Neurotransmitter localization in the neuroepithelial cells and unipolar neurons of the respiratory tract in the bichir, Polypterus bichir bichir G. ST-HIL

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Acta histochemica 110 (2008) 143—150

Neurotransmitter localization in theneuroepithelial cells and unipolar neuronsof the respiratory tract in the bichir,Polypterus bichir bichir G. ST-HIL

Giacomo Zaccone�, 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 23 April 2007; received in revised form 6 September 2007; accepted 27 September 2007

KEYWORDSnNos;Transmitters;Bichir;Unipolar neurons;Neuroepithelialcells;Evolution

AbstractImmunohistochemical localisation of neurotransmitters was used to determine thedistribution of unipolar neurons and neuroepithelial cells (NECs) in the respiratorytract of the bichir, Polypterus bichir bichir. NECs were commonly encountered inthe mucociliated epithelium of the lung. Unipolar neurons were located in thesubmucosal and muscle layers of the glottis. The results suggest the presenceof tyrosine hydroxylase (TH) and nNOS immunoreactivities in NECs. In addition,ACh-E/nNOS and TH/nNOS nerve fibers were also found associated with these cells.Unipolar neuronal cells showed a chemical code including the presence of 5-HT,ACh-E, peptides and P2� 2 receptors. The present findings indicate that nitric oxide(NO) is a primitive transmitter of neuroepithelial oxygen-sensitive chemoreceptorcells together with acetylcholine. The coexistence of ACh-E with other substances inthe unipolar neurons, but not with NO, may be a property of vagal postganglionicneurons since the emergence of the cranial autonomic pathways in the earliestvertebrates. It would be interesting to know about the provenance of the nerves incontact with NECs, which appear to have a complex innervation pattern.& 2007 Elsevier GmbH. All rights reserved.

Introduction

The family Polypteridae comprises two genera:Polypterus (bichirs) and the ropefish (Erpetoichthys),which are considered to be primitive with regard to

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teleost fishes (Poll and Gosse, 1995). Polypteridsretain several primitive characteristics, which areunknown in other recent actinopterygians, butoccur among fossil ray-finned fishes. During thelate Devonian, the group of palaeoniscoids posses-sing both lungs and gills gave rise to the modernforms of Polypterus, and the Holostei representedby the bowfin (Amia) and the gar (Lepidosteus)(Datta Munshi, 1999). All these fishes have lungs,which develop as pouches of the pharynx. In thebichirs, respiratory gas exchange is bimodal andoccurs chiefly across the gills and lungs. The liningsof the lung are mucoid in nature and have asurfactant produced by pneumocyte II types ofcells (Zaccone et al., 1989) found in the furrowedepithelium of the internal surface. This epitheliumalso contains a rich concentration of ciliated cells,mucous cells and neuroepithelial cells (Zacconeet al., 2007), the latter being considered asperipheral chemoreceptors involved in oxygen-sensing mechanisms in fish gills (Jonz and Nurse,2006).

In vertebrates, cardioventilatory events asso-ciated with various physiological situations havebeen poorly described in species living in aquaticenvironments. Environmental hypoxia plays a keyrole in driving compensatory responses, which aredriven by input from peripheral oxygen chemor-eceptors. Thus, these receptors are involved in theregulation of oxygen uptake during hypoxia (Jonzand Nurse, 2006). The gills are the main site ofoxygen chemoreception in developing aquaticvertebrates, and oxygen-sensitive neuroepithelialcells (NECs) are present in the gills in larval stagesof zebrafish and Xenopus laevis (Jonz and Nurse,2006; Saltys et al., 2006).

There is increasing evidence suggesting that themechanisms by which cells adapt to hypoxia involvemodifications in nitric oxide (NO) signalling. NO,besides having roles in regulating multiple biologi-cal functions (neurotransmitter function, media-tion of cell growth, apoptosis, platelet adhesionand vasodilation), is regarded as a regulator ofoxygen-sensitive phenotypes and a player in themaintenance of oxygen homeostasis, since lowoxygen levels characterise the normal microenvir-onment of cells in many tissues (Postovit et al.,2005; Campanucci and Nurse, 2007).

