The GABAergic-like system in the cyprid of Balanus amphitrite (=Amphibalanus amphitrite) (Cirripedia, Crustacea)

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The GABAergic-like system in the cyprid of Balanus amphitrite (=Amphibalanusamphitrite) (Cirripedia, Crustacea)Lorenzo Gallus a; Sara Ferrando a; Chiara Gambardella ab; Alberto Diaspro c; Paolo Bianchini d; VeronicaPiazza e; Giambattista Bonanno f; Marco Milanese f; Paola Ramoino g; Grazia Tagliafierro a

a LIBiOM, DIBIO, University of Genoa, Genova, Italy b Department of Animal Biology and Marine Ecology,University of Messina, Messina, Italy c The Italian Institute of Technology - IIT Genova (Italy), Via Morego,Genova (Italy) d IFOM-LAMBS/MicroScoBio, DIFI, University of Genoa, Genova, Italy e Istituto di ScienzeMarine, ICMM, CNR, Genova, Italy f Department of Experimental Medicine, Section of Pharmacology andToxicology and Center of Excellence for Biomedical Research, University of Genoa, Genova g DIPTERIS,University of Genoa, Genova, Italy

First Published on: 28 October 2009

To cite this Article Gallus, Lorenzo, Ferrando, Sara, Gambardella, Chiara, Diaspro, Alberto, Bianchini, Paolo, Piazza, Veronica,Bonanno, Giambattista, Milanese, Marco, Ramoino, Paola and Tagliafierro, Grazia(2009)'The GABAergic-like system in the cyprid ofBalanus amphitrite (=Amphibalanus amphitrite) (Cirripedia, Crustacea)',Biofouling,26:2,155 — 165

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The GABAergic-like system in the cyprid of Balanus amphitrite (¼Amphibalanus amphitrite)(Cirripedia, Crustacea)

Lorenzo Gallusa*, Sara Ferrandoa, Chiara Gambardellaa,b, Alberto Diasproc, Paolo Bianchinid, Veronica Piazzae,Giambattista Bonannof, Marco Milanesef, Paola Ramoinog and Grazia Tagliafierroa

aLIBiOM, DIBIO, University of Genoa, Viale Benedetto XV 5, I-16132, Genova, Italy; bDepartment of Animal Biology and MarineEcology, University of Messina, Salita Sperone 31, S. Agata, 98166 Messina, Italy; cThe Italian Institute of Technology – IITGenova (Italy), Via Morego, 30 16163-Genova (Italy); dIFOM-LAMBS/MicroScoBio, DIFI, University of Genoa, ViaDodecaneso 33, I-16146 Genova, Italy; eIstituto di Scienze Marine, ICMM, CNR, Via De Marini 6, I-16149 Genova, Italy;fDepartment of Experimental Medicine, Section of Pharmacology and Toxicology and Center of Excellence for BiomedicalResearch, University of Genoa, Viale Cembriano 4, 16148 Genova; gDIPTERIS, University of Genoa, Corso Europa 26, I-16132Genova, Italy

(Received 13 September 2009; final version received 3 October 2009)

In the present study, biochemical and immunochemicalmethods were used to investigate the presence and distribution ofGABA, glutamate decarboxylase (GAD), GABABR1 and GABAA g2 subunit receptors and the vesicular GABAtransporter (vGAT) in the cyprid ofBalanus amphitrite (¼Amphibalanus amphitrite). GAD65/67 immunoreactive neuroncell bodies and nerve fiberswere detected in the central nervous system. PairedGAD65/67 immunoreactive nerves runningfrom the posterior ganglion to the body and limb muscles were detected. Thin GABA-immunoreactive nerve terminalswere present on striated muscular fibers and in the antennules. Furthermore, GABA, GAD65/67, GABABR1 andGABAAg2 subunit receptors and vGAT were observed in the lateral compound eyes, and GABAAg2 subunit receptorimmunoreactivitywas seen in thenaupliar eye.These results suggest aneurotransmitter/neuromodulatory role forGABAin thoracic muscle contraction and regulatory functions in compound eyes and antennules of B. amphitrite cyprids.

Keywords: barnacle cyprid; GABA; GABA-related molecules; neuromuscular junctions; compound eyes

Introduction

The cirripedian crustacean Balanus amphitrite(¼Amphibalanus amphitrite) (Darwin, 1854) is a sessileacorn barnacle with aworldwide distribution (Clare andHoeg 2008; Carlton and Newman 2009). It is character-ized by six naupliar stages and a cyprid competent forsettlement. During settlement, the cyprid exploressubmersed rocks or ships’ hulls, walking by means ofits antennules; finally, using secretions of its cementgland, it binds irreversibly to the substratum andmetamorphoses into a sessile juvenile organism (Walker1971; Okano et al. 1996). Because of their sessile adultcondition and the ability of their cyprids to adhere to anykind of substratum, barnacles are considered one of themost successful forms of animal marine biofouling(Crisp 1984; Christie and Dalley 1987). The need toeliminate barnacles during the maintenance of ships’hulls, oil platforms and pipework carrying seawaterresults in great economic cost. Increased knowledge ofbarnacle biology is important in attempts to fight cypridsettlement and growth and to reduce biofouling.

