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Acta histochemica 111 (2009) 93—103

Distribution and neurotransmitter localization inthe heart of the ray-finned fish, bichir (Polypterusbichir bichir Geoffroy St. Hilaire, 1802)

Giacomo Zaccone�, Angela Mauceri, Maria Maisano, Alessia Giannetto,Vincenzo Parrino, Salvatore Fasulo

Section of Comparative Neurobiology and Biomonitoring, Department of Animal Biology and Marine Ecology, Faculty ofScience, University of Messina, Messina, Italy

Received 2 April 2008; received in revised form 29 April 2008; accepted 7 May 2008

KEYWORDSHeart;Neurotransmitters;Perikarya;Nerve fibers;Bichir;Fish;Polypterus bichirbichir

SummaryAnatomical and physiological studies of cardiovascular control are lacking in the ray-finned fish, the bichirs. The present immunohistochemical studies on the bichir(Polypterus bichir bichir) demonstrated the occurrence of intracardiac neurons andnerve fibers in the heart. Immunoreactivity to tyrosine hydroxylase (TH) andacetylcholinesterase (AchE) and various neuropeptides (substance P, galanin,vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activatingpeptide (PACAP)), including neuronal nitric oxide synthase (nNOS), was found in thenerve cell bodies lying close to the Sinus venosus and the sino-atrial region. The mainintracardiac localization of the nervous tissue is a network of nerve fibers,presumably corresponding to the postganglionic outflow giving rise to nerveterminals and the nerve cell bodies. In addition, the heart is innervated by extrinsicmonoamine-containing nerve fibers supplying the Conus arteriosus and Sinusvenosus, and substance P and galanin immunopositive fibers probably originatingfrom cranial and spinal ganglia. The adrenergic innervation of the heart of the bichiris similar to that of the teleosts, but further studies are required on nervous controlof the heart.& 2008 Elsevier GmbH. All rights reserved.

Introduction

The importance of the autonomic nervous systemin the regulation of the cardiovascular system infishes has been extensively reviewed (Laurent

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E-mail address: Giacomo.Zaccone@unime.it (G. Zaccone).

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et al., 1983; Morris and Nilsson, 1994; Olson, 1998;Donald, 1998). Except for relatively minor changes,the nervous control of the cardiovascular functionis similar in all the vertebrates. Air breathing inseveral groups of fishes has evolved independentlyfor the maintenance of oxygenation of the organsresponsible for the exchange of gases. Severalmechanisms have been developed to control theflow of the blood to respiratory organs. Thesecontrol mechanisms can be divided into local andremote processes, which are consistent with (a) alocal production of cardiac vasoactive substancesand (b) the release of endocrine hormones into thecirculation and their subsequent cardiovascularactions in target organs remote from the site ofproduction (Olson, 1998).The typical fish heart is avenous heart because it only pumps venous blood.It represents the main propulsive organ in the fishcirculatory system and at the same time ishomologous with the pulmonary or main heart inhigher vertebrates. The teleost heart has a singleventricle, which pumps blood into the ventralaorta, a single atrium and a Sinus venosus, whichis a reservoir for venous blood. The cardiac pace-maker is located at the sino-atrial junction,which is the location of intracardiac ganglion cells(see review: Kapoor and Khanna, 2004). The fourthcardiac chamber, the Bulbus arteriosus (BA) inteleosts and the Bulbus cordis in chondrichthyanfishes, represents the ventricular outflow tract, buthas important functions such as those involvingdepulsation (von Skramlik, 1935) and prolongationof aortic flow during diastole that provide constantperfusion of the gills through the ventral aorta(Bushnell et al., 1992). A coronary circulation isfound in the bichirs and consists of a cephaladcoronary artery arising from the subclavian artery,which supplies the compact myocardium of theConus arteriosus and the ventricle (Budgett, 1901).The duct of Cuvier and the hepatopulmonary veinform the Sinus venosus (Poll and Deswattines,1967). The Conus arteriosus of the bichirs isprovided with several longitudinal valves arrangedin six rows (Muller, 1844). The cardiovascularsystem of fishes shows varying degrees of develop-ment of the autonomic innervation (see review:Donald, 1998). Vagal cardiac innervation is found inall fishes except for the hagfishes. In all fishes, theparasympathetic innervation is cholinergic andinhibitory. There is now unequivocal evidence ofadrenergic sympathetic innervation of the heart inholosteans and in several teleost genera; however,adrenergic nerve fibers innervating the heart ofpleuronectid teleosts are absent (Santer, 1972).Although lungfish represent the first phylogeneti-cally important step in the transition from a single

