Ontogeny of serotonergic neurons inAplysia californica

Ontogeny of Serotonergic Neuronsin Aplysia californica

RENE MAROIS1 AND THOMAS J. CAREW2*1Interdepartmental Neuroscience Program, Yale University, New Haven, Connecticut 06520

2Departments of Psychology and Biology, Yale University, New Haven, Connecticut 06520

ABSTRACTAlthough the identity, projection patterns, and functions of serotonergic neurons in

juvenile and adult Aplysia are relatively well understood, little is known about thedevelopment of these cells. We have used light and electron microscopic immunocytochemis-try to investigate the genesis, differentiation, identity, and fate of the serotonergic cells in theembryonic, larval, and metamorphic stages of the life cycle of Aplysia. The results indicatethat the first serotonergic cells emerge at midembryogenesis and that a total of five cellsmakes up the entire serotonergic system by hatching. These cells are part of a newlydiscovered ganglion in Aplysia, called the apical ganglion. This serotonergic system of fivecells remains essentially intact throughout larval development. The apical ganglion, togetherwith its serotonergic cells, is resorbed at metamorphosis. A distinct set of serotonergic cells,which begins to emerge by the end of the larval period, is rapidly elaborated during themetamorphic and early juvenile periods to form the adult serotonergic system. These resultssupport the view that the larval and adult forms of the Aplysia nervous system consist ofentirely distinct sets of serotonergic cells, each adapted to the stage-specific morphologicaland behavioral characteristics of the animal. J. Comp. Neurol. 386:477–490, 1997.r 1997 Wiley-Liss, Inc.

Indexing terms: cell death; differentiation; immunocytochemistry; metamorphosis; mollusc

The relative simplicity of the nervous system of the marinemollusc Aplysia has been highly useful for elucidating thecellular basis of learning and memory (for reviews, see Carewand Sahley, 1986; Bailey and Kandel, 1995; Byrne and Kan-del, 1996). It is, therefore, surprising that the development ofsuch a well-studied neuronal system has been the subject ofrelatively few investigations (Kriegstein, 1977a,b; Schacher etal., 1979a,b; Jacob, 1984; Cash and Carew, 1989). This isdespite the fact that, in the past, invertebrate preparationshave represented powerful models of neuronal development(for reviews, see Chalfie and White, 1988; Anderson et al.,1980; Truman, 1984, 1992; Goodman and Doe, 1993;Shankland, 1995). Expanding our current knowledge of thedevelopment of the nervous system of Aplysia is importantfor two main reasons. The first is to include the molluscanphylum (and particularly the gastropod class) in theanalysis of the range of strategies utilized in neuronaldevelopment across the animal kingdom; there is alreadysome evidence that gastropods may use developmentalstrategies that are significantly different from most otherphyla (Marois and Carew, 1990; Moffett, 1995). A secondmotivation is to understand the development of learningand of its cellular correlates in Aplysia. Although there arenow several models for the study of the cellular andmolecular bases of memory in both vertebrates and

invertebrates, very little is understood about how any ofthese neuronal systems emerge during development.

As a first step toward addressing these two issues, wehave concentrated on the study of the development ofserotonergic cells in Aplysia. The neurotransmitter seroto-nin (5HT) has proven to be a useful marker for followingthe development of a subset of identifiable neurons in thecentral nervous system (CNS) of several gastropods (Crolland Chiasson, 1989; Goldberg and Kater, 1989; Kempf etal., 1991; Marois and Croll, 1992; Barlow and Truman,1992). However, all of these previous studies have beenlimited to the description of the development of serotoner-gic cells by using whole-mount immunocytochemistry. Thepresent study aims at extending our knowledge of thedevelopment of the serotonergic system by describing the

Grant sponsor: NSERC Predoctoral Fellowship; Grant sponsor: NSF;Grant number: IBN922117; Grant sponsor: NIMH Merit Award; Grantnumber: R01-MH-14-1083.

Rene Marois’ current address is Department of Diagnostic Radiology,Yale University School of Medicine, New Haven, CT 06520-8042.

*Correspondence to: Thomas J. Carew, Department of Psychology, YaleUniversity, PO Box 208205, New Haven, CT 06520-8205.E-mail: [email protected]

Received 4 October 1996; Revised 21 April 1997; Accepted 7 May 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 386:477–490 (1997)

r 1997 WILEY-LISS, INC.

ontogeny of serotonergic cells at the ultrastructural leveland by characterizing the genesis, target interaction, andfunction of the serotonergic cells in development.

The selection of serotonin as the focus of our developmen-tal study is also motivated by substantial experimentalevidence strongly implicating this neurotransmitter asone of the central modulatory inputs involved in simpleforms of learning in Aplysia: (Brunelli et al., 1976; Abramset al., 1984; Glanzman et al., 1989; Mackey et al., 1989;Mercer et al., 1991; Emptage et al., 1994; Clark andKandel, 1993). The central role of serotonin in learningmakes this neurotransmitter a highly attractive candidatefor studying the development of the cellular correlates oflearning in Aplysia (Marcus et al., 1994). Our initialstudies of the ontogeny of the serotonergic system ofAplysia have indicated that the basic adult pattern ofserotonergic expression is already established by the begin-ning of juvenile development (Nolen and Carew, 1994),several weeks prior to the emergence of several simpleforms of learning and memory (Rankin and Carew, 1988;Nolen and Carew, 1988; Wright et al., 1991). These resultsraise the questions of when the serotonergic cells firstemerge in development and what functions they mightsubserve.