B. amphitrite can be easily reared in the laboratory(Rittschof et al. 1992) and is commonly employed to

study antifouling strategies. The morphologies of adultbarnacle nervous systems have been studied (Gwilliamand Cole 1979; Gwilliam 1987), and many neuroactiveand related substances, including g-aminobutyric acid(GABA) (Koike and Tsuda 1980), histamine (Call-away et al. 1989), dopamine (Okano et al. 1996),serotonin (Callaway and Stuart 1989), pigment disper-sing hormone (PDH), crustacean cardioactive peptide(CCAP) (Webster 1998), the tetrapeptide FMRFamideand the acetylcholine (ACh) biosynthetic enzymecholine acetyltranferase (ChAT) (Gallus et al. 2001,2006a,b), have been detected. The anatomy of barnaclecyprids has also been extensively studied (Walker et al.1987; Walker 1992). The morphology of the nervoussystem of B. amphitrite was described by Harrison andSandeman (1999). In contrast to the adult, barnaclecyprids possess many sensory organs, including twoantennules with setae, two lateral compound eyes withtwo associated frontal filaments, and one naupliusmedian eye. The morphologies of these organs havebeen well described, and their involvement in sub-stratum recognition has been demonstrated (Fales1928; Hallberg and Elofsson 1983; Walker et al.

*Corresponding author. Email: [email protected] first two authors contributed equally to this study.

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Vol. 26, No. 2, February 2010, 155–165

ISSN 0892-7014 print/ISSN 1029-2454 online

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DOI: 10.1080/08927010903391193

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1987; Clare and Nott 1994; Callaway and Stuart 1999;Harrison and Sandeman 1999; Lagersson and Høeg2002). Various neuroactive substances and their relatedenzymes, including dopamine (Kon-ya and Endo1995), serotonin (Yamamoto et al. 1996; Gallus et al.2005), ChAT and the ACh lytic enzyme acetylcholi-nesterase (AChE) (Faimali et al. 2003), histamine(Stuart et al. 2002) and FMRFamide-like peptides(FLPs) (Gallus et al. 2009a), have been detected in thecyprid nervous system. Some of these substances wereimmunolocalized to cyprid sensory organs. Serotoninwas found in the compound eyes and antennules(Gallus et al. 2005), FLPs in the frontal filaments(Gallus et al. 2009a) and AChE activity in the setae ofantennules (Faimali et al. 2003). Although GABA hasbeen extensively studied in crustaceans, in particular indecapods (Miyata et al. 1997), its distribution inbarnacles has been described only in the adult. Besidesbeing present in ocelli, GABA immunoreactivity wasobserved in approximately 25 pairs of neurons in thebrain and in cirral nerves (Callaway et al. 1989). Nodata are currently available regarding the distributionof GABA-related molecules in the cyprid nervoussystem. GABA is synthesized by the enzyme glutamicacid decarboxylase (GAD), transported into vesiclesby the vesicular GABA transporter (vGAT) andreleased into the synaptic cleft in response to neuronalstimulation (Bowery et al. 2004; Schuske et al. 2004). Itcan act through ionotropic GABAA receptors, whichinduce fast inhibitory synaptic responses, as well asthrough metabotropic GABAB receptors, which play afundamental role in the reduction of presynaptictransmitter release and production of postsynapticinhibitory potentials (Bowery et al. 1980; Mezler et al.2001; Enna and Bowery 2004). These receptors are notspecific to organisms with a nervous system; they arealso found in ciliated protozoa (Ramoino et al. 2003,2004) and in sponges (Perovic et al. 1999; Ramoinoet al. 2007), both of which lack nerve and muscletissue.

In order to better understand the neurobiology ofB. amphitrite cyprids and the possible role of GABA inthe locomotion and sensory functions potentiallyinvolved in the settlement of these organisms, thepresence and distribution of GABA, GAD65/67, vGAT,and GABABR1 and GABAAg2 subunit receptors in B.amphitrite cyprids were studied via biochemical andimmunochemical methods. This improvement inknowledge related to B. amphitrite cyprid biologymay be useful to better understand, and possiblyprevent, settlement (Buckingham et al. 2005). Theresults obtained suggest that besides playing a neuro-transmitter/neuromodulatory role in thoracic musclecontraction, GABA exerts regulatory functions oncompound eyes and antennules in B. amphitrite

cyprids. Some of the results obtained with the secondharmonic method on striated muscle fibers have beenpreviously published in abstract form (Gallus et al.2009b).