to a double circulation in the vertebrates, theylack a sympathetic innervation of the heart(Olson, 1998). An extrinsic control of the cardio-vascular system by circulating catecholaminesreleased from the walls of the intercostal arteriesis of particular importance in these animals (Nilssonand Holmgren, 1992). This control has also beenreported for the elasmobranchs that lack adrener-gic innervation of the heart and possess no motorinnervation of their gill blood vessels. The sympa-thetic nervous system of teleosts consists ofsympathetic ganglia associated with the corre-sponding spinal nerves, a pair of sympathetic trunksconnecting the sympathetic ganglia and thesplanchnic nerves. A unique feature of the teleostsympathetic nervous system is that the sympathetictrunks extend into the cranial region and connectseveral cranial sympathetic ganglia that are asso-ciated with the cranial (trigeminal, facial, glosso-pharyngeal, vagal) nerves. The organization of thecranial sympathetic ganglia varies among species(Funakoshi and Nakano, 2007). When present in theteleost heart, the adrenergic nerve fibers can enterit along three pathways: (a) by entering the vagi viathe connections with the nerves of the cranialpostganglionic cells (Holmgren and Nilsson, 1976;Funakoshi and Nakano, 2007); (b) along the ante-rior pair of spinal nerves, and (c) along the coronaryarteries (Holmgren, 1977). The cardiovascularsystem and lungs of the bichirs have been thesubject of comprehensive anatomical studies,including accounts of the gross morphology of theheart of various species of the genus Polypterusincluding Herpetoichthys (Calamoichthys) calabar-icus (Magid, 1967; Poll and Deswattines, 1967). Todate, there is no evidence regarding the distribu-tion of autonomic nerves and neurons in the heartof the bichirs and very little is known about thesympathetic nervous system of the bichirs, exceptfor the labelling of sympathetic preganglionicneurons by choline acetyltransferase (ChAT) andthe sympathetic (adrenergic) innervation of lungmuscle in Polypterus bichir bichir (Funakoshi andNakano, 2007; Zaccone et al., 2006, 2007). Despitean earlier controversy regarding the origin of thebichirs, recent studies have concluded that thepolypteriforms have a basal position in the ray-finned lineage (Inoue et al., 2003) with respect tothat of lobe-finned fishes. They diverged from theancestral branch to the teleost fishes (Chiu et al.,2004; Hoegg and Meyer, 2005) and there is aproposal to include these in a separate group ofteleosts. This may be also supported by molecularphylogenetic analysis of natriuretic peptides in abichir species (Ventura et al., 2005), which arestructurally similar to teleost counterparts. Both

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lungfish and tetrapods are thought to be descen-dants of the lobe-finned fish. The aim of this studywas to investigate the presence of peptide andnon-peptide transmitters and neuronal nitricoxide synthase (nNOS) associated with nerves andintracardiac neurons in the heart of the bichir(Polypterus bichir bichir Geoffroy St. Hilaire,1802). Both single-labelling and double-labellingimmunofluorescence methods were used.

Material and methods

Animals and tissue preparation

Ten specimens of Polypterus bichir bichir(35–40 cm total length, six males and four females)were obtained from a local supplier, maintained inaquaria at 27 1C, and fed amphibian larvae andgoldfish. The experiments for this study wereconducted according to the Italian law on theprotection of animals. Adult specimens wereanesthesized with 0.01% ethyl 3-aminobenzoatemethanesulfonate (MS 222, Sigma-Aldrich). Thehearts were removed from the pericardial cavityand hand-perfused with 0.1 phosphate bufferedsaline (PBS), pH 7.4, then with 4% paraformalde-hyde in PBS and then immersed in this fixative for2–4 h. All specimens were dehydrated in anascending series of ethanol and routinely processedfor embedding in Paraplast as described by Zacconeet al. (2007).