The present study extends the characterization of thedevelopmental expression of serotonergic neurons fromthe juvenile stage, as established by Nolen and Carew(1994), to the earlier embryonic, larval, and metamorphicstages by investigating the genesis, ontogeny, identity, andfate of the 5HT cells in Aplysia. The companion paper(Marois and Carew, 1997a) describes the projection pat-terns and target tissues of the serotonergic neurons indeveloping Aplysia and provides the framework withinwhich the functions of these serotonergic cells can beexplored.

Some of the results presented in this study have beenpreviously reported in abstract form (Marois and Carew,1989; Marois et al., 1992, 1993).

MATERIALS AND METHODS

Mariculture

Egg masses and larvae were obtained from the Aplysiaresource facility of Miami University. They were main-tained and reared in large rotating bottles of filteredseawater according to the conditions described by Capo etal. (unpublished observations). Under these conditions,embryonic development lasts 9 days at 22°C. Larvae werefed Isochrysis microalgae. Competent larvae were inducedto metamorphose with the addition of the seaweed Gracil-laria. Occasionally, egg masses were obtained from breed-ing adults maintained in our mariculture facility at YaleUniversity and reared as described above. The larval,metamorphic, and juvenile animals were staged accordingto the method described by Kriegstein (1977a).

Bromodeoxyuridine incubation

Different sections of intact egg masses (each sectioncontaining at least 1,000 embryos) were exposed on differ-ent days of embryonic development to a Bromodeoxyuri-dine (BrdU) solution (Sigma, St. Louis, MO; 1 µM infiltered Miami seawater) for a 24-hour period. The eggmass strands were then rinsed several times in filtered

seawater and then returned to the normal maricultureconditions until time for processing.

Immunocytochemistry

Whole-mount immunocytochemistry (ICC). Ani-mals were first anesthetized in a MgCl2 solution isotonic toseawater for 5 minutes at room temperature (rt) followedby 8 minutes on ice. The CNS of juvenile Aplysia wasdissected in artificial seawater prior to ICC processing.Animals or isolated ganglia were then immersed in threechanges of ice-cold fix solution (4% paraformaldehyde inMillonig’s phosphate-buffered saline [PBS]) for 30 minutesand were then left in the fix solution at 4°C for anadditional 2.5 hours (total fixation time: 3 hours). Follow-ing three washes in PBS, Stage 1–6 larvae were exposed totrypsin (Type 1, Sigma, 0.1% in PBS for 5–15 minutes atrt), and all specimens were then immersed in 4% Triton-X100 in PBS for 1 hour, rinsed in PBS, exposed to 10% EDTAin PBS for 45 minutes at rt to decalcify the shell, andrinsed in PBS. This was followed by preincubation (2%goat serum (GS), 0.5% Triton-X 100 in PBS for 1 hour at4°C) and by 1° antibody (Ab) incubation (rabbit anti-serotonin, Incstar, Stillwater, MN; 1:650 in preincubationserum) for 2.5 days at 4°C on a shaker. The animals werethen rinsed in PBS, preincubated in 2% GS in PBS for 1hour at 4°C, and immersed in 2° Ab solution (fluoroisothio-cyanate [FITC]-linked goat anti-rabbit, Sigma, 1:50 inPBS with 2% GS, 0.5% Triton-X 100) for 2.5 hours at 4°C,and rinsed in PBS. The specimens were mounted in a 3:1glycerine:PBS solution and viewed under FITC optics(excitation range, 470–490 nm; dichroic mirror, 510 nm;long pass Barrier Filter, 520 nm) on a Nikon Optiphot-2microscope and photographed with Ilford XP2 400 orKodak Ektachrome 400. The results are based on theobservation of at least 100 animals for each stage.

The specificity of the serotonin antibody used in ourexperiments has been shown in other gastropod studies(Croll and Chiasson, 1989) and in juvenile Aplysia (Nolenand Carew, 1994). In addition, omission of the 1° Ab orpreincubation of the serotonin Ab with serotonin conju-gated to BSA (Incstar) eliminated the staining normallyobserved in hatching Aplysia (Fig. 1D).

Double-labeling whole-mount ICC. Procedures wereas described above until Trypsin incubation (0.05% in PBSfor 12 minutes at rt), followed by PBS rinses. The speci-mens were then exposed for 45 minutes at rt in 2 N HCl,followed by 1 M borate buffer for 5 minutes, and rinsed inPBS. The following primary and secondary preincubationsand incubations for serotonin were performed as describedabove. Following the rinses after the secondary incuba-tion, the animals were processed for detection of BrdU, asdescribed for 5HT, with the following exceptions: preincu-bation serum, 2% normal sheep serum (NSS), 0.5% Triton-X100 in PBS; 1° Ab solution, mouse anti-BrdU (Becton-Dickinson, San Jose, CA) 1:50 in preincubation serum,overnight at 4°C on shaker; 2° Ab solution, biotin-conjugated sheep anti-mouse (Cappel, Durham, NC) 1:50in preincubation serum for 2 hours at 4°C; and 3° Absolution, Texas Red-conjugated avidin (Cappel) 1:50 inpreincubation serum for 2 hours at 4°C. Specimens werethen rinsed and mounted as described above. Isolatedjuvenile ganglia were visualized with both FITC andRhodamine ITC optics (Excitation Filter, 530; BarrierFilter, 590) and photographed as described above. Double-labeled larvae were visualized with a Biorad MRC500

478 R. MAROIS AND T.J. CAREW

scanning laser confocal microscope. Serial optic sectionswere acquired at 1-µm intervals through the depth of thecluster of serotonergic cells. Images were photographedwith a film recorder using EKTAR 125 film. The resultswere based on the observation of at least 20 larvae and 10juveniles examined for each group.

