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Acta histochemica 112 (2010) 328—336

0065-1281/$ - sdoi:10.1016/j.

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Postganglionic nerve cell bodies andneurotransmitter localization in the teleost heart

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

Department of Animal Biology and Marine Ecology, Faculty of Science, Section of Comparative Neurobiology andBiomonitoring, University of Messina, Via Salita Sperone 31, I-98166 Messina, Italy

Received 12 November 2008; received in revised form 11 February 2009; accepted 13 February 2009

KEYWORDSHeart;Neuropeptides;nNOS;Innervation;Nerve cell bodies;Pacemaker;Teleosts

ee front matter & 2009acthis.2009.02.004

ing author. Tel.: +90 676ess: gzaccone@unime.i

SummaryA study was undertaken to determine the distribution of specific types of autonomicnerves and the presence of various transmitter substances in the heart of two teleostspecies: the mullet (Mugil cephalus) and the Nile catfish (Synodontis nigriventris).Large nerve trunks in the sinus venosus were shown to contain tyrosine hydroxylaseimmunoreactivity and indicate the location of adrenergic nerve fibers, which arealso associated with a coronary circulation to the ventricular myocardium in themullet heart. Fluorescence immunolabelling methods revealed that the atrium andthe outer and inner compact muscle of the ventricle have nerves in which substanceP and galanin (GA) are localized. It seems likely that the cell bodies (perikarya) ofthe substance P and GA-immunopositive axons are located at sites outside the heart.The GA-immunopositive nerve fibers may represent a population of axons ofintramural postganglionic nerve cell bodies. Most intracardiac nerve cell bodies arelocated in the sinus venosus and in the sinoatrial junction and reveal immunor-eactivity to substance P, GA, neuronal nitric oxide synthase (nNOS), vasoactiveintestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP).Furthermore, substance P immunoreactivity is present in the cardiac cellsintermingled with the substance P-immunopositive nerve fibers. A nerve plexusconsisting of a well-developed network of nerve fibers and nerve cell bodies maypossibly correspond to a cardiac pacemaker, but its function in fish cardiacregulation is unknown and remains to be elucidated.& 2009 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

5543; fax: +90 393409.t (G. Zaccone).

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Nerve cell bodies in teleost heart 329

Introduction

The teleost heart consists of four separateserially arranged chambers: the sinus venosus, theatrium, the ventricle and the bulbus arteriosus(Randall, 1968). The first chamber is the sinusvenosus, which receives blood from the pairedducts of Cuvier, the hepatic and anterior jugularveins. The ventral ducts of Cuvier also collect bloodfrom the anterior and posterior cardinal veins(Laurent et al., 1983; Farrell and Jones, 1992).The hepatic veins, in turn, open directly into thesinus venosus and participate in the formation ofthe chamber. The sinus venosus is distinguishedhistologically into three tunics: tunica intima,tunica media and tunica adventitia (Abraham,1969). The tunica media consists of longitudinallyarranged smooth muscle cells. Striated cardiacmuscles have also been reported together withthe smooth muscles (Kumar, 1997). The musclesaround the sinoatrial orifice are arranged circularlyand form the sinoatrial ring (Laurent, 1962; Nair,1973; Kumar, 1974). The atrium communicates withthe ventricle through a tubular structure, theatrioventricular funnel (Laurent, 1962).

The atrioventricular opening is surrounded bycircularly arranged muscle fibers that form theatrioventricular ring (Kumar, 1976). The mainintracardiac localization of nervous tissue is in asinoatrial plexus, which corresponds to the auto-matism (pacemaker) centre of the heart. Thisplexus consists of a well-developed network ofnerve fibers and postganglionic nerve cell bodies(Laurent, 1962), which are concentrated in thesinoatrial area (Zaccone et al., 2009a, b), but arealso distributed along the atrioventricular funnelup to the atrioventricular junction. Excitation ofthe fish heart is initiated from specialized pace-maker cells located in the sinus venosus, sinoatrialvalve, atrioventricular funnel or in the junctionalregion between the ventricle and the bulbusarteriosus (Saito, 1969).