Immunofluorescence labelling

The techniques for single and double immunola-belling were used to obtain information on thecoexistence of neurotransmitters in the same nervefibers and ganglion cells, as previously described byZaccone et al. (2007). Routinely deparaffinized andrehydrated sections were rinsed several times in

PBS. They were then consecutively incubated withthe primary antisera listed in Table 1, overnight at4 1C in a humid chamber. Different combinations ofdouble labelling were performed and differentsections were incubated with antisera againsttyrosine hydroxylase (TH)/nNOS, galanin (GAL)/5-hydroxytryptamine (5-HT) and substance P (SP)/acetylcholinesterase (AchE). After four rinsesin PBS, binding sites of the primary antibodieswere visualized by corresponding fluorescein iso-thiocyanate (FITC)-conjugated goat anti-mouse IgG(Sigma-Aldrich) and tetramethylrhodamine isothio-cyanate (TRITC)-goat anti-rabbit IgG (Sigma-Aldrich), both diluted 1:100; incubation was for2 h at room temperature.

Fluorescence microscopy

The preparations were viewed by fluorescencemicroscopy using a Zeiss Axio-Imager Z1 micro-scope, fitted with an AxioCam digital cameraintegrated with image analysis software AxioVision4.5, for image processing (Zeiss, Oberkochen,Germany). Sections were imaged using the appro-priate filter settings for the excitation of FITC(480–525 nm) and TRITC (515–590 nm). The co-localization of two markers in the same structure,or an overlap of closely apposed structures in thesame focal plane, resulted in a yellow mixed color.Image processing was carried out using AxioVision4.5 software (Zeiss, Oberkochen, Germany).

Control experiments

Negative controls for all immunohistochemicallabelling were performed by substitution of non-immune sera for the primary or secondary antisera.Specificity of the labelling of some peptides wasverified by incubating sections with antiserumpreabsorbed with the respective antigen (at aconcentration of 10–100 mg/ml). The preabsorption

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Table 1. Primary antibodies used

Antigen Animalsource

Distributor Dilution

Vasoactive intestinal polypeptide (VIP) Rabbit Biomeda, Milan (Italy) PredilutedAcetylcholinesterase (AchE) Mouse Sigma, St. Louis (USA) 1:505-Hydroxytryptamine (5-HT) Mouse Dako Cytomation, Milan (Italy) 1:50Substance P (SP) Rabbit Sigma, St. Louis (USA) 1:50Tyrosine hydroxylase (TH) Mouse Sigma, St. Louis (USA) 1:100Neuronal nitric oxide synthase (nNOS) Rabbit Biomol, Milan (Italy) 1:100Galanin Rabbit Peninsula labs (USA) 1:200Pituitary adenylate cyclase-activating polypeptide(PACAP) 38

Rabbit Peninsula labs (USA) 1:300

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procedures were carried out overnight at 4 1C. Thepurified antigens were obtained from Bachem andPeninsula and Auspep (vasoactive intestinal poly-peptide VIP, pituitary adenylate cyclase-activatingpolypeptide 38, PACAP), Biomol-DBA (nNOS) andSigma (5-HT, SP).

Results

Structure of the heart

The heart lies inside the pericardium in front ofthe pectoral region. It is four-chambered andpossesses a prominent pyramidal-shaped ventricle.The Sinus venosus receives blood from the duct ofCuvier, where blood is collected from the inferiorhepatopulmonary vein (Figure 1). The wall of theSinus venosus is rather smooth and a scattering ofmuscular strands is seen in this specialized myo-cardial (nodal) tissue and at the junction betweenthe Sinus venosus and the atrium. The atrium is a

small ovoid chamber bearing a few lobules. Itscavity has a somewhat thickened, spongy internallayer and extends over the ventricle in such a wayas to also cover the mid-part of the dorsal surfaceof the Conus arteriosus. The ventricle is a thick-walled chamber having a narrow lumen. Theventricular structure is characterized by branchingtrabecular sheets running towards the outer myo-cardium. This arrangement of cardiac muscle cellsis consistent with that found in fish with a Type IIventricle, showing two muscle layers (an innerspongiosa and an outer limited dense myocardium).The ventricle opens into a long Conus arteriosusthrough a ventriculo-bulbar aperture. The Conusarteriosus is a tubular structure embraced ventro-laterally by the ventricle. It possesses a thick wallconsisting of an outer layer comprised of connec-tive tissue, an intermediate muscular layer ofsmooth muscle cells arranged spirally and anintimal layer of endothelial cells. From the mus-cular Conus arteriosus, the blood enters the ventralaorta, from where it reaches the gills.

