NANC nerves in the respiratory air sac and branchial vasculature of the indian catfish, Heteropneustes fossilis

0065-1281/03/105/02-151 $ 15.00/0

NANC nerves in the respiratory air sac and branchial vasculature of the indian catfish,Heteropneustes fossilis*Giacomo Zaccone1**, Luigi Ainis1, Angela Mauceri1, Patrizia Lo Cascio1, Francesco Lo Giudice2,and Salvatore Fasulo1

1 Department of Animal Biology and Marine Ecology, Faculty of Science, University of Messina, and2 Department of Biomorphology and Technologies, Faculty of Medicine, University of Messina, Italy

Received 29 July 2002 and in revised form 11 December 2002 and 21 January 2003; accepted 21 January 2003

Summary

Gill and air sac of the indian catfish Heteropneustes fossilis harbour a nerve network comprisingan innervated system of neuroepithelial endocrine cells; the latter cells are found especially in thegill. A series of antibodies was used for the immunohistochemical detection of neurotransmittersof the neural non-adrenergic, non-cholinergic (NANC) systems such as the sensory neuropeptides(enkephalins), the inhibitory neuropeptide VIP and neuronal nitric oxide synthase (nNOS)responsible for nitric oxide (NO) production which is an inhibitory NANC neurotransmitter.NADPH-diaphorase (NADPH-d) histochemistry was used as marker of nNOS although it is not aspecific indicator of constitutively-expressed NOS in gill and air sac tissues. A tyrosine hydroxy-lase antibody was used to investigate adrenergic innervation. Nitrergic and VIP-positive sensoryinnervation was found to be shared by gill and air sac. Immunohistochemistry revealed the pres-ence of enkephalins, VIP, NOS and NADPH-d in nerves associated with branchial and air sacvasculature, and in the neuroendocrine cell systems of the gill. Adrenergic nerve fibers werefound in some parts of the air sac vasculature. The origin of the nerve fibers remains uncleardespite previous findings showing the presence of both NADPH-d and nNOS in the sensory sys-tem of the glossopharyngeal and vagus nerves including the branchial structure. Scarce faintlystained nNOS-positive neurons were located in the gill but were never detected in the air sac.These findings lead to the conclusion that a postganglionic innervation of the airways is absent.Mucous goblet cells in the gill were found to express nNOS and those located in the non-respira-tory interlamellar areas of the air sac were densely innervated by nNOS-positive and VIP-positivenerve fibers. Our immunohistochemical studies demonstrate that most arteries of the gill and airsac share a NANC (basically nitrergic) innervation which strongly suggests that they are homol-ogous structures.

Key words: NANC – neurotransmitters – nitric oxide synthase – nerve fibers – arteries – gill – airsac – H. fossilis

acta histochem. 105(2) 151–163 (2003)© Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/actahist

* Part of this study was presented at the meeting of European Comparative Endocrinologists, Bonn (Germany), August 26–30, 2002

** Correspondence to: Prof. Giacomo Zaccone, Department of Animal Biology and Marine Ecology, Section of Cell and Evolutionary Biology,Faculty of Science, University of Messina, Messina, Italy; tel: +39 090 391301; fax: +39 090 393409; e-mail: [email protected]

Introduction

A wide variety of teleost fishes have developed an air-breathing habit; whereas some teleosts use gills andskin, others have developed special accessory respirato-ry organs for aerial respiration. Heteropneustes fossilisis an air-breathing fish that utilizes a dual system for gasexchange, gills in water and the suprabranchial chamber(air sac) as an accessory organ (Munshi and Hughes,1992, 2001).