Sectioned tissue ICC. Embryos and larvae were pre-pared as above, with the following modifications: 2° Ab(goat anti-rabbit IgG, Cappel) 1:50 in 2% GS, 0.5% Tri-ton-X 100 in PBS for 2 hours at rt, and 3° Ab (rabbit

peroxidase anti-peroxidase [PAP], Cappel) 1:50 in 2% GS,0.5% Triton-X 100 in PBS for 2 hours at rt. Following thePBS rinses after the 3° Ab solution, the animals wereprocessed for HRP reaction (15 minutes in 0.05% DAB inPBS at rt, followed by 45 minutes in 0.005% H2O2, 0.05%DAB in PBS), rinsed in PBS, and dehydrated in an alcoholseries (50, 70, 80, 95, and 3 3 100% ethanol) and infiltratedin Epon (3 3 propylene oxide (PO); 2:1 PO:Epon; 1:2PO:Epon, and pure Epon). Some animals that had not beenprocessed for ICC were counterstained with the Richard-

Fig.1. Immunocytochemical (ICC) staining of serotonin in whole-mounted Aplysia embryos. A: Anterior view on Day 5 of embryogen-esis. Three cells are immunoreactive (arrows). B: Dorsal view on Day 5of embryogenesis. Anterior is on top. A bilateral pair of cells, anunpaired median cell and a dense central neuritic plexus (arrowhead)

are immunoreactive. C: Dorsal view at hatching. A second bilateralpair of serotonergic (5HT) cells is located medial to the first pair(arrowheads). D: Dorsal view of a control hatchling in which the 1° Abwas preincubated with BSA-conjugated serotonin. Scale bars 5 40 µmin A, 20 µm in B–D.

ONTOGENY OF SEROTONERGIC NEURONS IN APLYSIA 479

son’s stain solution (Richardson et al., 1960). All animalswere sectioned on a Sorvall MT-2 ultramicrotome. Sectionswere viewed under a Nikon Optiphot-2 microscope andphotographed with Kodak T-MAX 100 film. At least 10animals were analyzed at each developmental stage.

Ultrastructural microscopy

Immunoelectron microscopy. Embryos or larvae wereanesthetized as described above. They were fixed for 30minutes on ice and 3 hours at 4°C on shaker (fixative: 4%paraformaldehyde, 0.12% glutaraldehyde, 20% sucrose inMillonig’s phosphate buffer [PB]), rinsed, decalcified in10% EDTA in PBS (PB with 0.9% NaCl), exposed to 1%NaH2B4 in PBS for 1 hour at rt, rinsed several times inPBS, exposed to 0.05% trypsin for 15 minutes, rinsed inPBS, and freeze-thawed (incubation at 4°C for 2 hours incryoprotectant [25% sucrose, 10% glycerol in 0.1 M PB],followed by immersion in liquid N2-cooled isopentane, andby immersion in liquid N2, and rinsed several times inPBS). The 1°, 2°, and 3° Ab incubations were performed asfor sectioned tissue ICC except that the preincubationslasted 2 hours and no Triton-X 100 was present in thepreincubation and incubation solutions. The HRP reactionwas performed as for sectioned tissue ICC, except that ametal-enhanced DAB substrate was used (Pierce, Rock-ford, IL; 45–60 minutes incubation followed by PBS rinses).The animals were subsequently osmicated (2% OsO4 inPBS for 1 hour at rt on a shaker) and dehydrated andinfiltrated in Epon as described for sectioned tissue ICC.Serial silver and gold sections were cut on a Sorvall MT-2microtome and collected serially on either Formvar-coatedslot copper grids or Thin-200 copper grids (EMS, FortWashington, PA). The sections were viewed under a Phil-ips 300 or Zeiss EM-10 transmission electron microscopeat 80 kV.

Conventional electron microscopy. Following anes-thesia, animals were fixed in 2.5% glutaraldehyde, 20%sucrose in 0.1 M PB for 30 minutes on ice followed by 2.5hours at 4°C on shaker. Animals were osmicated, decalci-fied in 10% EDTA in PBS, dehydrated, infiltrated, andsectioned as described above. The grids were counter-stained for 12–15 minutes in 3% uranyl acetate and for 5minutes in 0.3% lead citrate and viewed as describedabove.

RESULTS

Ontogeny of the serotonergic neuronsin Aplysia

The life cycle of Aplysia is divided into five major phases(Kriegstein, 1977a): embryogenesis (lasting approximately9 days); a larval period (30 days, further divided in Stages1–6); metamorphosis (2–3 days, divided into Stages 7 and8); a juvenile period (3 months, Stages 9–12); and adult-hood. As early as Day 5 of embryogenesis, whole-mountICC revealed an unpaired median cell (UMC) and abilateral pair of cells that are immunoreactive for seroto-nin in the anterior region of the animal (Fig. 1A,B). Each ofthe three cells sends one anterior projection and onecentral projection that forms a dense plexus of serotoner-gic neurites (Fig. 1B,C). By Day 7, a second pair of bilateralcells is added slightly medially and posteriorly to the firstpair (Fig. 1C). This serotonergic pattern remains essen-tially unchanged for the rest of the embryonic period and

throughout the larval stages of development (Fig. 2A).Only at the last larval stage, Stage 6, are additionalserotonergic neurons (usually two) detected on each side ofthe five serotonergic cells (Fig. 2B).