There is still a lack of agreement regarding thepresence of a cardiac conducting system in fish, andwhether there is in fact nodal tissue equivalent tothat of homeothermic vertebrates that is respon-sible for the initiation and conduction of electricalimpulses. The cardiac conducting system in fishes isnot thought to be totally myogenic or neurogenic,but to consist of a complex combination of the two(Kumar, 1976). The pacemaker cells generatespontaneous action potentials, whose discharge isstrongly modulated by temperature, autonomicnerves and the purinergic system (Harper et al.,1995; Giles and Shibata, 1981; Zaza et al., 1996).According to Vornanen et al. (2002), the pacemaker

mechanisms in fishes have yet to be explored indetail; however, the characteristics of the pace-maker action potential of the fish heart are similarto those of other vertebrates (Campbell et al.,1992).

In most vertebrates, the autonomic nervoussystem influences heart rate and stroke volume.Sympathetic and parasympathetic innervation ofthe heart affects heart rate in the majority ofvertebrates by antagonistic action via beta-adre-nergic and muscarinic receptor stimulation, re-spectively. Intracardiac neuropeptides are thoughtto be implicated in modulation of heart function inmany vertebrate species, including fishes (Zacconeet al., 2008a, b), although details still need to beelucidated (for review see Beaulieu and Lambert,1998). The cardiac output in fishes is an integratedresponse to several factors that directly or indir-ectly influence heart rate and stroke volume(Johnsson et al., 2001). Nervous and humoralcontrol and local mechanisms are involved inregulating the blood flow; however, the modulationof heart rate in teleosts may be complex. It wasrecently shown in the bichir heart that the mainintracardiac innervation consists of a network ofnerve fibers and nerve cell bodies in the sinoatrialarea, containing a wide array of neurotransmitters,including neuronal nitric oxide synthase (nNOS;Zaccone et al., 2009a, b).

There is also evidence indicating that cardiovas-cular control in fishes may be integrated by thevenous system (Johnsson et al., 2001), since theveins of teleosts are innervated by neuropeptide-containing nerves responsible for the intrinsiccontrol of the heart. This is in contrast tothe case with hagfish, which are primitive verte-brates, where neither the systemic heart nor theportal heart receive external innervation and thecardiac output is influenced by the preload of theirhearts.

There is a general indication that the conductingsystem (the Purkinje system of higher vertebrates)is absent in fish hearts (Laurent et al., 1983). It isstill not clear whether the heartbeat of fishes isgenerated by means of muscles or nerves. Thereare relatively few physiological investigations con-cerning the occurrence of specialized pacemakermuscles or the specific identification of pacemakerfibers, although pacemaker cells have been demon-strated physiologically in cardiac myocytes in theatrioventricular funnel of the rainbow trout (Vor-nanen et al., 2002). A connection system composedof a myocardial continuity between the atrioven-tricular channel and the apex of the ventricle hasbeen localized in the heart of the zebrafish(Sedmera et al., 2003).

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The aim of this study was to investigate thepresence of various neuropeptides, nNOS, tyrosinehydroxylase (TH) and acetylcholinesterase (AchE)in the intracardiac neurons and nerves in the heartof the mullet (Mugil cephalus) and the Nile catfish(Synodontis nigriventris) by both single and doubleimmunolabeling procedures.

Material and methods

Animals and tissue preparation

Mullet (M. cephalus) were captured in Lake Faroon the Eastern coast of Sicily. The fish were keptindoors in aquaria supplied with circulating sea-water at approximately 23–25 1C. The Nile catfish(S. nigriventris) were obtained from a commercialfish farm and kept in aerated recirculating fresh-water at 23–25 1C. The fish were maintained inaquaria for 1–3 weeks before sacrifice. Adultspecimens were anesthetized by immersion indechlorinated freshwater or seawater containing a1:15,000 dilution of tricaine methanesulfonate (MS-222, Sigma-Aldrich, St. Louis, MO, USA), followedby pithing. The hearts were flushed in phosphate-buffered saline (PBS). The samples were fixed in 4%paraformaldehyde in phosphate-buffered saline(PBS) for 2–4 h, dehydrated in graded ethanolsand embedded in paraffin wax (Paraplast, McCor-mick Scientific, St. Louis, MO, USA).

Immunofluorescence labelling andmicroscopy

Routinely deparaffinized and rehydrated sectionswere rinsed several times in PBS and processed

Table 1. Primary antibodies used.