There is increasing evidence that teleost fishesexpress a wide variety of receptors for vasoactiveagents in their blood vessels, including arteries andveins. The neurotransmitter acetylcholine induces con-striction of isolated blood vessels in the teleostsOncorhynchus mykiss (Farrell and Johansen, 1995) andSalmo salar (Sverdrup et al., 1994). Blood vessels in O. mykiss, S. salar, Gadus morhua, Opsanus beta andSqualus acanthias are dilated in response to natriureticpeptides (Duff and Olson, 1986; Price et al., 1990;Evans, 1991; Evans et al., 1993; Sverdrup and Helle,1994; Takei et al., 1994; Tervonen et al., 1998). Vascu-lar smooth muscle in some species of teleosts andagnathan fishes have been found to express receptorsfor vasoactive substances such as acetylcholine,endothelin, NO, natriuretic peptides and prostanoids(Evans and Harrie, 2001). Vasodilation of swimbladdervessels in the eel (Anguilla anguilla) is induced byvasoactive intestinal polypeptide, NO, adenosine andprotons that may contribute to the effect of vagal activi-ty (Schwerte et al., 1999). A novel vasoactive intestinalpeptide (VIP)-like peptide, the pituitary adenylatecyclase-activating peptide (PACAP) and NO inhibitcontractions in the proximal intestine of G. morhua(Olsson and Holmgren, 2000). Both VIP and PACAPpeptides have been localized by immunohistochemistryin the gill arch of the teleost fish Carassius auratus (DiGirolamo et al., 1997, 1998). NOS, tyrosine hydroxy-lase and neuropeptides were recently localized in theperivascular nerves innervating large veins in G. morhua and O. mykiss (Johnsson et al., 2001).

Ultrastructural and physiological studies revealed acomplex vasculature of gills of teleosts by demonstratinga cholinergic innervation of the sphincter at the base ofthe efferent filament arteries and a functional control ofcholinergic vasoconstriction at this site (Bailly andDunel-Erb, 1986). Adrenergic vasomotor innervation ofbranchial vascular beds has been also reported in teleosts.Adrenergic fibres innervate both afferent and efferentarteries including gill arteries (Morris and Nilsson, 1994).

As far as we know, studies on the innervation of theaccessory air breathing organs of fish have not been per-formed yet. Neuropeptides in vasomotor nerves havebeen detected in the swimbladder of some teleostspecies by NANC innervation as shown by immunohis-

tochemistry (Sundin and Holmgren, 1989). Sparseinnervation by nNOS immunopositive nerves has beendemonstrated in the muscular layer of the swimbladderof C. auratus (Bruning et al., 1996). Preliminary studiesin our laboratory on the air-breathing fish, H. fossilis,showed the presence of a neuroepithelial cell system inthe gill (Zaccone et al., 1997). This system was reportedto be close to pseudobranch or carotid labyrinth in 6teleost species, including catfish (Srivastava et al.,1981). Our study (Mauceri et al., 1999) demonstratedthat the gill arteries were innervated by nitrergic fibers.Nitrergic nerves were also reported to terminate in theproximal portion of efferent filament arteries of the gillof a puffer fish, T. niphobles (Funakoshi et al., 1999).

The aim of the present study was to investigate pep-tidergic and NANC innervation of the air sac and thegill of H. fossilis and to compare air sac innervationwith innervation of the gill. Comparison may confirmdevelopment of the air sac by modifications of the gillin this species (Munshi, 1962).

Material and methods

Maintenance and killing of the fish used in this studyfollowed the guidelines of animal care and experimen-tation in compliance with current National laws andregulations. Adult specimens were anesthesized with0.01% MS-222 and the gill and air sac tissues weretaken and immediately fixed in 4% paraformaldehydein 0.1 M phosphate buffer (PB), pH = 7.4 for 2–3 h,dehydrated and embedded in paraffin. Serial sections, 6 µm thick, were cut in the sagittal and transverseplanes and mounted on glass slides for immunohisto-chemical and routine histological staining.

NADPH-d activity staining

The method to stain NADPH-d activity was modifiedafter Schuppe et al. (2001). Small pieces of tissue werefixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 60 min and incubated with 1 mM β-NADPH (tetrasodium salt; Sigma, St LouisMO, USA), 0.5 mM tetranitroblue tetrazolium (TNBT;Sigma) and 0.3% Triton X-100 in 50 mM Tris-HCl (pH 8.0) for 180 min at 25 °C in darkness. Tissues werethen dehydrated in an alcohol series, cleared in xyleneand embedded in paraffin. After deparaffinization andhydration of the sections, visualization of blue finalreaction product was performed with an Axiophotmicroscope (Zeiss, Oberkochen, Germany).