Materials and methods

Biological material

Cyprids were obtained from laboratory cultures(Rittschof et al. 1992) of brood stock of B. amphitrite(¼Amphibalanus amphitrite). Twenty to thirty adultbarnacles were reared in 700 ml beakers containingaerated filtered seawater at 28 + 18C, with a 16 h:8 hlight:dark (L:D) cycle. They were fed every 2 days with50–100 ml Artemia salina sp. at a concentration of 20larvae ml71 and 200–400 ml of Tetraselmis suecica at aconcentration of 2 6 106 cells ml71. The water waschanged three times per week and barnacles wereperiodically rinsed with fresh seawater to removeepibionts and other debris. Adults barnacles rearedunder such conditions produce nauplii throughout theyear. Nauplii were collected and reared in 500 mlbeakers on T. suecica (5 6 105 cells ml71) in 0.22 mmfiltered seawater at 28 + 18C with a 16 h:8 h L:Dcycle until they reached the cyprid stage.

Antibodies

The primary polyclonal antibodies rabbit anti-GABA,rabbit anti-GAD65/67 and rabbit anti-vGAT werepurchased from Chemicon International (Temecula,CA, USA); the polyclonal guinea pig anti-GABABR1subunit receptor antibody from Sigma (Steinheim,Germany); the polyclonal rabbit anti-vGAT antibodyfrom SynapticSystem (Goettingen, Germany); poly-clonal goat anti-GABABR1 subunit receptor fromSanta Cruz Biotechnology Inc. (Santa Cruz, CA,USA); polyclonal rabbit anti-GABAAg2 subunit fromAlomone Labs (Jerusalem, Israel). Secondary antibo-dies (goat anti-mouse conjugated with Alexa Fluor488, goat anti-rabbit conjugated with Alexa Fluor 488,and goat anti-guinea pig conjugated with Alexa Fluor488) were obtained from Molecular Probes, Invitrogen(Carlsbad, CA, USA). Immunogenic peptides used inproduction of rabbit anti-GAD65/67, guinea pig anti-vGAT and guinea pig anti-GABABR1 subunit recep-tor were purchased from Chemicon and the peptideused in production of rabbit anti-GABAAg2 subunitreceptor was purchased from Alomone.

Histological and immunohistochemical methods

About 500 zero- to 5-day-old cyprids were used. Theywere anesthetizedwith 3-aminobenzoic acid ethylmetha-nesulfonate salt (Sigma, USA) (1:1000 in seawater) and

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fixed in 4% paraformaldehyde in phosphate-bufferedsaline, pH 7.4 (PBS). Subsequently, the cyprids wererinsed with PBS, dehydrated and paraplast (Bioptica,Italy) embedded. Dewaxed transverse and longitudinalserial sections were preincubated with normal serum(1:50) and immunohistochemically treated overnight atroom temperature with the appropriate primary anti-body: rabbit anti-GAD65/67 (1:400), rabbit anti-GABA(1:400), guinea pig anti-vGAT (1:1000); guinea pig anti-GABABR1 subunit receptor (1:1000), or rabbit GA-BAAg2 subunit receptor (1:400). Treated sections wererinsed in PBS and incubated for 2 h at room temperaturewith the appropriate secondary antibody conjugatedwithAlexa Fluor 488 (dilution 1:300). Control experimentsincluded preabsorption of the primary antibody bothwith its immunogen (10 mg ml71 appropriate controlpeptide antigen for GAD65/67, vGAT, GABAAg2 andGABAB R1; 1 mM free GABA for GABA) and acrude extract from cyprids for 2 h at room tempera-ture. In a second set of controls, primary antibodieswere omitted and only the secondary antibodies wereapplied. No staining was detected in control experi-ments. Vertebrate brain tissue was used as a positivecontrol.

Histomorphology was assessed through hemato-xylin and eosin staining. Nuclei were counterstainedwith propidium iodide (PI, 2 mg ml71 in PBS for10 min) after pre-treatment with RNAase (10 mg ml71

for 30 min) or DAPI or 40,6-diamidino-2-phenylindole(1:10,000 in PBS for 5 min).

Image acquisition and analysis

Immunostained sections were examined in an OlympusBX 60 epifluorescence microscope equipped with aCCD Color ViewII Camera with analySIS software(Soft Imaging System GmbH, Germany). Furtherobservations were carried out using a LeicaDMI6000B-CS inverted microscope coupled to a LeicaTCS-SP5 AOBS spectral confocal scanning head(Leica Microsystems, Germany) (CLSM). The result-ing images were acquired, stored, and visualized with aLeica confocal software program using TIFF imageformats. The second harmonic generation (SHG) and2-photon excitation (2PE) microscopy were used todelineate muscle structure. Sections were observed on amultimodal nonlinear microscope based on the LeicaCLSM. The laser system used was a Ti:SapphireChameleon-Ultra (Coherent Inc, Santa Clara, CA,USA), tunable between 690 nm and 1040 nm andcharacterized by a pulse width of 140 fs delivered at arepetition rate of 90 MHz by means of a home-builtset-up (Bianchini and Diaspro 2008). From eachsection, z-stacks were performed in order to obtainthe 3-dimensional distribution of actin fibers. The

z-stacks were analyzed and processed using ImageJsoftware (US National Institutes of Health, Bethesda,Maryland, USA, http://rsb.info.nih.gov/ij/) (Rasband2006), thus obtaining the specimens’ volume recon-structions. Tables were assembled using the graphicsprogram Photoshop (Adobe, USA).