Substance antibody raised against Species antibodyraised in

Acetylcholinesterase (AchE) Mouse

Tyrosine hydroxylase (TH) Mouse

Neuronal nitric oxide synthase (nNOS) RabbitEndothelial nitric oxide synthase(eNOS)

Rabbit

Galanin (Gal) RabbitSubstance P (SP) Rabbit

Vasoactive intestinal polypeptide (VIP) RabbitPituitary adenylate cyclase-activatingpolypeptide (PACAP) 38

Rabbit

according to procedures previously reported byZaccone et al. (2008a). Sections were incubatedwith the primary antisera (Table 1) overnight at 4 1Cin a humid chamber. In this study we used bothsingle and double-immunolabelling procedures.Heart sections were double labelled for binding ofantibodies to tyrosine hydroxylase and nNOS, orantibodies to substance P (SP) and acetylcholines-terase. After several rinses in PBS, binding sites ofthe primary antibodies were visualized by thecorresponding fluorescing molecule, usingfluorescein isothiocyanate (FITC)-conjugated goatanti-mouse IgG (Sigma-Aldrich) and tetramethylr-hodamine isothiocyanate (TRITC)-goat anti-rabbitIgG (Sigma-Aldrich), both diluted 1:100. Incubationwas for 2 h at room temperature. The preparationswere viewed in a Zeiss Axio-Imager Z1 fluorescencemicroscope fitted with an AxioCam digital cameraintegrated with AxioVision 4.5 image analysis soft-ware (Zeiss, Oberkochen, Germany). Sections wereimaged using the appropriate filter settings for theexcitation of FITC (480–525 nm) and TRITC(515–590 nm). Negative controls for all immunohis-tochemical labelling were performed bysubstitution of non-immune sera for the primaryor secondary antisera. Specificity of the labelling ofsome peptides was verified by incubatingsections with antiserum preabsorbed with therespective antigen (at a concentration of10–100 mg/ml). The preabsorption procedures werecarried out overnight at +4 1C. The followingpurified agents were used: vasoactive intestinalpolypeptide (Bachem Torrance, CA, USA; Peninsula,San Carlos, CA, USA), adenylate cyclase-activatingpolypeptide 38 (Auspep, Louisville, KY, USA; Ba-chem, Torrance, CA, USA) and nNOS (Biomeda,Milan, Italy).

Supplier Working dilution

Sigma-Aldrich, St. Louis,MO, USA

1:50

Sigma-Aldrich, St. Louis,MO, USA

1:100

Biomol, Milan, Italy 1:100Biomol, Milan, Italy 1:100

Biomol, Milan, Italy 1:200Sigma-Aldrich, St. Louis,MO, USA

1:50

Biomeda, Milan (Italy) Supplied predilutedPeninsula Labs, San Carlos,CA, USA

1:200

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Nerve cell bodies in teleost heart 331

Results

Structure of the heart

In the two fish species studied, the four cardiacchambers are arranged in a typical teleost pattern.The sinus venosus is relatively small and is no morethan a simple confluence of the common cardinalveins (ducts of Cuvier). It communicates with theatrium through a circular atrioventricular ostium.The atrium lies dorsally to the ventricle andappears to be much smaller than the ventricle. Asinoatrial canal and an atrium proper characterizethe atrium. The former is a thick-walled tube andthe latter a thin-walled spongy cavity. The funnel-like portion of the atrium is essential for atrialfilling. The atrium communicates with the ventriclethrough the atrioventricular funnel. The ventricleis a large muscular chamber with an apex project-ing inferiorly and its base lying superiorly, as

Figure 1. Proposed scheme for the innervation and distributthe teleost heart. Afferent sensory SP – immunopositive nervCAN and may arise from sensory neurons within the vagaimmunopositive fibers may be intracardiac neurons or neuron2000). SP, substance P; TH, tyrosine hydroxylase; GA, galanin;VIP, vasoactive intestinal polypeptide; PACAP, pituitary adeautonomic nucleus; SV, sinus venosus; A, atrium; V, ventricle1994).

previously described in other teleost species (forreview see Kumar, 1997). It has a sac-like shape inthe mullet and a pyramidal shape in the Nilecatfish. The ventricular myocardial architecture issimilar in the two species. The muscle layersconstituting the ventricle wall are differentiatedinto the epicardium, myocardium and endocar-dium. The myocardial fibers are packed in orderlyarranged bundles (compacta) that enclose an innerspongiosa. A coronary circulation to the ventricle isfound in the mullet. The epicardium of the catfishis supplied by vessels.