Sinus venosus

The thin-walled Sinus venosus with sparse cardi-ac muscle cells and the sino-atrial ostium are themajor sites of pacemaker cells i.e., nerve cellbodies (ganglion cells). These cells have roundprofiles or may be pear-shaped (Figure 2A) and arefound isolated or clustered into small groups in thesino-atrial region. Six major populations of nervecell bodies could be distinguished by the double-labelling and single-labelling methods, which re-vealed the combinations of neurotransmitter im-munoreactivity; TH, AchE (Figure 2A), galanin (GA),SP (Figure 2A), vasoactive intestinal polypeptide(VIP) (Figure 2B), PACAP and nNOS (Figure 2C) inimmunopositive nerve cell bodies. Some nerve cellbodies in the Sinus venosus show a somaticrelationship between nerve terminals and thesoma of these cells. These preganglionic axonswere TH/nNOS immunoreactive. VIP and PACAPimmunoreactivity was observed in the ganglioncells in the Sinus venosus. Figure 2F shows thepresence of a VIP immunopositive neuron asso-ciated with a nerve terminal forming some varic-osities on the soma.

Sino-atrial junction

The nerve cell bodies in the sino-atrial tissuecontained immunoreactivity to GA (Figure 2D), nNOS,and TH (Figure 2E) and were contacted by nNOS-immunoreactive preganglionic axons (Figure 2E).

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IA

Aff I

Aff II

Aff III

Aff IV

HPV

DC

PA

V

A

CA

Figure 1. A schematic diagram of the heart of Polypterusbichir bichir. IA, hyoid artery; Aff I, the afferent branchialartery of the first arch; Aff II, the afferent branchial arteryof the second arch; Aff III, the afferent branchial artery ofthe third arch; Aff IV, the afferent branchial artery of thefourth arch bearing a single hemibranch; CA, conusarteriosus; A, atrium; V, ventricle; DC, ductus cuvierii;PA, pulmonary artery; HPV, hepatopulmonary vein. Adaptedfrom Poll and Deswattines (1967).

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The TH and nNOS double-labelling method revealsthe presence of both TH and nNOS-immunopositivenerve terminals in close proximity to some neurons.The AchE and SP double-labelling method showsthat few nerve cells are AchE immunopositive,while the majority of these cells in both the Sinusvenosus and the sino-atrial region are labelled withantibodies directed against SP. Furthermore, SPimmunoreactivity in preganglionic axons or boutonsassociated with these cells is found, similar to thatin the nerve cell soma (Figure 2F). Double labellingof sections for GA and 5-HTshows only the presenceof GA-immunoreactivity in the nerve cells scatteredalong the sino-atrial region. Single labelling withantibodies to VIP and PACAP shows the presence ofthe nerve cells in the sino-atrial tissue. These cellsare contacted by VIP and PACAP-immunoreactivepreganglionic axons.

Intracardiac distribution of nerve fibers

Bundles of nerve fibers showing GA-immunoreac-tivity were found in association with the cardiacmuscle in the Sinus venosus (Figure 3A). Myocardialcells are also supplied with adrenergic nerve fibersshowing TH-immunoreactivity. Double labellingwith antibodies against TH and nNOS showed THand nNOS immunoreactivity co-localised in thenerve fibers (Figure 3B) in contact with some nervecell bodies. The sino-atrial tissue is innervated byadrenergic nerve fibers forming a fine plexussurrounding groups of muscle cells. After doublelabelling with antibodies against AchE and SP, AchE-immunoreactivity was only localized in largebundles of varicose nerve fibers running across theSinus venosus and the atrium (Figure 3C). Single-labelling methods with antibodies against VIP and

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Figure 2. (A–F) Immunohistochemical labelling for neurotransmitters and nNOS in the Sinus venosus and sino-atrialtissue of the heart in the bichir. (A) Double labelling for AchE and SP in the Sinus venosus showing the presence ofSP-immunopositive axons (arrowheads) associated with two ganglion cells. (B) VIP immunoreactivity in a ganglion celland axons (arrowhead) bearing varicosities near to the nerve cell soma. (C) nNOS immunoreactivity in a ganglion cell(arrowhead) and the extensive network of nerves (arrows) in the Sinus venosus. (D) A GAL-immunopositive ganglioncell in the sino-atrial tissue. (E) Immunolabelling for nNOS and TH in the sino-atrial region. Combination of the greenand red channel showing the presence of nNOS-immunopositive axon (arrowed) lying near to the soma of a nNOS-immunopositive nerve cell body. (F) A group of SP immunoreactive nerve cell bodies in the sino-atrial tissue. Scalebar, 20 mm.