SDS-PAGE and immunoblotting

Samples (50 mg of B. amphitrite cyprid protein or 10 mgof rat cerebral cortex protein) were subjected toSDS-polyacrylamide gel electrophoresis (SDS-PAGE)on 7%–12.5% polyacrylamide gel and electrophoreti-cally transferred to nitrocellulose membranes (Laemmli1970). Membranes were saturated with 3% nonfat milkand incubated at room temperaturewith the appropriateprimary antibody: rabbit anti-GAD65/67 antibody(1:1000), rabbit anti vGAT antibody (1:2000), goatanti-GABABR1 receptor antibody (1:200), or rabbitanti-GABAAg2R antibody (1:200). After extensivewashing, membranes were incubated with the appro-priate secondary antibody conjugated with IgG perox-idase (1:2000); immunoreactivity was detected using achemiluminescence system (Amersham, UK).

HPLC

The amount of GABA present in B. amphitrite cypridswas determined by HPLC. The analytical methodinvolved precolumn derivatization with o-phthalalde-hyde followed by separation on a C18 reverse-phasechromatography column (Chrompack,Middleburg, TheNetherlands; 10x4.6 mm, 3 mm; 308C) coupled withfluorometric detection (excitation wavelength 350 nm;emission wavelength 450 nm). Solvents and the gradientprogram were as follows: solvent A, 0.1 M sodiumacetate (pH ¼ 5.8):methanol 80:20; solvent B, 0.1 Msodium acetate (pH ¼ 5.8):methanol 20:80; solvent C,sodium acetate (pH ¼ 6.0):methanol, 80:20. The gradi-ent started at 20%methanol, pH ¼ 6.0 and increased to55%methanol, pH ¼ 5.8 over 19 min. The flow ratewas0.9 ml min71. The amount of GABA in the biologicalsamples was determined by interpolating the peak area ofthe unknown sample using a curve obtained usingdifferent standard GABA solutions. Areas were normal-ized using homoserine as an internal standard. Thesensitivity of the assay was about 0.1 pmol.

Results

B. amphitrite cyprid morphology

Figure 1a is a micrograph of a B. amphitrite cypridshowing the antennules and the thoracic appendagesextending from the bivalve carapace (Figure 1a). It ispossible to observe one of the two lateral compound

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eyes, the median naupliar eye and the oil cells. Thenervous system consists of brain and posterior gang-lion, joined together with two paired circumesophagealconnectives; neuronal cell bodies are positioned in thecortex and surround the neuropil (Figure 1b). Accord-ing to Harrison and Sandeman (1999) the brain issubdivided into protocerebrum and deutocerebrum(Table 1a–f). The protocerebrum presents two opticlobes connected to compound eyes and frontal filamentcomplex, one dorso-frontal region connected to thenaupliar eye, and one median protocerebral region(Table 1e,f). The frontal filament complex consists ofone frontal filament vesicle and one frontal filament(Table 1e,f). The deuterocerebrum is connected to theantennules via two antennular nerves. The posteriorganglion comprises seven fused parts corresponding toneuromeres: the laterally elongated anterior portion(also referred as subesophageal ganglion) and theposterior portions, deriving from the remaining sixsubdivisions, the thoracic ganglia. The alimentarycanal consists of a cuticle-lined foregut and hindgutwith an interposed midgut, subdivided into dilatedanterior and narrow posterior portions (Table 1a).

Immunoreactivity

A detailed description of the anatomical organizationof GABA-like immunoreactivity in the central and

peripheral nervous system of B. amphitrite is presentedbelow.

Brain

Numerous intensely GAD65/67 immunoreactive (IR)neuronal cell bodies and nerve terminals were presentin the brain (Table 1a,e; Figure 2a,b). The IR neuronalcell bodies were localized in the brain cortex, both

Table 1. (a–d): Schematic drawing of different lateral planesof the cyprid in sagittal section, modified from Gallus et al.(2009a). (a): Section passing through the median sagittalplane of the CNS; (b–d): sections passing through the lateralthoracic muscle plane. (e,f): Schematic drawing of the cypridCNS in a frontal plane, modified from Harrison andSandeman (1999) and Gallus et al. (2009a). The drawingshows the distribution of GAD65/67 immunoreactivity (IRneuronal cell bodies: large black elliptic point; IR nervefibers: dashed line), GABA (IR nerve terminals on musclefibers and immunoreactivity in the eyes: black stars), vGAT(IR cells: small black point), GABABR1 subunit receptor (IRnerve terminals: black quadrangle). Antennula (ant); brain(b); cement gland (cg); compound eye (ce); deutocerebrum(dc); frontal filament (ff); frontal filament vesicle (ffv);hindgut (hg); midgut (mg); naupliar eye (ne); neuropil (np);optic lobe (ol); oil cells (oc); ommatidium (om); posteriorganglion (pg); protocerebrum (pc); muscular bundles (MB);inner insertion of the abductor muscles (am).