In a companion paper (Zaccone et al., 2007),immunohistochemical evidence has demonstratedthe involvement of several neurotransmitters in theinnervation of the lung in the bichir, Polypterusbichir bichir. The arrangement of the cranialautonomic pathways in this species is not known.In teleost fishes, in general, the preganglionic fibersrun to ganglia in or near the target organs,

synapsing with postganglionic neurons. In fish, thepostganglionic neurons in the branchial rami of theglossopharyngeal and vagus nerves innervate arange of targets in gills (Nilsson, 1983; Gibbins,1990, Gibbins et al., 1995). In the present study, wewere interested in determining whether the mono-polar neurons lying in the nerve plexuses andsubmucosal layers of the glottis of the speciesstudied are homologous with the ganglion cells inthe branchial and pharyngeal rami of the vagus infishes. In particular, the aim of the present studywas also to investigate neurotransmitter co-locali-sation in the NECs and their associated innervationin the respiratory tract of the bichir, Polypterusbichir bichir. This study was also to compare, interms of some transmitter substances, types ofnerves providing innervation to the lung, with thosefound in chemosensory neuroepithelial cells ofhigher vertebrates.

Materials and methods

Animals and tissue preparation

Five specimens of bichir, Polypterus bichir bichir(35–40 cm in length), were obtained from localsuppliers and were anaesthesised with 0.01%Tricaine (ethyl 3-aminobenzoate methanesulpho-nate salt, Sigma-Aldrich, St. Louis, MO, USA). Theventral glottis with right and left lung wereperfused with 4% paraformadehyde in 0.1M phos-phate buffer (pH 7.4), then fixed in the samefixative for 2–4 h at 4 1C. The specimens were thendehydrated and routinely processed for embeddingin paraffin wax (Paraplast, Histo Line Laboratories,Milano, Italy). The structure of the glottis and thelungs was investigated using the standard haema-toxylin–eosin (H–E) staining method.

Immunofluorescence labelling

Sections cut from tissue blocks were dewaxed inxylene and rehydrated through a graded ethanolseries. Following several rinses in 0.1M phosphate-buffered saline (PBS), pH 7.4, they were incubatedwith primary antibodies detailed in Table 1, over-night at 4 1C in a humid chamber. Binding sites ofprimary antibodies were visualized by incubationwith corresponding fluorescein isothiocyanate(FITC)-conjugated goat anti-mouse IgG (Sigma) andtetramethylrhodamine isothiocyanate (TRITC)-goatanti-rabbit (Sigma), both diluted 1:100, at roomtemperature.

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Fluorescence microscopy

A Zeiss Axio Imager Z1 microscope integratedwith Axio Vision 4.5 and an AxioCam digital camera(Zeiss, Jena, Germany) was used for image acquisi-tion. Sections were imaged using the appropriatefilter settings for the excitation of FITC(480–525 nm) and TRITC (515–590 nm).

Controls

Negative controls for immunohistochemical la-belling were performed by substitution of non-immune sera for the primary antisera. Specificity ofsome peptide labelling was verified by incubatingsections with antiserum pre-absorbed with10–100 mg/ml respective antigen, overnight, at

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Table 1. Details of primary antibodies used

Antigen Animal source Distributor Dilution

Tyrosine hydroxylase (TH) Mouse Sigma, St. Louis, USA 1:100Neuronal nitric oxide synthase(nNOS)

Rabbit Biomol, Milan, Italy 1:100

Acetylcholinesterase (AchE) Mouse Sigma, St.Louis, USA 1:50P2� 2 receptor Rabbit Alomone Labs, Jerusalem, Israel 1:1005-Hydroxytryptamine (5-HT) Mouse DakoCytomation, Milan, Italy 1:505-HT3 receptor Rabbit Sigma, St. Louis, USA 1:100Pituitary adenylate cyclaseactivating polypeptide 38 (PACAP)

Rabbit Peninsula Labs., San Carlos, CA,USA

1:300

Substance P (SP) Rabbit Sigma, St. Louis, USA 1:50

Figure 1. Haematoxylin–eosin (H–E) staining. (A) Transverse section of the glottis (G) opening into the right lung (RL).(B) A view of the lung wall showing the respiratory epithelium (RE), the furrowed epithelium (FE) and the striatedmuscle (M). (C) A view of the lung epithelium showing the respiratory epithelium (RE) consisting of a layer of flat cellsand the ciliated furrowed epithelium (FE) containing mucous goblet cells (GC) and ciliated cells (arrow). Scale bar,A–C ¼ 20 mm.

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4 1C. PACAP was provided by Bachem (Bubendorf,Switzerland), nNOS enzyme, SP and 5-HT werepurchased from Biomol (Milan, Italy) and Sigma,respectively.

Statistical analysis

The immunopositive cells were analyzed usingAxio Vision Release 4.5 software package. Thissoftware identifies positive cells and the data were

statistically processed by Anova system (GraphPadSoftware Inc., San Diego, CA, USA).