Immunostaining

For immunohistochemical staining, sections were pro-cessed for the indirect immunoperoxidase method as

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previously reported (Zaccone et al., 1996; Mauceri et al., 1999) using heterologous antisera. Details of theantisera used are presented in Table 1. After inhibitionof endogenous peroxidase by 1% H2O2 in PBS for 30 min, sections were incubated in the presence of pri-mary antisera (see Table 1). Incubation with primaryantisera was carried out overnight at +4 °C in a moistchamber. The antigen-antibody complexes were visual-ized using goat-anti-mouse IgG (1:100; Chemicon,Temecula CA, USA) or a goat-anti-rabbit Ig peroxidaseconjugate (1:100; Sigma). Peroxidase activity wasdemonstrated by incubation for 5–12 min in a solutionof 0.015% 3-3′-diaminobenzidine in 0.01 M Tris buffer,pH 7.6, that contained 0.005% H2O2.

Specificity of peptide immunostaining was verifiedby the following controls: 1) incubation of sections withnormal serum instead of specific antisera; 2) incubationof sections with antiserum preabsorbed with the respec-tive antigens (10–100 µg/ml). The preabsorption proce-dures were carried out overnight at +4 °C. Peptideswere purchased from Sigma. Purified nNOS was a giftfrom B. Mayer (Institute of Pharmacology and Toxicol-ogy, University of Graz, Austria).

Results

Respiratory air sac

The air-breathing organ originates from the supra-branchial chambers extending backwards into the trunkmyotomes adjacent to a muscular sheath derived fromthe cucullaris muscle (Munshi et al., 1986; Munshi andHughes, 1992, 2001). Respiratory epithelium is devel-oped by fusion of gill filaments and lamellae. The gilllamellae that are structurally adapted for gas exchange

cover the mucosal surface of the respiratory air sac. Onthe basis of their vascularization, 2 distinct regions ofthe lamellae can be distinguished: a respiratory distalpart and a non-respiratory proximal (basal) part.Mucous cells and epithelial cells are usually inter-spersed between non-respiratory interlamellar areas(Figs. 1, 2). Vascular areas form respiratory islets andlanes are non-vascular areas. The vasculature consistsof a large extension of the afferent branchial artery witha circulation in parallel with the gill arches. The blood iscollected by efferent islet vessels and efferent lateralislet vessels which open into the main efferent respira-tory sac vessels (Fig. 1). The major vessels possess anadventitia and media with several layers of smoothmuscle cells (Fig. 4a).

The nerve supply of the respiratory air sac is paral-leled with that of the gill since the afferent branchialartery that runs into the main ridge of the respiratory airsac produces a series of lateral blood vessels corre-sponding to the afferent filament vessels of the gills.These vessels supply blood to the lamellae of respirato-ry epithelium, which is collected by the main efferentsac vessels (Fig. 1). A close distribution pattern ofnerves within arterial walls of the afferent-efferent sys-tem, as indicated above, is present.

Enkephalin immunostaining. Antibodies against leu-5-enkephalin labeled single cells (presumably neuroep-ithelial endocrine cells) scattered in the non-vascularareas. Antibodies against met-5-enkephalin show asparse to dense innervation in the walls of lateral efferentislet vessels and efferent islet capillaries and the mainefferent vessels of the respiratory air sac (Figs. 3, 4b).Large numbers of mainly single fibers were stained posi-tively and formed a perivascular plexus in the adventitia(Fig. 4b). Nerve cell bodies were not observed.

Tyrosine hydroxylase. Tyrosine hydroxylase immunos-taining was not detected in the musculature of thecucullaris muscle. In nerves running along the main effer-ent vessels, tyrosine hydroxylase innervation was verysparse; only a few single fibers were found. These fibersformed a perivascular plexus which penetrated into thelayer of the tunica media (Fig. 5).

nNOS immunostaining and NADPH-d activity stain-ing. A dense plexus of nitrergic nerve fibers was locatedin the submucosal layer (Fig. 6). nNOS immunostainingwas detected in nerves running in the walls of afferentand efferent arteries of the respiratory islets and in thecucullaris muscle (Fig. 7). NOS-containing nerve cellbodies in the neural plexus and neuroepithelial endocrinecells were never detected with nNOS antibodies.