Distribution of immunoreactive nerve fibers

In fish, the classic division of the autonomicnervous system into parasympathetic and sympa-thetic nerves is oversimplified by Nilsson (1983).Figure 1 shows the sources, the distribution ofautonomic and peptide-containing nerve fibers,

ion of autonomic nerves and related neurotransmitters ine fibers innervate the atrium. These fibers project to thel nerve outside the heart. The origin of cells of GA-s lying in the rostral portion of the CAN (Funakoshi et al.,Ach, acetylcholine; nNOS, neuronal nitric oxide synthase;nylate cyclase-activating polypeptide 38; CAN, central; BA, bulbus arteriosus (redrawn from Morris and Nilsson,

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and the presence of known or putative neurotrans-mitters for each main class of neurons in the heartof fishes. The general pattern of the microscopicinnervation of the heart of the two speciesexamined in the present study is similar to thatrecently reported in the bichir (Zaccone et al.,2008a). Nerve branches showing tyrosine hydro-xylase immunoreactivity (Figure 2A) enter the sinusvenosus, sending out large nerve trunks in thetunica adventitia (subepericardial space). Galanin(GA)- immunopositive nerve fibers penetrate intothe tunica media. Smooth muscle layers are alsorichly innervated by GA and neuronal nitric oxidesynthase-immunopositive nerve fibers. The latterare found passing through the visceral pericardium.Fine varicose substance P-immunopositive fibersare present in the wall of the sinus venosus andatrium. In the atrium the SP-immunopositive axons

Figure 2. Immunohistochemical localization of neurotransmaxons associated with mullet muscle fibers (M) in the sinus(arrows) in the atrial myocardium (M) of the mullet; C–E,compacta (C) and spongiosa (S) of the ventricular myocardiuvaricose axons on the outer layer (arrows) are seen branchingF, SP-immunoreactive nerve terminals (arrows) in the compacbar on all micrographs ¼ 20 mm.

branch and form small bundles (Figure 2B) andsingle fibers, which run between cardiac musclefibers and the pericardium. The density of fluor-escent nerve fibers is lower in the atrium than inthe sinoatrial tissue.

Bundles of GA-immunopositive nerves were pre-sent in the outer compact layer of ventricularmyocardium. Varicose GA-immunopositivefibers were also discernible in the compacta andthe spongiose of the ventricular myocardium(Figures 2C–E). There were a few large nervebundles showing SP-immunoreactivity extendingfrom the adventitia of the bulbus into the periph-eral compact and inner layers of the ventricle(Figure 2F).

The endocardial endothelial cells in the ventriclehave immunoreactivity for eNOS. The compactlayer of the myocardium contained coronary blood

itters in the fish heart. A, TH-immunopositive varicosevenosus. B, bundles of SP-immunopositive nerve fibersimmunohistochemical labelling for galanin (GA) in the

m in the catfish (S. nigriventris); the GA-immunoreactiveacross the spongy trabeculate myocardium (arrowheads);ta (C) of the ventricular myocardium in the catfish; scale

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Nerve cell bodies in teleost heart 333

vessels, which bore a perivascular plexus oftyrosine hydroxylase-immunopositive varicosenerve fibers. In the catfish species (S. nigriventris)TH-immunopositive nerve fibers could be discernedin the epicardial vessels.

The sinus venosus and the atrium of the two fishspecies are supplied by vasoactive intestinal pep-tide (VIP)- and pituitary adenylate cyclase-activat-ing peptide (PACAP)-immunopositive axons. VIP-and PACAP-immunopositive varicose axons ranalong the atrial muscle fibers, whereas musclefibers in the sinus venosus were associated withvery few axons. VIP-immunopositive nerve fiberswere seen supplying the valves located at thesinoatrial junction.

Postganglionic nerve cell bodies

A highly schematic illustration, Figure 3, showsthe location of nerve cell bodies at the sinoatrialjunction (marked by shaded area). These cells lie atthe site of specialized cardiac pacemaker tissue(nodal tissue) in many fish (Laurent et al., 1983;Kapoor and Khanna, 2004). Comparable resultswere obtained for the mullet and the catfishspecies. In the mullet, a ganglion consisting ofadrenergic and nNOS-immunopositive (nitrergic)nerve cell bodies lies in the muscle layers of thesinus venosus (Figures 4A, B). Double immunolabel-ling with antibodies against TH and nNOS show THand nNOS immunoreactivity co-localised in thesecells. Single-labelling methods with antibodies toPAPAC also revealed the presence of this ganglionamong the muscle layers in the sinus (Figure 4C).