Metamorphosis appears to be characterized by dramaticand swift modifications in the serotonergic pattern, includ-ing cell loss (Fig. 2C). The fate of the larval 5HT cellsduring this short metamorphic period (48 hours) could notbe resolved at the individual level with the whole-mountICC technique (but see below).

By the beginning of the juvenile phase at Stage 9, thereis a bilateral cluster of five cells as well as a single largecell located anteroventrally to each cluster (Fig. 2D). Acomparison based on the number, position, and relativecell size of this expression pattern with that obtained witholder juvenile and adult Aplysia (Nolen and Carew, 1994)indicates that the cluster of five serotonergic neuronscorresponds to the posterior cell cluster (PCC) of thecerebral ganglion, whereas the large cell corresponds tothe metacerebral giant cell (C1) of the anterior cell cluster(ACC) of the cerebral ganglion.

Genesis of the serotonergic neurons

Because the whole-mount ICC approach could not deter-mine which, if any, of the larval serotonergic neuronscorrespond to the identified adult serotonergic neurons, wehave used a double-labeling technique to address thisquestion. The thymidine analog BrdU can serve as amarker of terminal cell division (Gratzner, 1982) and wasused here to label the 5HT cells at the time of their genesis.Different portions of a single egg mass were exposed ondifferent days of embryonic development to 1 µM of BrdUsolution in seawater for 24 hours and then returned tonormal seawater for 2 weeks. The larvae were thenprocessed for confocal laser double-ICC for the detection ofboth BrdU- and serotonin-positive cells. Animals exposedto BrdU for 24 hours on the first 2 days of embryogenesisdid not show any double-labeled serotonergic cells 2 weekslater (data not shown). By contrast, the unpaired mediancell (UMC) of animals exposed to BrdU on Day 3 ofembryogenesis was BrdU immunoreactive, whereas thetwo bilateral pairs of 5HT cells were not (Fig. 3A). TheUMC and an adjacent cell (Fig. 3A) appeared to be the onlytwo BrdU-positive cells in the anteromedial region of theanimal. Exposure of animals to BrdU on Day 4 of embryo-genesis resulted in BrdU immunolabeling of the twobilateral pairs of 5HT cells while the unpaired median cellwas BrdU negative (Fig. 3B). A similar, albeit weaker,staining pattern also occurred in some of the animalsexposed to BrdU on Day 5 of embryogenesis (Fig. 3C).Thus, the unpaired median cell is born on the thirdembryonic day, whereas the two bilateral pairs of 5HT cellsare generated on Day 4 or 5.

Other animals of the same egg strand exposed to BrdUwere reared until Stage 11 of juvenile development, whenserotonergic neurons of the cerebral ganglia can be unam-biguously identified. No serotonergic neurons of the cere-bral ganglia (or of any other ganglia) were found to beBrdU positive, regardless of whether these correspondedto animals exposed to BrdU on the third, fourth, or fifthday of embryogenesis (Fig. 4). However, other cells in thecerebral and other ganglia were BrdU positive (Fig. 4B,C),indicating that the absence of double labeling is not aresult of a technical difficulty with BrdU ICC in juvenilesor of the dilution of BrdU during further growth and


Fig. 2. ICC staining of serotonin in whole-mounted larval, metamor-phic, and juvenile Aplysia. A: Dorsal view of a midstage larva (Stage3). Anterior is on top. Only five serotonergic cells are detected. B: Frontalview at end of larval development (Stage 6). One to three more cells areadded lateral to the five cells (arrowheads). Serotonergic (5HT) cellsare also detected in the pedal ganglia (PG). C: Frontal view atmetamorphosis (end of Stage 7). Fewer cells stain in the region of the

cerebral ganglia (CG). D: Frontal view at the beginning of juveniledevelopment (Stage 9). A bilateral cluster of five cells corresponding tothe posterior cell cluster (PCC, small arrows) and a single cellcorresponding to cell C1 of the anterior cell cluster (ACC, arrowhead)of each cerebral ganglia are stained. e, eye; r, radula; v, velum. Scalebars 5 40 µm in A, 20 µm in B–D.


development of the nerve cells. Thus, these results suggestthat none of the five larval serotonergic cells persist asadult serotonergic neurons.

Identity and fate of the serotonergic cells

To determine the identity and fate of the larval seroton-ergic cells, we have followed their ontogeny during larvaland metamorphic development with the use of the immu-noperoxidase technique applied to serially sectioned tis-sue. This technique facilitated determining the conse-quences of metamorphosis on the serotonergic pattern andprovided a finer level of tissue localization than whole-mount ICC.

Serial sections of Stage 1 larvae show that the 5HT cellsare not part of the cerebral ganglia but instead are locatedabove and between them (Fig. 5A). Ultrastructural exami-nation of this region reveals a highly specialized set ofapproximately 15 cells, of which 5 are serotonin positive,surrounding a central neuropil (Fig. 5B,C). Comparison ofthis structure with previous studies indicates that itcorresponds to the apical ganglion (AG, also called apicalsense organ), a highly conserved marine invertebratestructure with presumed sensory function (Bonar, 1978;Chia and Koss, 1984; Tardy and Dongard, 1993; see Maroisand Carew, 1997b, for detailed description of this gangli-onic structure in Aplysia).