Immunostaining of nNOS was present in severalmucous cells located in the non-respiratory interlamel-

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Table 1. List of primary antibodies that have been used in the pre-sent study. All antibodies were raised in rabbit except for the tyro-sine hydroxylase antibody that was raised in mouse

Primary antisera tested Source Working dilution

Anti-neuronal nitric Transduction Labs, 1:500 oxide synthase (nNOS) Lexington KY, USA

Anti-leu-5-enkephalin Sigma, St.Louis MO, USA prediluted

Anti-met-5-enkephalin Peninsula Lab, San 1:500Francisco CA, USA

Anti-vasoactive intestinal Biomeda, MI, Italy predilutedpolypeptide (VIP)

Anti-tyrosine hydroxylase Sigma 1:1000

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Fig. 1. Comparison of vascular pathways in air sac and gill of Heteropneustes fossilis. Adapted with permission from Munshi and Hughes,1992. A. The suprabranchial chamber (SBC) extends backwards into the trunk region in the form of a sac (AS) embedded in trunk myotomes(MYO). G, gill. B. Cross section of AS showing the dorsal ridge containing afferent (AASV) and efferent (EASV) vessels. RE, respiratory epithe-lium. C. Afferent and efferent vascular supply of respiratory lamellae (RL) of a respiratory islet (RI). ARIV, afferent respiratory islet vessel; ERV,efferent respiratory islet vessel. D. Cross section of a series of lamellae (G) of a respiratory islet. PC, pillar cell; MC, mucous cell; E, epithelialcell; RC, red blood cell; WBC, white blood cell; B, basement membrane. E. Arterioarterial (respiratory) and arteriovenous (nutritive andosmoregulatory) pathway of the fish gill filament. GA, gill arch; GR, gill ray; ABA, afferent branchial artery; EBA, efferent branchial artery; AFV,afferent filament vein; EFB, efferent filament vein; GL, gill lamellae; EFA, efferent filament artery; AVA, arterio-venous anastomosis; VS, venoussystem; GF, gill filaments.

Fig. 2. Cross section of 2 lamellae (L) of a respiratory islet. Mucous cells (M) are present in the non-respiratory interlamellar areas. EIV, effer-ent lateral islet vessels; C, cucullaris muscle. H&E staining. Magnification, ×320.

Fig. 3. Met-5-enkephalin-immunopositive submucosal nerve fibers run along the arterial wall of an efferent lateral islet vessels (EIV). Theyform a perivascular plexus. E, epithelium. Magnification, ×470.

Fig. 4. a) Histological sections of a main efferent respiratory air sac vessel (ESV) showing the muscular layer in the media (M) and the adventitia(A). H&E staining. Magnification, ×320. b) Met-5-enkephalin-immunopositive nerve fiber plexus occur throughout the circumference of a largebranch of a main efferent respiratory air sac vessel (ESV). The perivascular plexus is mainly distributed in the adventitia. Magnification, ×320.

Fig. 5. Tyrosine hydroxylase immunopositivity in nerve fiber plexus of an efferent respiratory air sac vessel (ESV). The perivascular plexus(arrowhead) penetrates the layer of the tunica media (M), but appears to be discontinuous. Magnification, ×320.

Fig. 6. nNOS immunopositivity is present in nerve fibers (N) in connective tissue. P, pigment cells; EP, epithelium. Magnification, ×470.

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lar areas. A fine nerve plexus consisting of thin fiberswas located in the proximity of mucous cells. Some ofthe immunostained fibers were in contact with mucouscells at different levels (Figs. 8, 9).

Some NADPH-d-positive nerve fibers were presentin walls of afferent and efferent vessels of the air sacincluding lateral efferent islet vessels. NADPH-d activ-ity was also localized in nerve fibers of the cucullarismuscle.

VIP immunostaining. VIP immunostaining wasobserved in nerve fibers of connective tissue of the airsac (Figs. 10, 11). In the core of this tissue, thick bun-dles were easily distinguished. Nerve fibers were alsoobserved along arterioles or capillaries in the connec-tive tissue core. VIP-positive fibers ran regularly andformed a perivascular plexus in vascular walls of affer-ent, efferent and lateral vessels and in the main efferentvessels of the air sac (Figs. 10, 12). Nerve plexuses withlower density were also found in the cucullaris muscle(Fig. 11). Some nerve fibers of the connective tissuepenetrated the basal lamina of the epithelium and werein contiguity with some mucous cells (Fig. 11).