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PACAP showed VIP and PACAP immunoreactivity inthe muscle fibers in the Sinus venosus and theatrium. Four regions can be distinguished in theventricle: (a) the atrio-ventricular funnel, (b) two

layers composed of inner spongiosa and outercompacta of the ventricle, (c) the epicardial layerand (d) the endocardial myocardium. TH-nNOSdouble labelling showed dense innervation of the

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Figure 3. (A–H) Immunohistochemical labelling for neurotransmitters and nNOS in the myocardial muscles.(A) Immunohistochemical double labelling for GA and 5-HT in the Sinus venosus. Combination of both channelsrevealing that the GAL immunoreactivity is present in nerve fibers (arrows) associated with cardiac muscle. (B) A richnetwork of nNOS-immunopositive nerve plexuses (arrows) in the myocardium of the Sinus venosus. (C) AchE-immunopositive varicose axons (arrows) in the atrial muscle. (D) A dense network of nNOS-immunopositive nerve fibers(arrows) associated with the atrio-ventricular funnel. (E) Bundles of TH-immunopositive nerve fibers (arrows) in thespongy layer of the ventricle. (F) Immunolabelling for TH and nNOS in the outer and spongy layer of the ventricle.The red channel revealing the TH-immunopositive nerve fibers entering the inner spongy layer (arrow).(G) TH-immunopositive nerve fibers (arrowhead) surrounding a superficial coronary vessel in the epicardial layer.(H) Overview of the Conus arteriosus with neural plexuses surrounding the myocardial layer (M), immunolabelled withantibodies directed against AchE and SP. Combination of both channels revealing two different populations of AchE-immunopositive fibers (arrowheads) and SP-immunopositive fibers (arrows) in the muscle layer.

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atrio-ventricular funnel. A plexus giving rise tonumerous nerve terminals showing nNOS immunor-eactivity was present (Figure 3D). The epicardium,the compact subepicardial myocardium and spongylayer were richly innervated by bundles of TH-immunopositive nerve fibers. The fibers formed aplexus surrounding the cardiac muscle myofibrilorganization (Figure 3E, F). TH-immunopositivenerve fibers were observed running around thesuperficial coronary vessels in the epicardial layer(Figure 3G). GA-5HT double labelling demonstrateda rich plexus of GA-immunopositive nerve fibers inthe epicardial layer, but there was no TH-immuno-positivity associated with the coronary arteries inthe compact myocardium of the ventricle. Gang-lionic structures interpreted by Laurent (1962) asreceptor cells (‘‘formations receptrices parfaite-ment lisses’’) are found in the endocardial area ofthe ventricle. These cells showed nNOS, GA and5HT immunoreactivity. nNOS-immunopositive nervefibers were also observed in the myocardium of thisarea.

Innervation of the Conus arteriosus

The Conus arteriosus is supplied by nerve fibersbelonging to separate populations as demonstratedby the double-labelling experiments with antibo-dies directed against SP and AchE. SP-immunopo-sitive nerve fibers ran in the smooth muscle of theadventitia–media layer (Figure 3H), and werelocated in close proximity to AchE immunopositivefibers, which showed a conspicuously differentdistribution compared to the SP-immunoposi-tive fibers. GA and 5HT double labelling demon-strated a plexus of two separate nerve fiberpopulations associated with the adventitia–medialayer. TH/nNOS double labelling showed a denseplexus of TH-immunopositive nerve fibers in theadventitia–media layer, and there were nNOS-immunopositive neurons close to the smoothmuscle. VIP and PACAP single labelling showed thepresence of these neurons in the muscle layer ofthe Conus arteriosus.

A summary of the distribution of the observedimmunoreactivities for neurotransmitters in theheart of the bichir is shown in Table 2.

Discussion

The bichirs are both water-breathers and air-breathers and use their gills and lungs for gaseousexchange. They depend in anoxic water exclusivelyon pulmonary ventilation, which involves a shut-