The serotonergic pattern observed in Stage 1 remainsunchanged until the last larval stage, Stage 6, when one tothree (generally two) additional serotonergic cells aredetected lateral to the AG and inside the cerebral ganglia(Fig. 6A). Within 24 hours of the induction of metamorpho-sis (Stage 7), the animals have shed their velar cilia andthe velum is resorbed (compare Fig. 6A and 6B). At thisstage, the 5HT cells of the AG display strong evidence ofdestruction, as indicated by cellular swelling, apparentvacuolization, and disruption of membrane integrity (Fig.6B,C). These effects were specific to the AG, as theserotonergic neurons of the cerebral ganglia remain intact(Fig. 6B). Following a further 24 hours of metamorphosis(Stage 8), the animals have begun feeding with the adultbuccal structure (Kriegstein et al., 1974; Kriegstein, 1977a).At this stage, the corresponding location of the AG isdevoid of serotonergic or of any other cells (Fig. 7A). One or

Fig. 3. Birthdating of the larval serotonergic neurons. Confocallaser microscopic images of serotonin and BrdU immunocytochemistryin Stage 2 larvae. Green indicates a 5HT-positive cell, red indicates aBrdU-positive cell, and yellow a serotonin- and BrdU-positive cell. A:Exposure to BrdU on Day 3 of embryogenesis. Anterior is to the right.The two bilateral pairs of 5HT cells are BrdU negative (small arrows),whereas the unpaired median cell (UMC, open arrow) is doublelabeled. A cell adjacent to the UMC is also BrdU positive (arrowhead).B: Exposure to BrdU on Day 4 of embryogenesis. Anterior is to thelower right corner. Both bilateral pairs of 5HT cells are double labeled(arrows), whereas the UMC is BrdU negative (green). C: Exposure toBrdU on Day 5 of embryogenesis. Anterior is to the right. The lateralpair of 5HT cells is doubled labeled (arrows), whereas the medial pairis weakly double labeled (arrowheads). The UMC is BrdU negative(white arrowhead). Note that the relative differences of immunostain-ing among the serotonergic cells are more meaningful within adevelopmental age than those between the developmental ages (e.g.,between A and C). This is due to the fact that the backgroundintensities associated with BrdU ICC changed across development,with Day 3 embryos consistently showing high background levels (andhence yellow-tainted BrdU-negative 5HT cells, as in A). Scale bars 520 µm in A–C.


two additional 5HT cells are detected in the cerebralganglia such that each cerebral ganglion contains three tofour serotonergic cells (Fig. 7A). Cells can only be clearly

distinguished as belonging to the ACC or PCC followingone additional day of development, at the beginning of thejuvenile phase (Stage 9), when the PCC contains four cells

Fig. 4. ICC staining of serotonin and BrdU in whole-mountedjuveniles exposed to BrdU at Day 4 of embryogenesis. Similar resultswere obtained with Day 3 and Day 5 animals. A: Serotonin ICC of theC1 cell of the ACC and the five cells of the PCC in the cerebralganglion. B: BrdU ICC of the same preparation shows that none of the5HT cells are BrdU positive, although other cells are BrdU positive(arrows). One of the BrdU-positive cells is adjacent to the cells of the

PCC (arrowhead in A, B). C: Another specimen double exposed to 5HTand BrdU ICC shows that none of the serotonergic cells of the ACC andPCC clusters are double labeled, although nearby cells are BrdUpositive (indicated by the red circular pigmentation, arrows). Theyellow tinge observed over the entire cerebral ganglia is due to thehigh background intensity associated with BrdU ICC. Scale bars 520 µm in A,B, 40 µm in C.


while the ACC contains the C1 cell and a smaller cell (Fig.7B). The cerebral ganglia and the interconnecting commis-sure have dramatically enlarged by the beginning of Stage

9 (Fig. 7B). Comparison of the number, size, and position ofthe 5HT cells between Stages 8 and 9 suggest that the5HT-immunoreactive cells first observed in the cerebral

Fig. 5. Localization of the serotonergic cells in semithin andultrathin sections of Stage 1 larvae stained with the immunoperoxi-dase technique. A: Semithin cross section showing the unpairedmedian cell and a bilateral pair of cells (arrowheads) located immedi-ately dorsal to each cerebral ganglion (CG). The dotted lines indicatethe dorsal boundary of the CG. B: Semithin horizontal section showinga bilateral pair of serotonergic cells (arrowheads) in the anterior

region of the animal. Anterior is on top. C: Ultrathin horizontal sectionin the region of the five serotonergic cells shows the unpaired mediancell and the two bilateral pairs of serotonergic cells (asterisks) as partof the apical ganglion. Four nonserotonergic cells with ciliary bundles(ci) are interspersed between the 5HT cells. Anterior is on top. m,muscle fiber; n, neuropil; O, oesophagus; V, velum. Scale bars 5 10 µmin A,B, 1 µm in C.


ganglia at Stage 6 belong to the PCC and that cell C1 of theACC is detected only by the end of metamorphosis. Asummary of the ontogeny and fate of the serotonergicneurons in embryonic, larval, metamorphic, and juvenileAplysia is provided in Figure 8.