Single VIP-positive cells in the respiratory part of thelamellae were detected. nNOS-positive nerve fiberswere observed to run along the circular musculature ofthe air sac.

Gill

H. fossilis has 4 pairs of gills which are provided withstiff gill rakers. The gills have a basic organization con-sisting of gill filaments with their surface enlarged bythe development of lamellae. Gill lamellae are made of2 epithelial layers on a supporting basement membraneand a series of pillar cells; some of these are embeddedin the epithelium of gill filaments. The pillar cellsenclose large blood spaces.

When the gills are processed for peptide immunohis-tochemistry, numerous neuroendocrine cells are foundscattered throughout the gill filament and lamellarepithelium (Zaccone et al., 1997). As for other teleosts,2 major vascular pathways have been found in gill fila-ments of the species studied: the arterio-arterial (respi-ratory) and arterio-venous pathways (Munshi andHughes, 1992). The respiratory pathway consists of onafferent filament artery which branches of a series ofafferent lamellar arterioles, which open into the vascu-lar networks of the lamellae. Blood is collected into ashort efferent lamellar arteriole which opens into theefferent filament artery and subsequently into the effer-ent branchial artery.

The arterio-venous pathways originate from the post-lamellar circulation, i.e. the primary efferent filamentarteries. Its function includes both ionic and gaseousexchange in addition to nutrition of filament tissue(Munshi and Hughes, 1992; Fig. 1). The branchialnerves in each gill arch originate from 3 major nervebundles: the gill raker nerve, the anterior gill filamentnerve and the posterior gill filament nerve. Both anteri-or and posterior gill filament nerves run along the effer-ent filament arterial system and originate from themetatrematic and protrematic ramus of the branchialnerve respectively (Nilsson, 1984).

Enkephalin immunostaining. Two regions have beenstudied in longitudinal sections of gill arches, the innerpart containing the bony arch and the outer part consist-ing of the gill filament area.

In the first region, single varicose fibers and largebundles of varicose fibers form a dense network of leu-and met-enkephalin fibers in the anterior and posteriorfilament nerves. Nerve bundles of the filament nerves inthe gill bar were observed running along the branchialarch skeletal cartilage in the connective tissue core (Fig. 13). Positive fibers were present around the effer-

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Fig. 7. nNOS-immunopositive nerve fibers are present around vascular walls of efferent lateral islet vessels (EIV). P, pigment cells; EP, epithe-lium. Magnification, ×470.

Fig. 8. A complex nerve fiber network innervates epithelium of the air sac, especially mucous goblet cells (G). nNOS immunopositivity is pre-sent in nerve fibers around mucous goblet cells. Magnification, ×470.

Fig. 9. nNOS immunostaining is present in small nerve fibers around mucous goblet cells (arrowheads) of air sac epithelium. G, mucous gob-let cells. Magnification, ×470.

Fig. 10. VIP immunopositivity is present in nerve fiber bundles (arrowheads) in the media (M) of a large efferent vessel of the air sac (ESV).Magnification, ×470.

Fig. 11. VIP-immunopositive nerve fibers are present in connective tissue and close to efferent islet capillaries (arrow). A few fine varicosenerve fibers occasionally penetrate the epithelium and are present around mucous goblet cells (G).VIP immunostained nerve plexuses (arrow-heads) are present in lower density in the cucullaris muscle (M). Magnification, ×470.

Fig. 12. VIP immunostaining of nerve fibers is present in connective tissue and around the vascular wall of a large efferent lateral islet ves-sel (EIV). Magnification, ×470.

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ent branchial artery. Some fibers ascended towards thesecond gill region at the level of subepithelial interfila-ment areas in a perpendicular direction. Nerve fibersthat were strongly and selectively stained for leu- andmet-enkephalin were distributed along the wall of effer-ent filament arteries (Fig. 16) and efferent lamellar arte-rioles in regions related to basal and middle portions ofthe filaments. Numerous fine varicose met-enkephalin-positive nerve fibers were found in areas related to thebranchial muscles (constrictor branchiales) that areinvolved in movement of the gill filaments.

Numerous neuroepithelial endocrine cells were pre-sent in the interlamellar and lamellar epithelium (seeZaccone et al., 1997 for reference).