All the nerve cell bodies in the two species ofteleost were immunopositive for vasoactive intest-inal polypeptide (VIP; Figure 4D), pituitary adeny-late cyclase-activating polypeptide (PACAP 38), GA,SP and nNOS (Figures 4E, F). The acetylcholine

Figure 3. Structure of the teleost heart showing the arrangpresumed specialized cardiac tissue area where ganglion cellregion. DC, ductus cuvierii; S, sinus venosus; PM, pacemakventricle; B, bulbus arteriosus (modified from Laurent, 1962)

esterase (AchE) and SP double-immunolabellingmethod showed that the levels of AchE immunor-eactivity were weaker and more variable, making itdifficult to confirm that all the nerve cell bodiescontain AchE. The majority of these cells werelabelled with antibodies to SP. Single nerve cellsand cell clusters were also visible close to thesinoatrial border. These cells were intermingled inthe nerve plexuses (Figure 4E, F). No fluorescentlylabelled ganglion was observed in the sections ofthe sinus venosus of the heart of the catfish speciesafter double immunolabelling for TH and nNOS andsingle-immunolabelling methods with antibodiesagainst the neuropeptides.

In most, if not all, nerve cell bodies in the twospecies of teleost fish, perineuronal baskets or anyneural contacts were recognized as an indication ofinnervation of these cells.

Discussion

The anatomy and physiology of the autonomicnervous system in fishes have been extensivelystudied (Nilsson 1984a, b; Holmgren and Nilsson,1982; Morris and Nilsson, 1994). Cardiac innerva-tion shows a pattern of evolutionary specializationincreasing in the dipnoans, elasmobranchs andteleosts. There has been an evolution from apredominantly endocrine-based control in cyclos-tomes, to a dual endocrine/neural control inteleosts (Donald, 1998), unlike the myxinoid heart,which receives no extrinsic innervation (Fange etal., 1963). The teleosts also exhibit an intrinsiccontrol of their hearts, since adrenergic nervesinnervate systemic blood vessels of most species.Chromaffin cells, present near the great veins or inthe heart, are common in all fishes and arehomologs of the adrenal medulla (Olson, 1998).

ement of the various chambers and the location of thes are intermingled in nerve fibers to form the pacemakerer; A, atrium; AV, atrial valve; VV, ventricular valve; V,.

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Figure 4. A, B, immunohistochemical double labelling for neuronal nitric oxide synthase (nNOS) and tyrosinehydroxylase (TH) in the sinus venosus in the heart of the mullet (M. cephalus); single channels revealing that a ganglion(G) is present in the muscle layer (M), consisting of several cell bodies giving nNOS (green channel) and TH (red channel)immunoreactivity; C, D, a ganglion (G) in the muscle layer (M) of the sinus venosus in the heart of the mullet; it iscomposed of dense clusters of (C) PACAP- and (D) VIP-immunopositive nerve cells; PACAP-immunopositive axons(arrows) are also associated with muscle layers; E, F, numerous immunoreactive axons (arrows) in the sinoatrial area inthe heart of the catfish (S. nigriventris); these axons and the nerve cell bodies (arrowheads) display SP-immunoreactivity; A, atrium; SV, sinus venosus; scale bar on all micrographs ¼ 20 mm.

Zaccone et al.334

The fish heart is innervated by a pair of largenerve trunks, which enter the heart via the cardinalveins and carry both cranial autonomic fibers ofvagal origin and spinal sympathetic autonomicfibers (Donald, 1998; Zaccone et al., 2009a, b) thatjoin the vagus nerve within the cranium (commonlytermed the ‘vagosympathetic nerve’). Tonic inputto the heart is provided by adrenergic nerves,whereas humoral mechanisms may contribute tomyocardial cell function and/or depend to varyingdegrees on the species (Olson, 1998). The excita-tion of the heart is also initiated by a group ofautonomous pacemaker cells, leading to the acti-vation of all the cells of the cardiac muscle duringcontraction (Vornanen et al., 2002).