Figure 1. B. amphitrite (¼A. amphitrite) cyprid. (a) Cypridwith extended antennules and limbs. Lateral view.Bar ¼ 100 mm. (b) Sagittal section of cyprid showing thehistomorphology of the entire body. Hematoxylin–eosin.Bar ¼ 100 mm. Antennula (ant); carapace (car); compoundeye (ce); oil cells (oc); posterior ganglion (pg); striated muscle(sm); thorax (th).

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protocerebrum and deutocerebrum, and were oftenorganized in clusters. In the protocerebrum, a pairedcluster was present ventrally near the emergence of theantennular nerves, while another was localized dorsallynear the optic lobes. In the deutocerebrum, one clusterwas situated dorsally between the protocerebral andthe deutocerebral neuropile, and another cluster waslocalized ventrally. No IR neuronal cell bodies wereseen in the optic lobes. Dense meshes of GAD65/67 IRnerve fibers run along the brain neuropil, but only afew were seen in the neuropil of the optic lobe. In thebrain, as in the posterior ganglion, neuronal cell bodiesappear very similar to each other in shape anddimension, being triangular or olive-shaped with amajor axis 5 to 7 mm in length (Figure 2d). Theimmunoreactivity appears diffuse throughout thecytoplasm. No immunoreactivity was observed withthe remaining antisera.

Posterior ganglion

Various clusters of GAD65/67 IR neuronal cell bodieswere localized throughout the cortex of the posterior

ganglion. One pair of clusters of neuronal cell bodieswas situated ventrally in the anterior portion andtwelve pairs were found in the remaining posteriorportion, six ventral and six dorsal (Table 1a,e; Figure2a,c,d). From these clusters, paired nerve roots extendto the corresponding ipsilateral thoracic limbs (Table1c,d). Dense meshes of GAD65/67 IR nerve fibers werepresent along the entire posterior ganglionic neuropil.No immunoreactivity was observed using the otherantisera.

Thoracic muscles

In the thoracic and limb muscles (Table 1b,c),numerous nerve terminals reactive to GABA (Figure

Figure 3. Thoracic GABA immunoreactivity. (a, c, e, f):Bar ¼ 20 mm; (b, d): Bar ¼ 10 mm. (a–d): sagittal sections,(e–f): frontal sections. (a): Posterior ganglion, muscles andproximal portion of limbs. (b): Enlargement of a section ofimage a. (c): Lateral section of thorax, showing muscles. Thecuticle is autofluorescent. (d): Particular of the proximalportion of limbs. (e): Gut, posterior ganglion and muscles inlongitudinal view. Few IR epidermal cells (asterisk); GABAIR neuromuscular nerve terminals (arrow); muscle fibers(mf); immunoreactivity in the antennules (þ); cuticle (CUT);posterior ganglion (PG); posterior ganglion neuropil (PGnp).The striations of muscle fibers are clearly visible with secondharmonic generation microscopy.

Figure 2. (a–d): CNS GAD65/67 immunoreactivity. (a–c):Bar ¼ 20 mm. (a): CNS sagittal section, (b): brain, sagittalsection, volume rendered; z axes; (c): Posterior ganglion,frontal section, z axes. (d): Bar ¼ 15 mm. (d): Brain andposterior ganglion, frontal section. optical tract (OpT);protocerebrum neuropile (PCnp); deutocerebrum neuropile(DCnp); median protocerebral neuropile (MPnp); posteriorganglion (PG); anterior portion of the PG (apAG);circumoesophageal connective (circ). Arrows: GAD65/67 IRcells clusters in the brain; Arrow head: GAD65/67 IR cellclusters in the PG; asterisk: antennular nerve emergence.

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3a–f), GABABR1 subunit receptor (Figure 4a,b),GABAAg2 subunit receptor (Figure 4c,d) and vGAT(Figure 5a–d) antisera were found. These nerveterminals were localized to muscle cells and ramifiedparticularly on muscle nuclei (Figure 3b,d). Thearrangement of striated muscular fibers was clearlyoutlined in false color (blue) by the use of secondharmonic generation (SHG) and 2-photon excitation(2PE) microscopy techniques (Figures 3a,b,d–f, 4a,band 5b,c) and by phase contrast (Figure 4c,d). Acluster of vGAT IR cells was also observed in theinner insertion of the carapax abductor muscles(Figure 5d).