DISCUSSION

Our results reveal an unsuspected level of complexity inthe establishment of the serotonergic system in Aplysia:1) the larval serotonergic neurons do not correspond to anyof the adult serotonergic cells; 2) these larval cells are notpart of the cerebral ganglia but instead are in a highlyconserved structure called the apical ganglion (see Maroisand Carew, 1997b); 3) the apical ganglion, together withthe larval serotonergic cells, are resorbed at metamorpho-sis; and 4) the emergence of the adult serotonergic cells ofthe cerebral ganglia begins only at the end of larvaldevelopment.

Genesis and differentiation of the larvalserotonergic system

One interesting observation arising from our studies isthat birth order does not predict the order of differentia-tion of the serotonergic neurons in larval Aplysia. Specifi-cally, the unpaired median cell is born on the third day ofembryogenesis, whereas the two lateral pairs of serotoner-gic cells are generated on the fourth (and fifth) day ofembryonic development. By contrast, the whole-mountimmunocytochemical results indicate that both the UMCand the anterior pair of cells take on their serotonergicphenotype on Day 5 of embryogenesis, whereas the poste-rior pair of serotonergic cells do not emerge before Day 7.In addition to differentiating synchronously, the threeanterior cells also have similar morphological characteris-tics that are distinct from those of the posterior pair of 5HTcells (Marois and Carew, 1997b). These results suggestthat the three anterior and the two posterior serotonergiccells are subject to different developmental programs.

A dissociation between birth date and time of differentia-tion has also been observed in the moth Manduca, wherepostembryonic neurons are generated throughout the lar-val period but differentiate simultaneously at metamorpho-sis (Booker and Truman, 1987a,b). Interestingly, transmit-ter identity in these cells is lineage-derived, whereas thetemporal emergence of this transmitter phenotype is un-der hormonal control (Witten and Truman, 1991a,b). Like-wise, the simultaneous differentiation in Aplysia embryosof the three 5HT cells born at different times is more easilyexplained by an extrinsic control of the onset of differentia-tion. These results suggest that extrinsic control of trans-mitter differentiation may be prevalent when differentia-tion is temporally dissociated from neurogenesis.

Interestingly, the BrdU results also suggest that theserotonergic cells may be among the first neurons gener-ated in the entire CNS of Aplysia. This is particularly trueof the unpaired median cell, which, together with anadjacent cell, appear to be the only two cells in the apical

Fig. 6. Semithin sections of the serotonergic cells in the first phaseof metamorphosis. A: Horizontal section of a Stage 6 larva competentfor metamorphosis. There are five serotonergic cells in the apicalganglion (asterisk) and two 5HT cells in each cerebral ganglion(arrowheads). Anterior is on top. B: Horizontal section of an animal 24hours after induction of metamorphosis (Stage 7) showing disintegrat-ing serotonergic cells in the apical ganglion (arrowheads) and intact5HT cells of the cerebral ganglia (arrows). Note the absence of thevelum (open arrow). C: Cross section of a Stage 7 animal showing thedisintegration of serotonergic cells in the apical ganglion (arrows). Theunpaired median cell (arrowhead) is still not completely resorbed inthis preparation. e, eye; M, larval retractor muscle; O, oesophagus; V,velum. Scale bars 5 20 µm in A,B, 10 µm in C.


region of the animal that are terminally differentiated byembryonic Day 3. On Day 4, most BrdU-positive cells aretightly clustered with the serotonergic cells and are there-fore probably in the AG. On Day 5, the BrdU-positive cellsare mostly localized around the lateral pair of serotonergiccells, and some may therefore be in the region of thecerebral ganglia. These results suggest a spatiotemporal

pattern of neurogenesis where the cells of the apicalganglion, and particularly the UMC, are generated firstduring Days 3 and 4 of embryogenesis, whereas the cells inthe cerebral ganglia are generated later. Consistent with atemporal dissociation in neurogenesis of the apical andcerebral ganglia, these two neural structures arise fromseparate developmental lineages; the apical ganglion isderived from a rosette of cells in the apical plate, whereasthe cerebral ganglia originate from cells of the cephalicplates of the molluscan gastrula (Raven, 1958; Verdonkand van den Biggelaar, 1983). Although these resultsindicate that the serotonergic cells of the AG develop veryearly, recent work on a few different gastropods, includingAplysia, suggest that a few FMRFamide-containing cellslocated posteriorly near the visceral region emerge evenearlier than the serotonergic cells, casting doubt on thegeneral model of an anterior-posterior gradient of neurogen-esis (Croll and Voronezhskaya, 1995, 1996).

Impact of metamorphosison the development of the serotonergic

system in Aplysia

A major finding of this study is that there are virtuallyno commonalities between the serotonergic system of thelarval and adult phases of the life cycle of Aplysia, reinforc-ing the idea that each of the phases has a specializednervous system subserving distinct functions and innervat-

Fig. 7. Semithin sections of the serotonergic cells in the secondphase of metamorphosis. A: Horizontal section showing two to threeserotonergic cells in each cerebral ganglion (CG) of a Stage 8 animal.Anterior is on top. Note the absence of any apical ganglion structure atits presumed location (arrow). B: Horizontal section through thecerebral ganglia at the completion of metamorphosis and the begin-ning of juvenile development (Stage 9) showing the C1 cell of the ACCand two cells of the PCC. Anterior is on top. BM, buccal mass; cc,cerebral commissure; o, oesophagus. Scale bars 5 20 µm in A,B.

