the rhinophoral ganglionthrough which the afferent fibers pass. The abundant TH-lir neurons appear to be sensory cells, possibly mediatinglight touch and/or sensitivity to water currents. TheFMRFamide-lir cells and the closely associated tangles offibers and endings in the rhinophoral nerves may beinvolved in the processing of afferent signals fromolfactory receptors. In contrast, examination of the largerand less abundant FMRFamide-lir cells lateral to thebases of the oral tentacles yielded no clues regarding theirpossible functions. In addition to permitting visualizationof novel cells and fibers, the identification of transmittersemployed by the different elements also leads to theformulation of specific tests using agonists and antago-nists to examine the sufficiency and necessity of thevarious cell types in the perception of different sensoryqualities.

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Acknowledgements We thank Annette Kolb-Klussman for criti-cally reading an earlier version of this report.

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