Results

The lungs of Polypterus bichir bichir arise from aventral glottis and pass dorso-posteriorly aroundthe alimentary canal into the coelom. The glottisopens into the right lung (Figure 1A). The inner

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Figure 2. (A–D). Neuroepithelial cells (NECs) and associated innervation in the bichir lung. (A) A tyrosine hydroxylase(TH)-immunoreactive NEC (green) in the mucociliated epithelium of the lung, with a slender apical process (arrow)facing the airway lumen. (B) The same NEC as in A showing nNOS immunoreactivity. (C) nNOS-immunopositive nervefibers (arrowheads) running towards the apical end of a NEC. (D) Overlay of TH immunoreactivity and nNOSimmunoreactivity showing nerve varicosities (arrowhead) towards the apical pole of a NEC. AL, airway lumen; MC,mucous goblet cell, NEC, neuroepithelial cell. (E—F). Cell bodies of glottic unipolar neurons in submucous localisations.(E) Neurons showing 5-HT immunoreactivity. The single axon is marked by an arrowhead. (F) The same neurons labelledto show P2� 2 receptor immunoreactivity. The single axon is marked by an arrowhead. (E) epithelium lining the glottis.Scale bars ¼ 20 mm.

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surface of the lungs is smooth except for a patternof longitudinal striations termed furrows. Therespiratory epithelium is a layer of flat cells liningthe luminal surfaces between furrows (Figures 1B,C). The furrowed epithelium contains rich concen-trations of neuroepithelial cells (NECs), (Zacconeet al., 2007) and mucous goblet cells and ciliatedcells (Figure 1C) (Graham, 1997). NECs are alsorecognizable in the epithelium lining the glottis andfrom a thin apical process that extends to theairway lumen as shown by immunoreactivitiesof TH and nNOS (Figures 2A, B). Double immuno-labelling for nNOS-Ach-E and nNOS-TH revealsa close association of varicose nerve fiberswith NECs. The overlap signal (yellow orange)reflects co-localization of nNOS and TH(Figure 2D) in the nerves. Figure 2C shows nNOS-immunopositive nerve endings running toward theapical pole of a NEC.

An extensive innervation of the submucosa ispresent in the lung, and several nerves containing arich variety of neurotransmitters project to theepithelium (Zaccone et al., 2007). A collection ofunipolar neurons was noticed in the submucosal andthe muscular layers of the glottis. These neuronscontain P2� 2 and 5HT (Figures 2E, F). Table 2summarises the distribution of the observed im-munoreactivities for neurotransmitters in the NECs,NEC-associated nerve ending and unipolar neurons.

The number of positive cells showing the colo-calization of neurotransmitters was tabulated(Figures 3A–E). The number of positive cells issignificantly different in A, B, C (po0.01), com-pared with that in D and E (p40.05). The meannumber of nNOS immunopositive cells for sampledarea is lower for nNOS (po0.001; n ¼ 7) comparedwith that for AchE (po0.001; n ¼ 35).

The nNOS-TH labelling shows that the meannumber of nNOS immunopositive cells is alsomuch lower (po0.001; n ¼ 1) compared with thatfor TH (po0.001; n ¼ 42). The mean number ofAchE positive cells is lower (po0.001; n ¼ 11) in

comparison with that for SP (po0.001; n ¼ 35). Nosignificant differences are noted for the P2� 2

receptor positive cells and 5-HT-immunopositivecells, both occurring in small numbers. The meannumber of 5HT3- and 5HT-immunopositive cellsshows that there is no significant difference intheir density.

Discussion

We cannot rule out the possibility that theTH-nNOS immunopositive axons showing directcontacts with NECs represent terminals of eithervagal or spinal components of the selectiveinnervation of these cells. The association ofnNOS-positive nerve endings with NECs may justifytheir interpretation as sensory nerve endings,which were observed in the apical cell pole ofmammalian NEB cells (Van Lommel and Lauweryns,1993). Further studies are required to establishthe physiological functions of these innervationpatterns, although the neurochemical characteri-zation of nerves has been established. Sympatheticpostganglionic adrenergic neurons are thought tocontain NOS and have specific targets in teleosts(Funakoshi and Nakano, 2007). It is possible thatNECs are the targets of sympathetic adrenergicganglion cells. NECs contain AchE, TH, SP, nNOSand the P2� 2 receptor. The nerve endings alsocontain the same transmitters except for P2� 2.It is remarkable that both NECs and nerve terminalscontain identical substances. The wide expressionpatterns of nNOS in the NECs and afferent path-ways may be correlated with its modulatoryinfluence on the regulation of presynaptic trans-mitter release. Some of these substances havebeen found in the carotid body (CB) and inpulmonary neuroepithelial bodies (NEBs). NEBcorpuscular cells were found to display P2� 2 andP2� 3 purinergic receptor subunits (Fu et al.,