down of branchial exchange (Lomholt and Glass,1987) and, concomitantly, a perfusion of the lungsthat constitutes a larger fraction of total cardiacoutput. The creation of a double circulatory loop ispresent to some degree based on anatomy, but isnot as developed as in the obligate air-breathinglungfishes (Brauner and Berenbrink, 2007). Theorigin of these primitive fishes may be near tothe ancestral group of the ray-finned fishes and thelobe-finned lineages. There is virtually no informa-tion regarding cardiovascular anatomy and thecontrol systems that regulate the heart and thevasculature of these fishes. Most studies oncardiovascular control in primitive fishes arelimited to the control of cardiac output, as wellas control of blood flow through the gills to air-breathing organs and the gastrointestinal tract(Farrell, 2007). The heart of the bichir speciesstudied here consists of four separate chambersarranged in series, consisting of the Sinus venosus,the atrium, the ventricle and the Conus arteriosus.The hepatopulmonary vein and the ducts of Cuvierare fused over a short distance, forming the Sinusvenosus. The ventricle is composed of spongytrabeculate myocardium which is invested with athin layer of densely arranged muscle fibers,the cortical, denser myocardium. Type II ventricleis found and the myoarchitecture accounts fora trabecular arrangement versus compact type(Tota et al., 2006). The epicardial layer is suppliedby a coronary vasculature. As reviewed by Donald(1998), the cardiovascular system of fishes shows avaried degree of development of the autonomicinnervation. Cardiac control in lungfish resemblesthat in other vertebrates, in that the heart receivesan inhibitory, cholinergic vagal innervation. How-ever, in lungfishes there is only an adrenergiccontrol by humoral catecholamines (mainly epi-nephrine) released from chromaffin tissue in thewalls of the intercostal arteries and azygos vein(Nilsson and Holmgren, 1992). Similar to lungfishes,the heart of the elasmobranchs is innervated byparasympathetic cholinergic nerves (Laurent et al.,1983). The innervation of the teleost heart waslong thought to be exclusively vagal in origin(Randall, 1968), but both monoaminergic andadrenergic components have been localized insome teleost species (Donald and Campbell, 1982;Holmgren, 1977). Teleost fish exhibit both nervousand humoral control as well as intrinsic control oftheir hearts. Using immunohistochemistry, we havedetected for the first time the localization of bothpeptide and non-peptide neurotransmitters in theheart of the bichir. The immunolabelling studiesindicated that the main intracardiac localization ofneurons is the sino-atrial plexus consisting of a

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network of nerve fibers and nerve cell bodies(ganglion cells). The sino-atrial junction is regardedas the site of the main pacemaker region of theteleost heart (Saito, 1973). The atrium and theatrio-ventricular funnel probably receive postgan-glionic innervation from the cardiac ganglion cells.This raises the possibility that there are intrinsiccircuits involving several different populations ofintracardiac neurons, which can be detected byimmunohistochemistry. Clearly retrograde-label-ling techniques are needed to demonstrate thatthe axons of the various cell populations project toother heart regions. Also, functional studies mayhelp to clarify if these neurons are responsible fornegative inotropic and chronotropic actions in thebichir.

Immunolocalization of neurotransmitters inthe ganglion cells

Using different combinations of antibodies indouble labelling, we showed the coexistenceof various neurotransmitters in the nerve cellbodies in the Sinus venosus and sino-atrial area.A prominent feature is the relationship betweenthe nerve terminals and the somata of someganglion cells in the Sinus venosus and sino-atrialtissue. Many axons immunopositive for THwith nNOS are found associated with the nervecell bodies. Some AchE and SP-immunopositiveganglion cells showed the presence of synapticboutons or a pericellular array of axons containingSP immunoreactivity. The present study shows thepresence of several populations of intracardiacnerve cell bodies using both single- and double-labelling immunofluorescence techniques. In thecardiac neurons of four species of teleost fishes,

immunoreactivity to VIP has been demonstrated(Davies et al., 1994), and GA, SP and calcitoningene-related peptide has been detected in theaxons of these neurons. Our study showed that theintracardiac neurons contain, in addition to AchEand TH, an array of neuropeptides such as GA, SP,VIP, PACAP; furthermore, nNOS was detected. Inthis study, the co-localization of VIP (and/or PACAP)and nNOS was not tested. A coexistence ofVIP/PACAP and NOS has been found in the myen-teric neurons of most vertebrate species. Severalstudies also demonstrated that VIP and PACAPstimulate the production of nitric oxide (NO) ingastric muscle cells, as well as affecting the levelsof cAMP and cGMP in these cells (Murthy et al.,1997).The distribution of VIP and PACAP-immunor-eactive nerve cell bodies and fibers in the sino-atrial tissue agree with previous findings in the fishgut (Olsson and Karila, 1995). The effects of theseneuropeptides on the cardiovascular system of thefish have not been investigated, but preliminarystudies performed on the cod intestine suggest arelaxing effect on gut smooth muscle (Olsson andHolmgren, 2000). Neural NOS has been demon-strated in the intracardiac ganglion cells of thegoldfish Carassius auratus (Bruning et al., 1996) andmammalian species (Tanaka et al., 1993). In thelight of the present results, it seems that NO mightbe involved in the regulation of the heart rate ofthe bichir, but its involvement in the cardiacactivity of this species is not known. In mammals,NO is released by a rich variety of cardiac tissues,i.e. myocardiocytes, endothelial cells (both vascu-lar and endothelial), interstitial cells, coronaryvessels and myocardial neurons (Balligand, 2000;Hare, 2003). Tota et al. (2006) have also studiedthe role of NO in modulating mechanical perfor-mance in teleost hearts.