Cephalic sensory organs

In the compound eye (Table 1a–f), GABA (Figure 6a),GAD65/67 (Figures 3f and 6b), GABABR1 subunit(Figure 7a,b), vGAT (Figure 7c,d) and GABAAg2R(Figure 7e) immunoreactivities were present in cen-trally located cytoplasmic granules. It was not possibleto determine whether these granules belonged topigment cells and/or to retinular cells. In the com-pound eyes, the radial arranged ommatidia arecentrally localized; each consists of three crystalline

Figure 5. Thorax, vGAT immunoreactivity. (a, c, d):bar ¼ 10 mm, (b): bar ¼ 15 mm, i: bar ¼ 15 mm. (a–c):sagittal sections, (b): frontal section, d: transverse section.(a–c): vGAT IR nerve terminals on striated muscles (arrows);i: particular of vGAT IR epidermal cells (asterisk); (d):vGAT IR nerve terminals in abductor muscles (arrows).

Figure 4. Thorax. (a–c): bar ¼ 25 mm; (d): bar ¼ 10 mm; i:bar ¼ 10 mm. (a, c–d): sagittal sections, (b): frontal section.(a): GABABR1 subunit receptor (GBR1) IR limb nerve fibers(arrows); GABABR1 subunit receptor IR epidermal cells(asterisk); (b): GABABR1 subunit receptor IR limb nervefibers (arrows); (c): GABAAg2 subunit receptor (GAg2) IRnerve fibers of limbs (arrows); (d): GABAAg2 subunitreceptor IR nerve fibers in dorsal striated muscles (arrows);i: particular of GABAAg2 subunit receptor IR epidermal cells(asterisk). Cuticle (CUT); muscular fiber (mf); posteriorganglion neuropil (PGpn); posterior ganglion (PG).

Figure 6. Compound eye. (a, b): bar ¼ 10 mm a–b: sagittalsections. (a): GABA immunoreactivity (arrows); (b): GAD65/67

immunoreactivity in the compound eye (arrows). Crystallinecone cells (cc); crystalline cone cell nuclei (arrow head); cuticle(CUT); distal pigment cell nuclei (dpcn); epidermal cells (ec);epidermal cell nuclei (ecn); rhabdom of the retinular cells(asterisk); retinular cell nuclei (rcn); striated muscle (sm).

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cone cells, two retinular cells with rhabdoms and twodistal pigment cells. The rhabdom of the retinular cellsappeared immunonegative to all the antisera used(Figure 6a–c and 7a–e).

In a few cyprids, GABAAg2 subtype receptorimmunoreactivity was detected in ommatidia cells ofthe median naupliar eye (Figure 7d). Faint GABAimmunoreactivity was found in the antennules (Figure4f), while GABA, GABABR1 and GABAAg2 subunitreceptor and vGAT immunoreactivities were alsoobserved in the cytoplasm of some epidermal cells(Figures 3e,f, 4a,i and 5i).

HPLC and immunoblot analysis

The amount of GABA in B. amphitrite cyprids, asmeasured by HPLC, is 5000 pmol mg71 protein. OnWestern blots of cyprid proteins, a single band with anapparent molecular weight of 65–67 kDa was detectedusing an antibody to GAD, while another band withan apparent molecular weight of 55–60 kDa was seenusing an antibody against vGAT (Figure 8, line 1). TheGABAA g2 receptor antibody reacted with twoproteins with estimated molecular weights of approxi-mately 39 and 54 kDa, while the GABAB R1 antibodyevidenced a single band with molecular weight ofabout 150 kDa (Figure 8, line 1). The apparentmolecular weights of the cyprid proteins reactingwith the various antibodies used were consistent withthe molecular weights of immunoreactive proteins inrat cerebral cortex synaptosomes assayed under thesame experimental conditions (Figure 8, line 2).

Discussion

In this article, the presence of GABA and the presenceand anatomical distribution of immunoreactivity forGABA and GABAergic molecules in B. amphitritecyprids are demonstrated for the first time. Immunor-eactivity to GABAergic molecules was observed inCNS, compound eyes, thoracic muscles, epidermalcells, antennules and naupliar eyes. No immunoreac-tivity was seen in gut or in cement glands.

Central nervous system

GABAergic systems have been described in variousinvertebrates (Elekes and Florey 1987; Fischer andParnas 1996a,b; Fabian-Fine et al. 2002; Schuske et al.2004; Buckingham et al. 2005; Kononenko and Zhukov

Figure 8. Western blots of proteins from the B. amphitritecyprids (line 1) and rat cerebral cortex synaptosomes (line 2).Single bands with apparent molecular masses ofapproximately 55, 65 and 150 kDa were detected whenvGAT, GAD65/67, GABABR1 receptor subunit antibodies,respectively, were used. Double bands with apparentmolecular masses of approximately 39 and 54 kDa weredetected using GABAAg2 receptor subunit antibody. Theposition of the molecular weight marker is shown on the left.