Tyrosine hydroxylase. Tyrosine hydroxylase immunos-taining was not detected at all in the gills.

nNOS immunostaining and NADPH-d activity stain-ing. As has been previously reported for the speciesstudied (Mauceri et al., 1999), an extensive distributionpattern of nNOS-positive nerve fibers was found in the

Fig. 13. Met-5-enkephalin immunostaining is present in nerve bundles of an anterior gill filament nerve in the gill bar. Branchial muscles (M)are also innervated by a network of met-enk-containing fibers. Magnification, ×470.

Fig. 14. nNOS immunopositive interlamellar neuroepithelial endocrine cells in a longitudinal section in the middle of a gill filament. Inter-lamellar neuroendocrine cells are adjacent to lamellar arteries (asterisk). Varicose nerve fibers are present beneath filament epithelium inner-vating neuroendocrine cells (arrows). Magnification, ×320.

Fig. 15. Nitrergic nerve fibers (arrowheads) are present beneath the cell layers of gill lamellae in close association with the pillar cell system(P). Magnification, ×320.

Fig. 16. Positive met-enk-nerve fibers are present along filament vasculature. Magnification, ×470.

Fig. 17. VIP-immunopositive nerve fibers (arrowheads) along filament vasculature and neuroendocrine cells (NE) in filament and gill epitheli-um. Magnification, ×470.

Fig. 18. Nitrergic nerve fibers surround efferent filament arteries (asterisks) in the middle of the filament as detected with nNOS immuno-staining. Magnification, ×470.

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walls of efferent filament arteries and neuroepithelialendocrine cells, some of which appeared in the prox-imity of efferent filament and lamellar arteries (Fig. 18). Most of the endocrine cells were also inner-vated by nitrergic nerves (Fig. 14).

Positive nNOS nerve fibers were found in both anteri-or and posterior gill filament nerves branching alongefferent filament arteries. In addition, fine varicose nervefibers were found to penetrate the pillar cells system ofgill lamellae (Fig. 15). nNOS-positive nerve fibers werealso traced along the wall of efferent and afferentbranchial arteries. Fig. 19 shows perivascular nervesshowing nNOS immunostaining in the vascular wall ofthe efferent filament artery at the top of the filament.

Neurons were located in the parenchyma beneath thegill epithelium and at the level of efferent lamellar arter-ies, as was shown by Bailly et al. (1992) in variousspecies. These neurons were hardly positive for nNOS.

Many mucous cells were found in the epithelial lay-ers of the edge of gill arches facing the buccopharynxand were positive for nNOS, but not for NADPH-d

activity. Intraepithelial nNOS- positive nerve fiberswere never observed.

NADPH-d-positive nerve fibers showed a distribu-tion pattern that was similar to that of nNOS-positivenerve fibers. However, large numbers of neuroen-docrine cells adiacent to efferent lamellar arteries andfilament arteries showed a comparatively strongNADPH-d activity (Fig. 20). Neurons were not stainedfor NADPH-d activity.

VIP immunostaining. The main part of the filamentvasculature showed a dense distribution pattern of VIP-positive nerve fibers (Fig. 17). Positive varicose fiberswere mainly found around vascular walls of efferent fila-ment arteries and efferent lamellar arterioles (Fig. 17). Alarge number of VIP-positive neuroepithelial endocrinecells were found in the interfilament and lamellar epithe-lium (Fig. 21). Most of the labeled cells were isolatedcells and sometimes extended longitudinally above sidesof the efferent filament arteries and the basal parts ofefferent lamellar arteries (Fig. 21).

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Fig. 19. Area of an efferent filament artery (EFA). The nNOS-immunopositive filament nerve fibers (arrowheads) innervate thewall of a large vessel. Neuroendocrine cells (NE) are present in gilllamellae. Magnification, ×470.

Fig. 20. NADPH-d-positive varicose nerve terminals (arrows) arepresent in the vicinity of an efferent filament artery. Neuroepithelialendocrine cells (NE) are also stained. MC, mucous goblet cells. Mag-nification, ×320.

Fig. 21. VIP immunopositivity is present in cytoplasm of neuroen-docrine cells (NE) in filament epithelium. Some cells are in closeproximity of lamellar arterioles (arrows). Magnification, ×470.

Scarce VIP-positive neurons were located in subep-ithelial areas of the base or middle sections of the fila-ment.