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Table 2. Immunoreactivities for neurotransmitters in the heart of the bichir

Neurotransmitter Nerve cellbodies

Boutons Nerves in cardiacmuscle

Conus arteriosus(muscle layera)

Tyrosine hydroxylase + + + +Neuronal nitric oxide synthase (NO) + + + �

Acetylcholinesterase + � + +5-Hydroxytryptamine substance P � � � +Vasoactive intestinal polypeptide + + + �

Pituitary adenylate cyclase-activatingpolypeptide 38

+ + + �

Galanin + � + +

Key: +FITC-labelled structures; � No fluorescence.Some combinations of double labelling were performed. The antibody pairs (TH–nNOS, GAL–5-HT and SP–AchE) are used. Singleimmunohistochemical-labelling techniques were also performed using both VIP and PACAP 38 antibodies. Possible cases of co-localization are not tested for the latter antibodies.aNerve cell bodies embedded in the myocardium show immunoreactivity for nNOS, VIP and PACAP 38.

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Immunolocalization of neurotransmitters inthe intracardiac nerves

Large bundles of varicose nerve fibers showing GAimmunoreactivity were seen running in the Sinusvenosus in association with the myocardial cells.TH-nNOS immunopositive axons were also presentin the cardiac muscle in the Sinus venosus,including an array of axons terminating in theganglion cells in the sino-atrial tissue. It is knownthat specific cranial nerves contain fibers from thecranial sympathetic ganglia (Sarnat and Netsky,1981). In addition, Funakoshi et al. (1995, 1996a, b)showed that preganglionic fibers in the cranial partof the sympathetic nervous system in the puffer fishoriginate in the central autonomic nucleus, and inthe file fish, NO is synthesized in the spinalsympathetic preganglionic neurons. The aboveresults may indicate that TH and nNOS-immunopo-sitive nerves provide innervation of the adrenergicneurons in the sino-atrial region. We do not knowthe origin of these nerves. Our findings indicatethat the non-adrenergic (nNOS positive), presumedto be of vagal origin, and adrenergic nerveterminals may show some influence on the pace-maker cells and on the cardiac muscle. The presentresults are consistent with earlier pharmacologicalstudies by Holmgren and Nilsson (1976) andHolmgren (1977), which suggested that acetylcho-line and adrenaline are released from the sympa-thetic postganglionic cells in the cod, Gadusmorhua. According to Laurent et al. (1983), itmay be argued that besides the inhibitory choli-nergic vagal control of the heart, the neural controlby adrenergic nerves constitutes a third phyloge-netic step. The most primitive feature is theintracardiac source of catecholamines in endogen-ous chromaffin cells in cyclostomes and dipnoansand the release of these substances into the bloodfrom the exogenous chromaffin tissue, which islocated outside the heart in elasmobranchs andteleosts. The present study shows that the adre-nergic nerve fibers can enter the heart of the bichiralong the Sinus venosus and the coronary arteries.GA and nNOS immunoreactivity occur in the nervecell bodies of the sino-atrial tissue. Varicosenerve fibers with GA and nNOS-immunopositivenerve fibers are also found in this area. The originof this group of fibers is not known. Presumably,these fibers project from the neuronal cell bodiesof the sino-atrial area to all the chambers of theheart and to the pericardium. The SP-immunopo-sitive boutons found on the nerve cell bodies mayresemble those described by Davies et al. (1994),which they interpreted as terminals of eithersensory or postganglionic sympathetic neurons.