Fig. 8. Summary diagram of the ontogeny of serotonergic cells inAplysia. Three serotonergic cells emerge on Day 5 of embryogenesis,with two more added to the apical ganglion (AG) by the beginning oflarval development (S1). By the end of larval development (S6),additional serotonergic (5HT) cells are detected in the cerebral ganglia(CG). During metamorphosis, the AG cells are resorbed while other5HT cells are added to the cerebral ganglia. By the beginning ofjuvenile development (S9), the adult pattern of serotonergic expres-sion, consisting of the ACC and PCC clusters, is discernable. cc,cerebral commissure.


ing different targets (Marois and Carew, 1990, 1997a). Thelarval serotonergic neurons are born during embryogen-esis and are destroyed together with the AG at metamor-phosis. The adult serotonergic pattern is assembled moreprogressively, with the first cells differentiating at the endof larval development and others added throughout themetamorphic and juvenile period (see also Nolen andCarew, 1994). Thus, the two serotonergic systems areseparated both in space (apical ganglion vs. cerebralganglion) and in time (larval stage vs. juvenile stage). Thisseparation is not absolute, because some of the adultserotonergic cells differentiate at the end of the larvalperiod. The seemingly precocious development of thesecells might suggest a role for them in metamorphosis,before they assume their adult functions. Although bathapplication of serotonin is known to induce metamorphosisof the larvae of the prosobranch Ilyanassa (Levantine andBonar, 1986; Couper and Leise, 1994; Leise, 1996), nocomparable effect has been observed in Aplysia or in manyother gastropods (Morse et al., 1979; Hadfield, 1984;Marois and Hofstadter, unpublished observations). Alter-natively, the precocious establishment of the 5HT cells inthe cerebral ganglia may be occurring in anticipation ofthe swift functional changes accompanying metamorpho-sis. Metamorphosis in Aplysia brings about profoundmorphological, physiological, and behavioral changes inonly 48 hours (Kriegstein et al., 1974; Kriegstein, 1977a;Marois and Carew, 1990). For instance, the animal’smeans of locomotion switches from cilia-propelled swim-ming to crawling; similarly, its change of diet from phyto-plankton to seaweed necessitates very different feedingmechanisms (Kriegstein et al., 1974). These rapid physi-ological and behavioral changes must put a heavy burdenon the dynamics of the requisite underlying neuronalreorganization. One way to alleviate the metamorphic loadmay be to preassemble components of the neuronal circuitsbefore metamorphosis. In fact, such developmental strat-egy seems to underlie the acquisition of new behaviorsduring insect metamorphosis (Levine and Truman, 1983;Weeks and Levine, 1990; Truman, 1992).

The disintegration of the larval serotonergic neuronsand of the apical ganglion during metamorphosis of Aply-sia is the first clear example of neuronal cell death in theCNS of gastropods, if not of all molluscs (Marois andCarew, 1990; Barlow and Truman, 1992). The disintegra-tion of the serotonergic cells and of the AG should not besurprising, given the resorption at metamorphosis of thevelum, the major target tissue of the AG and its serotoner-gic cells (Marois and Carew, 1990, 1997a). The presentimmunocytochemical results could not conclusively deter-mine which of the various cell death mechanisms (Clarke,1990) is responsible for the destruction of the AG and itsserotonergic cells, although apoptosis has been attributedto be the mechanism of cell attrition most prevalent duringanimal metamorphosis (Wyllie et al., 1980).

Comparison of Aplysia metamorphosiswith other systems

The patterns of neuronal reorganization observed dur-ing metamorphosis of Aplysia show both similarities anddifferences with the mechanisms at work during metamor-phosis of other holometabolous animals, especially insects(see Harris, 1990). As in Aplysia, both cell differentiationand cell death characterize the metamorphic period ofDrosophila and Manduca (Truman, 1983; Weeks and

Truman, 1985; Booker and Truman, 1987a,b; Prokop andTechnau, 1991; Truman, 1992). Some insect neurons alsoshow evidence of remodeling of their axonal and/or den-dritic arbors during metamorphosis (Truman and Reiss,1976; Nuesch, 1985). Interestingly, neuronal remodelinghas not been observed during Aplysia metamorphosis.Perhaps the short metamorphic period of Aplysia (48hours) heavily constrains the extent to which neuronalreorganization can occur. Indeed, in contrast to Aplysia,metamorphosis in the moth lasts 17 days from the pupal tothe adult stage (Truman, 1992), which is ample time for aslow reorganizational process to occur.

Development of the adult serotonergicneurons of Aplysia

The first serotonergic cells of the adult nervous systemdifferentiate at the last stage of larval life, and they appearto be members of the posterior cell cluster (PCC; Nolen andCarew, 1994). The adult complement of 5 PCC cells isalready established by the beginning of the juvenile pe-riod. Unfortunately, it was not possible to resolve theidentity of the individual cells during larval and metamor-phic development. This information would be very interest-ing, given that the CB1 neuron of the PCC is a serotonergiccell thought to participate in synaptic facilitation (Mackeyet al., 1989). Nevertheless, it is clear that this facilitatoryneuron is not implicated in the early establishment of thenervous system.

The first cell of the anterior cell cluster (ACC) todifferentiate, the metacerebral giant cell (C1), does so atthe end of the metamorphic period (late Stage 8). Othersmaller cells join the C1 cell only at the beginning ofjuvenile development (Stage 9). C1 is a multitargetedserotonergic neuron that modulates the neuronal andmuscular activity associated with feeding (Weiss et al.,1978; Schwartz and Shkolnik, 1981). Because adult feed-ing behavior emerges at Stage 8 (Kriegstein, 1977a), it isunlikely that C1 is involved in the functional developmentof this behavior. Rather, it probably acts from the outset asa neuromodulator of the feeding behavior.