Discussion

Previous studies of gill innervation in teleosts were pri-marily focussed on cholinergic and adrenergic nerves(see for reference, Morris and Nilsson, 1994). Teleostvasculature has a double innervation by cranial andspinal autonomic nerves. The cranial autonomic (vagal)nerves consist of vasomotor fibres to the gills innervat-ing the oral and branchial apparatus. Physiological stud-ies have shown a cholinergic vasoconstrictor control ofthe filament sphincters which are a thickened portion ofthe efferent filament artery as was described by Smith(1977) and Dunel and Laurent (1977). A NANC compo-nent of the cholinergic innervation of teleost gill fila-ments has been suggested by Petterson and Nilsson(1979). Adrenergic nerves innervate both afferent andefferent arteries of the gill and the central venous sinus(Nilsson and Holmgren, 1992). Because the air sac isderived from the gill, the same nerves as in the gill wereexpected to be found in the air sac. The present studyshows that all immunostaining patterns as observed inthe air sac are also present in the gill of H. fossilis,except for tyrosine hydroxylase-positive nerves that arepresent only in the air sac.

The gill contains neuroepithelial cells in addition tonerve terminals. Numerous nerve terminals run alongthe efferent filament vasculature and efferent lamellararteriole. The latter is located in a strategic position tocontribute to filament outflow and perfusion of the vas-cular compartment of the filament (Bailly et al., 1989).Few neurons are present in the submucosa, they are lim-ited to the base or middle sections of filaments. The dif-ferent neuromodulators as found in nerve componentswith immunohistochemistry suggest a functional com-plexity of fish gill.

Our study demonstrates for the first time an extensivedistribution pattern of VIP-positive axons in the gillefferent filament arterial system. It is possible that theVIP-positive axons represent part of a vagal postgan-glionic innervation of the gill. Few VIP-positive neu-rons were observed in distinct locations. VIP has beenshown to mediate vasodilation of smooth muscle in pul-monary arteries of mammals (Barnes et al., 1986) and aNANC neural vasodilation has been demonstrated aswell (Hamaski et al., 1983).

VIP positivity is widely by spread in fish (Lundinand Holmgren, 1989; Schwerte et al., 1999; Johnsson et al., 2001). The presence of VIP-positive andenkephalin-positive fibers close to vessels of gill fila-ments may be compatible with both excitatory and

inhibitory innervation of the gill vasculature. Largenumbers of neuroendocrine cells that were positive forVIP, nNOS and NADPH-d activity were demonstratedin the proximity of efferent filament and lamellar arter-ies. This area is also innervated by nNOS-positive fiberswhich have been found in vascular nerves in most majorvessels of the gill. The adrenergic component of the gillinnervation is not present, as indicated by the absence oftyrosine hydroxylase-positivity. A functional role maybe assigned to both the excitatory and inhibitory neuralNANC system of the gill in relation to the autonomicregulation of regional blood flow in the gill. Variousreceptors in the gill are sensitive to blood pressure,mechanical displacement and oxygen and carbon diox-ide tension (Burleson et al., 1992; Burleson and Sma-tresk, 2000). Sensory information from these receptorsis transmitted via the glossopharyngeal (IX) andbranchial branches of the vagus (X) nerves to cardio-vascular and ventilatory centres in the brain system. Invivo physiological measurements of blood flow duringventilation are needed to study vasodilator and vasocon-strictor mechanisms.

The origin of neuropeptide-containing nerves includ-ing the nNOS/NO-producing fibers in the gill remainsto be clarified by further investigations. As has beenreported in other fish species (Funakoshi et al., 1999),nitrergic and NADPH-d-positive nerve terminals in theefferent filament artery of Takifugu niphlobes areperipheral processes of sensory neurons of the IX and Xnerves, which pass through the branchial rami and playa role in mechanoreception. Gill mechanoreceptors aresensitive to position and movement of the arches, fila-ment and rakers (Burleson and Smith, 2001). Sensoryinformation of these receptors is of fundamental impor-tance in the control and coordination of gas exchange(de Graaf et al., 1987). Previous studies (Burleson andSmith, 2001) showed that both the IX and X nerves areactive during ventilation in Ictalurus punctatus. Thevascular resistance playing a role in the control of bloodpressure and blood distribution is an integrated responseto nervous, humoral and endothelial substances (seeBurnstock, 1993). Recent investigations have demon-strated distribution patterns of neuropeptide-containingnerves in heart and vasculature of teleosts which mayact on smooth muscle cells of vessel walls and affect thevenous tone (Johnsson et al., 2001). The neurochemicaland physiological investigations mentioned above onvasculature and mechanoreception show a direct partic-ipation of sensory neuropeptides and NO in the process-ing of mechanosensory information in gills of fishes.