Both the outer myocardium and the spongiosa ofthe heart of the species studied show that theseregions are richly innervated by TH-immunopositivenerve fibers. The adrenergic innervation of theventricle is also dependent on the presence of aperivascular supply of TH-immunopositive nervesaround the coronary arteries found in the epicardiallayer. This is in line with the adrenergic supply tothe compact myocardium, which is often associatedwith coronary arteries (Donald and Campbell, 1982;Holmgren, 1977). We do not know the origin of theadrenergic nerve fibers in the ventricular myocar-dium of the bichir. If these represent a cardiacsource of adrenergic innervation, it could beargued that they emanate from the vagus andcould reach coronary vessels along the sino-ventricular nerve bundles (Laurent et al., 1983).The muscle cells of the atrio-ventricular funnel arerichly innervated by nNOS-immunopositive nervefibers. The origin of these nerves is unknown.Laurent et al. (1983) suggested that the innervationof the atrio-ventricular funnel indicated that someneural elements are postganglionic cranial auto-nomic fibers. Another interesting feature is thepresence of cholinergic nerves associated with themyocardial cells of the atrium. According toYamanouchi et al. (1973) and Yamauchi (1980),these nerves represent a part of the cranialautonomic cholinergic system responsible for thecardio-stimulatory effects of the vagal stimulation.We do not know if the nerve terminals arisefrom the axons of AchE, VIP and PACAP immunopo-sitive ganglion cells located in the Sinus venosusand sino-atrial region since VIP is often co-localizedin postganglionic parasympathetic nerves (Davieset al., 1994).

Immunoreactive nerves in the Conusarteriosus

This study is the first anatomical report showing arich population of nerve terminals in the Conusarteriosus. Double labelling with antibodies againstSP-AchE, GAL-5HT and TH-nNOS revealed thecapacity of these nerve terminals to store andrelease many types of transmitters, includingpeptides, at the level of the muscle layer. Thecomplexity of this innervation could be consistentwith the possible auxiliary functions of this cardiacstructure that represents the ventricular outflowtract like the Bulbus cordis (Conus) of chondrichth-yans, holostean and dipnoan fishes (Farrell, 2007).The muscle layer of the Conus arteriosus isinnervated by SP-immunopositive nerves, butthe origin of these peptidergic projections is

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not known. These fibers might originate from theSP-immunoreactive perikarya found in cranialand spinal sensory ganglia (Funakoshi et al.,2000). The origin of GA and monoaminergicimmunopositive nerves entering the Conus arter-iosus is not known. Holmgren (1977) reported thepresence of amine-containing fibers reachingthe heart, not only in the cardiac branches of thevagus, but also above the 1st and 2nd spinal nerves.While GA-immunopositive fibers running throughoutthe heart may originate from the GA-immunoposi-tive intracardiac neurons, the aminergic fibers arelikely to be of spinal autonomic origin (Holmgren,1977). A rich innervation of the medial layer of theConus arteriosus by TH-immunopositive nerves wasalso shown. Intrinsic cell bodies showing nNOS andPACAP immunoreactivity were also observed in thesmooth muscle. These results are also consistentwith the presence of adrenergic fibers in the BA ofteleost fishes (Farrell and Jones, 1992). In contrast,no cholinergic nerves have been reported. How-ever, the BA may contain adrenergic and choliner-gic receptors since agonists to these receptorsincreased and decreased, respectively, the compli-ance of the BA in Oncorynkus mykiss (Farrell,1979). Evans et al. (2003) demonstrated the effectsof a rich variety of vasoactive substances on the BAof the eel, Anguilla rostrata. Our data suggest thatthe Conus arteriosus is controlled by severalneurotransmitters and paracrine substances and,therefore, may be more than a simple ‘‘wind-vessel’’ or ‘‘pressure chamber’’ (von Skramlik,1935; Johansen and Burggren, 1980). In conclusion,from a morphological point of view, the heart ofthe bichir species studied shows a pyramidalshape that is typical of teleosts and air-breathingfishes (Icardo et al., 2005; Munshi et al., 2001).Contrary to cyclostomes and lungfishes, wherethere are limited physiological studies of cardio-vascular control, there is no information onthe control of the cardiovascular system inthe Polypterids. Several of the transmitter sub-stances immunolocalised in the ganglion cellsand nerves innervating the bichir heart in thisstudy are known to produce vascular effects andto regulate the teleost cardiovascular system(Johnsson et al., 2001). The future challenge is toprovide pharmacological evidence for the sametransmitters in cardioregulation and intrinsic con-trol in the bichirs.

Acknowledgement

Thanks are due to Dr. John Donald for criticalreading of the manuscript.

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