The main intracardial localization of the nervoustissue in fishes consists of a network of nerve fibers

and nerve cell bodies (Laurent, 1962; Laurentet al., 1983; Zaccone et al., 2009a, b), whichcorresponds to the pacemaker locus of the heart.In the older literature (e.g. Keith and Flack, 1907;Keith and Mackenzie, 1910), nodal tissue wasreported at the base of the venous valve, whichhas been confirmed by later studies on thesinoatrial myocardium of the loach (Yamauchi,1969), catfish (Laurent, 1962) and trout (Yamauchi,1969). In particular, in the loach (Misgurnusanguillicaudatus), neuromuscular contacts havebeen observed and transmission may occur via anintermediate cell, the internucial cell, interposedbetween the postganglionic axon and the musclecell. The internucial cells represent a cell type thathas a specialized role in the distribution andmodulation of an autonomic control of the fish

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Nerve cell bodies in teleost heart 335

myocardial cells (Laurent et al., 1983). This patternof organization is restricted to the sinoatrial(nodal) tissue in the loach, and has never beenobserved in other fish species. This part of fishheart is also thought to play a key role in initiatingand controlling the heartbeat (Skramlik, 1935;Laurent, 1962). Despite the lack of recognition ofnodal tissue or conduction pathways in the heart ofa large number of species, the distribution of thenerve cell bodies found in some species (Davies etal., 1994; Zaccone et al., 2009a, b) may correspondto a more specific pacemaker region in the fishheart. Future studies should include ultrastructure,molecular composition and electrophysiologicalproperties of the pacemaker cells located in thesinus venosus, the sinoatrial junction and theatrioventricular funnel in order to elucidate thisspecific region of the heart in fishes.

A cholinergic vagal influence on the pacemakercells can be postulated by the innervation patternof some intracardiac parasympathetic neuronslocated in the heart of some fish species (Davieset al., 1994; Zaccone et al., 2009a, b). Associatedwith a majority of intracardiac VIP-immunopositivecell bodies were VIP-immunopositive axons thatformed pericellular baskets (Davies et al., 1994).Furthermore, both TH- and nNOS-immunopositivenerve terminals were found in close proximity tosome neurons. SP-immunoreactivity in preganglio-nic axons or boutons is associated with nerve cellbodies in the heart of the bichir (Zaccone et al.,2009a, b). AchE-, VIP- and PACAP-immunopositiveaxons were found to supply the sinus venosus andthe sinoatrial region. Although neuropeptides andnNOS are reliable markers for both parasympa-thetic nerve cell bodies and nerves in the fishheart, we still do not know the functional implica-tion of these substances or their effects on cardiacmuscle or the nature of the terminals of eithersensory or postganglionic sympathetic neuronsinnervating the intracardiac neurons. If SP-immu-nopositive nerves represent terminals of thesensory neurons, these nerves presumably arisefrom cells outside the heart.

SP-immunoreactive neurons are seen withinmotoneuronal columns of the vagal nerves in thefilefish, Stephanolepis cirrhifer (Funakoshi et al.,2000). In the species studied here, we did not findthe occurrence of fluorescently labelled perineur-onal baskets or any indications of fluorescentlylabelled innervation of nerve cell bodies. Thepresent study shows that the adrenergic nervefibers can enter the heart along the sinus venosusand the coronary arteries. These arteries are seenrunning through the compacta of ventricularmyocardium in the mullet. Vessels that are limited

to the pericardium are discernible in the catfish.SP-immunopositive nerve cell bodies are located inextensive nerve plexuses in the atrium. We do notknow if these plexuses are provided with innerva-tion by the vagus nerve or by the axons of SP-immunopositive intracardiac cell bodies of post-ganglionic neurons.

GA-immunopositive axons show a prominentdistribution in the compacta and spongiosa of theventricular and myocardium. Presumably, thispopulation of GA-immunoreactive nerves originatesfrom the intracardiac GA-immunopositive neurons.Funakoshi et al. (2000) also argued that GA-immunoreactive neurons lie in the rostral portionof the central autonomic nucleus (CAN) in therostral spinal cord. The neurons make a predomi-nant contribution of preganglionic fibers to coelo-mic viscera and some cranial part of the body viathe sympathetic ganglia. Consequently, some GA-immunoreactive axonal projections may arise fromcells outside the heart such as SP-immunoreactivenerves entering the atrium.

VIP has been regarded as the exclusive marker ofpostganglionic neurons in fish heart (Davies et al.,1994). However, it appears that in the two speciesstudied here and also in Polypterids (Zaccone et al.,2009a, b), different transmitter substances are alsocandidates among a variety of neuropeptidestested. SP and GA are regarded as the mainneuropeptides found in the intracardiac neuronsof other vertebrates (Morris and Nilsson, 1994).Although these peptides are thought to mediatenegative inotropic and chronotropic actions in theheart of mammalian species (Morris and Nilsson,1994), as yet there have been no studies performedthat have demonstrated the roles of these peptidesin vagal neurotransmission and their specific con-tribution in identifying pacemaker fibers in fishes.

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