We have demostrated expression of nNOS in mucose-cretory cells in epithelial layers of the gill arches facingthe buccopharynx, but nNOS-positive intraepithelialnerve fibers were not found. In the gastrointestinal tract,NO may affect muscle tone as well as endocrine and

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exocrine secretion (Whittle, 1994). The role of NO pro-duced by mucosecretory cells is not known yet. Studiesin mammals have indicated a possible role for NO in thesecretion of mucus or bicarbonate, which are bothimportant mechanisms in mucosal defense againstmicroorganisms in the stomach (Brown et al., 1992).

The air sac is structurally derived from the gill. Thegill lamellae show a structural adaptation for aerial gasexchange. Moreover, the main ridge of the respiratorysac containing the fourth afferent arch and efferent ves-sels (Fig. 1) represents the gill arch region of thebranchial arch (Munshi et al., 1986; Munshi and Hugh-es, 2001). Unfortunately, functional studies have notbeen performed of the innervation of vascular musclesand lamellar structures of the respiratory islets of the airsac in the species studied in comparison with that inteleost gill vasculature.

The innervation of the air sac is unlike that of theswimbladder and lungs in fish. In the swimbladder,parasympathetic cholinergic fibres are responsible forstimulation of lactic acid production by the gas gland.Sympathetic adrenergic fibres produce a complex ofresponses in the muscularis mucosae and vasculaturepromoting gas resorption (Campbell and McLean,1994). The lungs in lungfish are innervated by vagusnerves that provide a cholinergic constrictor innervationto the vasculature (Campbell and McLean, 1994). Thepattern of innervation of the vasculature in the air sac issimple: all innervation that has been identified byimmunohistochemistry is both excitatory and inhibito-ry, and is represented by a NANC system of fibers.Mucous cells are also innervated by nNOS and VIPnerve fibers. NANC parasympathetic neurons may notbe involved. The immunohistochemical identificationof i-NANC-positive parasympathetic neurotransmitters(VIP and NO/nNOS) in the neural supply of mucousgoblet cells has been shown for the first time in airwaysof lower vertebrates. Mucus-secreting elements in air-ways have a nervous control (Rogers, 1997). The effectof NO on the production of mucus in mammalian air-ways is unkown. Functional studies have not beenundertaken to determine directly the involvement ofinhibitory nerves in the control of mucus production.However, a number of peptides and receptor types withinhibitory effects on secretion have been identified(Rogers, 1997). The inhibitory effect of NO has beenfound only in vitro (Belvisi et al., 1992). The contribu-tion of both e-NANC and i-NANC pathways (mediatedby enkephalin-, VIP- and nNOS-immunopositivenerves) to regulate neurogenic secretion of mucous cellsin air sac awaits further investigations.

Immunohistochemistry revealed only a sparse distri-bution pattern of adrenergic fibres in vascular muscles.Furthermore, the simple innervation of air sac apparent-ly comprises solely a NANC-positive neural system

similar to that of the gill, which suggests that theseorgans have similar physiological roles. InhibitoryNANC innervation is also not a feature of the swim-bladder function which is under vagal tonic control(Schwerte et al., 1999).

NOS is present in the visceral sensory system of thevagus nerve in the pufferfish (Funakoshi et al., 1999).The nitrergic innervation of gill and air sac is not medi-ated by parasympathetic postganglionic fibres (see Zac-cone et al., 2003 for review). The blood flow in theseorgans may be under the control of the vagus nerve.However, the relationship between neuroepithelialendocrine cells and filament neurons has to be takeninto account in future studies.

In conclusion, vagal NANC (perhaps basically nitr-ergic) and vagal excitatory autonomic elements seem tobe present in the gill and air sac of H. fossilis. The pre-sent study serves as a basis for further physiologicalstudies on NANC innervation of teleost gill and acces-sory respiratory organs.

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