Comparison of the serotonergic systemin the CNS of gastropods with divergent

life histories

This study offers a rare opportunity to compare thedevelopment of an identified subset of neurons in relatedspecies with widely divergent life cycles. Similar compari-sons between direct and indirect modes of developmenthave generally been limited to the level of brain structureor cell class (Raff, 1987; Fritzsch, 1990; Helluy et al., 1993).However, the present comparison must still be consideredtentative, given that most of the studies in related specieswere solely based on whole-mount ICC, which is limited inits resolution. The opisthobranch Phestilla sibogae, whoselife cycle greatly resembles that of Aplysia, has a serotoner-gic system very similar to the one observed in the presentstudy (Kempf et al., 1991; Kempf and Page, 1995). Thelarval serotonergic organization is also remarkably simi-lar in the phyletically distant prosobranch Haliotis rufe-scens (Barlow and Truman, 1992). Although the embryonicand larval periods of Haliotis are considerably compressedin comparison with Aplysia, the developmental emergenceof the serotonergic pattern relative to distinct morphoge-netic events is nearly identical. Thus, not only is thenumber of 5HT cells comparable with Aplysia both at the


beginning and end of the larval period, but at least two ofthe larval pairs of cells appear to be clear homologues.Interestingly, the conservation of the serotonergic patternbetween Haliotis and Aplysia does not extend to thejuvenile and adult stages. The two clusters of serotonergiccells in the cerebral ganglia of adult Aplysia do not haveclear homologues in Haliotis (Barlow and Truman, 1992).These results reinforce the idea that the different stages ofthe life cycle of marine invertebrates are subject to indepen-dent evolutionary pressures (Freeman, 1982; Horn et al.,1982; Wilmer, 1990; Wray, 1995a,b). The present resultsindicate that these evolutionary processes are reflected atthe cellular level in the nervous system.

The developmental pattern of serotonergic cells is moredivergent in gastropod species that undergo direct develop-ment. Unlike Aplysia, whose embryos pass through larvaland metamorphic stages before reaching the juvenilestage, the development of the pulmonates Lymnaea andHelisoma proceeds directly from the embryonic to thejuvenile stages of development. The direct nature of thelife cycle of Lymnaea stagnalis is reflected in its serotoner-gic development: the establishment of the adult patternbegins at 35–40% of embryogenesis and is progressivelyelaborated with further development (Croll and Chiasson,1989; Marois and Croll, 1992). Despite the striking differ-ences in the developmental establishment of the serotoner-gic pattern between Aplysia and Lymnaea, the adultserotonergic system of these two species shows strongsimilarities. Two of its five serotonergic clusters are clearhomologues of the Aplysia ACC and PCC clusters (Crolland Chiasson, 1989; Nolen and Carew, 1994), and thesetwo clusters develop first in Lymnaea (Marois and Croll,1992). An intermediary developmental serotonergic pat-tern between Aplysia and Lymnaea may be observed inanother pulmonate, Helisoma trivolvis. Although its adultserotonergic configuration is virtually identical to that ofLymnaea (Croll and Chiasson, 1989; Goldberg and Kater,1989), Helisoma has a pair of transiently detected embry-onic serotonergic cells with morphological characteristics(cell shape and position) and functional characteristics(ciliary innervation) similar to the larval 5HT cells ofAplysia (Goldberg and Kater, 1989; Diefenbach et al.,1991; Voronezhskaya and Elekes, 1993; Marois and Carew,1997a,b). Because freshwater pulmonates are direct devel-opers derived (via land snails) from marine ancestors (proso-branchs) with an indirect mode of development (Morton, 1979;Russell-Hunter, 1979), it is possible that these Helisomaneurons may be vestigial homologues of the larval opistho-branch and prosobranch serotonergic neurons.

The elaboration of a transiently expressed serotonergicsystem during embryonic and/or larval development of allgastropods examined so far appears to most stronglycorrelate with the morphological differentiation of larval-specific organs. The transient serotonergic system is mostcomplex in organisms with an elaborate velum (Aplysia,Phestilla, and Haliotis), and minimally present (Helisoma)or altogether absent (Lymnaea) in species with an atro-phied velum. Interestingly, although the embryonic andlarval serotonergic patterns of Aplysia resemble more thatof the prosobranchs than the pulmonates, the converse istrue for the adult serotonergic patterns. Thus, phyloge-netic relationships per se are not the determining factor ofeither the adult or developing serotonergic pattern. Rather,functional ecology may play a more important role (seeWray, 1995b). This conclusion is supported by a prelimi-

nary study of the serotonergic pattern in Melampus, apulmonate with a true larval stage, that indicates a strongresemblance to the serotonergic system of larval opistho-branchs (Moffett, 1992, 1995).

ACKNOWLEDGMENTS

We thank Isabel Gauthier for assistance with the draw-ings and Paul Hofstadter for excellent technical assis-tance. This work was financially supported by an NSERC(Canada) predoctoral fellowship to R.M. and by NSF grantIBN9221117 and NIMH Merit Award R01-MH-14-1083 toT.J.C.

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Ontogeny of serotonergic neurons inAplysia californica