Figure 7. Compound and naupliar eyes. (a, c, e):bar ¼ 10 mm, (b, d, f): bar ¼ 15 mm. (a–e): sagittal sections,(f): frontal section. (a, b): GABABR1 subunit receptor (GBR1)immunoreactivity in the compound eye. (b): 3D representationof the compound eyewith the adjacent optic lobe; (c, d): vGATimmunoreactivity in the compound eye (arrows), (d): 3Drepresentation of ce and the adjacent optic lobe; (e):GABAAg2subunit receptor (GAg2) immunoreactivity in the compoundeye (arrows), (f): GABAAg2 subunit receptor (GAg2)immunoreactivity in the naupliar eye. Crystalline cone cells(cc); crystalline cone cell nuclei (arrow head); cuticle (CUT);distal pigment cell nuclei (dpcn); epidermal cells (ec); epidermalcell nuclei (ecn); naupliar eye (ne); rhabdom of the retinularcells (asterisk); retinular cell nuclei (rcn); striated muscle (sm).

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2005) but it is very difficult to compare the barnacle CNSto that of other crustaceans, due to its considerablesessile adaptation (Webster 1998). At the same time,very little information is available on the neurobiologyof crustacean larval stages, though the complexity of thebarnacle cyprid CNS, which is well organized forcoordinating settlement behavior, has been described(Harrison andSandeman1999; Semmler et al. 2008). It isalso not easy to compare the barnacle cyprid with theadult barnacle, even considering that they are the samespecies. Differing distributions of serotonin and FLPsimmunoreactivities have been demonstrated in adultand cyprid barnacles. In the adult, serotonin IR cells arelocalized to the posterior ganglion (Callaway and Stuart1999), while in the cyprid, serotonergic cells are moreabundant in the brain, where they are probably linkedwith cephalic sensory organs (Gallus et al. 2005). In theadult, FLPs IR cells are abundant in the posteriorganglion andare probably related to feeding,while in thecyprid, FLPs immunoreactivity is present in the entireCNS and in the frontal filament vesicle associated withthe compound eye. In the adult barnacle, GABA IRneurons were found in the brain and GABAergic nervefibers in cirral nerves (Callaway and Stuart 1989), whilein the B. amphitrite cyprid, GAD65/67 IR neurons andaxons were present throughout the CNS. Furthermore,the distribution of GABA immunoreactivity in thecyprid brain seems to be linked with vision (Kolodziejc-zyk et al. 2008) and with the control of movement of theantennules. The latter suggestion is supported by thepresence of GABA immunoreactivity in antennules.GABA innervation in antennules may be derived fromsome clusters of GAD65/67 IR neuron cell bodies foundin the deterocerebrum. To resolve this question, aspecific study of antennule muscle innervation isrequired, since almost 29 muscles, controlled by themost complex nervous system of cirripedes, have beenobserved (Lagersson and Høeg 2002). In the cypridposterior ganglion, as in other crustaceans, the distribu-tion of GAD65/67 IR neurons appears linked to thecontrol of limbmovement (Koike and Tzuda 1980). It isimportant to keep in mind that limbs in the cyprid areused during exploration and settlement, while in thesessile adult, the rhythmicmovement of the cirri is linkedto feeding and that these represent two very differentkinds of movement. Taken together, the foregoingstudies show that there are great differences in theanatomical localization and organization of the GABAsystem in the two developmental stages.

Thoracic muscle

In B. amphitrite cyprids, two types of muscle fiber wereidentified (Lagersson 2002). The first fiber type islocalized to the antennular system, while the second

fiber type is associated with the swimming behaviorgenerated by limbs. The antennular muscle fibers havethe characteristics of ‘slow’ fibers, whereas the thoracicfibers display the properties of ‘fast’ muscle fibers. Thedifference between the thoracic and antennular musclesemphasizes the specialized morphology of the B.amphitrite cyprid, which is linked to their unique rolesin settlement (Lagersson 2002). The thoracic distribu-tion of GABA, GABABR1 and GABAAg2 subunitreceptor immunoreactivity seen in this study is inaccord with that reported for other crustaceans. TheGABA IR nerve fibers observed innervating thethoracic and limb muscles of B. amphitrite cypridappear to be derived from GAD65/67 IR neuron cellbody clusters present in the posterior ganglion neuro-meres. As in vertebrates, nerve terminals at neuromus-cular junctions ramify near the nuclear region of musclefibers. The similar distribution of GABA, GABABR1and GABAAg2 subunit receptors observed at the levelof neuromuscular junctions allows the present authorsto hypothesize that in B. amphitrite cyprids, the samescheme proposed for GABAergic crustacean muscularinnervations pertains (Fischer and Parnas 1996a,b;Murphy et al. 1998; Feinstein 2001).

The crayfish claw muscle provides a model for thestudy of neuromuscular control of motor function(DeMill and Delaney 2005). This claw opener muscle isinnervated by a single excitatory axon that releasesglutamate and a single inhibitory axon that releasesGABA. The presynaptic excitatory terminal containsGABAA, GABAB, glutamate, 5HT and dopaminereceptors; the presynaptic inhibitory terminal containsglutamate andGABAB receptors (Feinstein et al. 2003),and the postsynaptic muscle membrane has bothglutamate and GABA B receptors (Dudel and Kuffler1961; Miwa et al. 1990; Feinstein et al. 1998; Parnaset al. 1999; Feinstein 2001). It can thus be presumedthat in the B. amphitrite cyprid, neuromuscular junc-tion GABAB receptors and GABA are localized topresynaptic inhibitory nerve terminals, while GA-BABR1 and GABAA receptors are present on pre-synaptic excitatory nerve terminals and inhibitoryGABAA receptors reside on the postsynaptic mem-branes of muscle fibers. The presynaptic excitatoryterminals may be glutamatergic. The intense vGATimmunoreactivity observed at B. amphitrite cypridneuromuscular junctions supports this hypothesis.Nevertheless, modulation at this nerve terminal mustinvolve some other neurotransmitters, since this mod-ulation can be complex and can involve other receptors,as demonstrated for crustacean neuromuscular junc-tions. Glutamate (Feinstein 2001), as well as ACh andserotonin (Harzsch andWaloszek 2000; Feinstein 2001;Faimali et al. 2003; Gallus et al. 2005), are goodcandidates for this role.

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Cephalic sensory organs

Compound eyes

The morphology of barnacle cyprid compound eyeshas been well studied (Fales 1928; Elofsson 1966, 2006;Walley 1969). In the present results, the immunoloca-lization of GAD65/67, GABA, vGAT, GABABR1 andGABAAg2 subunit receptors antisera in the B.amphitrite cyprid compound eye corroborates thehypothesis that GABA is involved in cyprid eyefunction. Immunoreactivity is localized in cytoplasmicgranules of ommatidia cells and retinular and/orpigment cells. These results are consistent with thoseof Callaway et al. (1989) using photoreceptor cells ofadult barnacle ocelli and with the observations ofKoike and Tsuda (1980) on the cellular synthesis ofGABA in photoreceptor cells of lateral ocelli of B.eburneus. As hypothesized for adult barnacles, GABAmay be a bioactive molecule involved in eye function.No GABA-like immunoreactivity was detected in theB. amphitrite cyprid frontal filament vesicle, a structurethat is homologous to the crustacean eyestalk X-organ(Elofsson and Lake 1971; Gallus et al. 2009a), despitethe fact that in the crayfish X-organ, GABA transpor-ter (GATs) immunoreactivity was detected at neuronlevel (Garduno et al. 2002). In any case, the function ofthe eye in these two species is not comparable, sincecyprid represents an immature condition.

Epidermal cells

The immunoreactivities observed in epidermal cellscould be due to a non-neuronal role of GABA that isrelated to secretion or proliferation (Parducz et al.1992; Nguyen et al. 2001; Owens and Kriegstein 2002).

Conclusion

This article provides novel information about thepresence and immunohistochemical distribution ofGABAergic system molecules in the cyprid of B.amphitrite. The relevance of this neurotransmitter inthe cyprid form of this species was demonstrated by itsdetection in many neuronal and non-neuronal ele-ments. Indeed, immunoreactivity for GABA was seennot only in thoracic appendages, as has already beendemonstrated in crustaceans, but also in antennules,which play a fundamental role in settlement. GA-BAergic molecules were also detected in ommatidiacells in the compound eyes, suggesting their modula-tory role in vision. The epidermal GABA immunor-eactivity observed suggests the possible involvement ofGABA in epidermal cell functions such as the secretionof signal molecules and of cuticolar components suchas the glycoprotein settlement-inducing protein

complex (SIPC) recently demonstrated by Dreannoet al. (2006).

In agreement with Buckingham et al. (2005), it issuggested that the GABAergic system may represent apossible target from the antifouling perspective. Thefindings support the presence of a muscle GABAergicinnervation in the cyprid of B. amphitrite, as previouslydescribed for other crustaceans (DeMill and Delaney2005). It is known that GABAA receptors are thetargets of antagonists pesticides such as Dieldrin and,in Drosophila melanogaster, a point mutation in thesereceptors underlies most cases of resistance to insecti-cides (Buckingham et al 2005). Thus, studies on theinteraction between the GABAA receptor and pesti-cides could lead to identification of substances activeon some species and inactive, or less active, on others,due to the different sensibility of their GABAA

receptors. These putative new GABAergic insecticidescould be used as selective antifoulants (Rittschof et al.2003; Gisselmann et al. 2004; Buckingham et al. 2005;Murosaki et al. 2009; Raveendran and Limna Mol2009). Currently the study of the genetics of B.amphitrite is in progress (Bacchetti et al. 2009).

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