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Table 2. Immunoreactivities for neurotransmitters in the glottis and the lung of the bichir

Neurotransmitter Glotticneurons

NEC NEC associatednerve endings

Tyrosine hydroxylase (TH) – + +Neuronal-nitric oxide synthase (nNOS) – + +Acetylcholinesterase (AchE) + + +P2� 2 Receptor (P2� 2) + – –

5-HT3 Receptor (5-HT3) + – –

5-Hydroxytryptamine (5-HT) + + –

Pituitary adenylate cyclase activating polypeptide (PACAP) + + –

Substance P (SP) – + –

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Figure 3. (A–E) Statistical analysis of the immunopositive cells showing the colocalization of the neurotransmitters in the glottis and the lung of Polypterus bichir bichir.(A) nNOS-AchE. A lower number (po0.001; n ¼ 7) of nNOS positive cells is noted in comparison with that for TH (po0.001; n ¼ 35). (B) SP- AchE. A lower number(po0.001; n ¼ 11) of AchE positive cells is found in comparison with the number of SP-immunopositive cells (po0.001; n ¼ 35). (C) nNOS-TH. Significant differences arefound between the number of nNOS positive cells (po0.001; n ¼ 1) and the number of TH positive cells (po0.001; n ¼ 42) per sampling area. (D) P2� 2-5-HT.No significant differences are found between the number of P2� 2 receptor positive cells (p40.05; n ¼ 3.5) and the number of 5-HT positive cells (p40.005; n ¼ 3.8).(E) 5HT3–5HT. No significant differences are found between the number of 5-HT3 receptor positive cells (p40.005; n ¼ 9.6) and the number of 5-HT positive cells(p40.05; n ¼ 9.8).

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2004). Both TH and SP imunoreactivities have beenlocated in the afferent neurons of CBs (Ichikawaet al., 1993; Finley et al., 1992). Acetylcholine issynthesized in the glomus cells of CBs (review: VanLommel, 2007). Regarding the neurotransmittersand receptors, there are a number of similaritiesamong NECs, NEBs and CBs (review: Van Lommel,2007). Ach-E, TH and SP, while present in the CBsand absent in NEBs, are reported for the first timein the NECs of the lung of our bichir species.Serotonin, NO and VIP are candidate neurotrans-mitters in fish gills (Saltys et al., 2006; Zacconeet al., 2003). It is not known if different speciesstore different transmitter substances. These sub-stances should be regarded as neurotransmittersconveying stimuli from NEB cells or NECs to theassociated nerves and vice versa, however, aresponse of the putative transmitter in pharmaco-logical tests is not available. Neurons and nervefibers are found in submucous localization in theglottic region and form ganglionic plexuses. Theseneurons are likely to be homologues of the neuronsof the branchial rami of the teleost vagus (Gibbinset al., 1995). Lungs develop as a ventral evagina-tion of the foregut at the levels of the branchialarch region of the pharynx. A characteristic featureis also the expression acetylcholine in the glotticneurons. It is a property of cranial parasympatheticneurons from early stages in the evolution of thevertebrates (Gibbins et al., 1995).

Colocalization of AchE-PACAP, P2� 2-5-HT and5-HT–5-HT3 within the same neuron indicates apotential modulation of a target response, whetherit be predominantly excitatory or inhibitory. Manyof the transmitters found in the glottic neuronshave been reported in autonomic neurons ofdifferent vertebrate groups. Acetylcholine hasbeen considered the classical transmitter of mostpostganglionic neurons. The presence of purinergicand 5-HT3 receptors besides 5-HT is reported forthe first time in autonomic neurons of fish airways.The present results do not show the presence ofnNOS in glottic neurons. It is suggested that purinescould participate in inhibitory responses, perhapsby enhancing other inhibitory neurotransmitters(Pelleg and Burnstock, 1990), such as PACAP.

The nervous regulation of the function of theglottis still needs to be elucidated. This preliminarystudy shows the mucosal neurons do not contactglottic NECs. Added to the different neurotrans-mitter spectra of NEC-associated nerve endingsand glottic neurons, this indeed seems to arguefor a vagal, rather than intrinsic, nerve supply ofglottic NECs.

A challenge is to study the evolutionary history ofthe glottic neurons, which should be considered to

be homologous with the vagal postganglionicneurons innervating the airways and at least theupper part of the oesophagus in mammals. Thisshould help differentiate them from the neurons ofthe enteric nervous system.

In conclusion, we propose that the presence ofthe various transmitters located in the glotticneurons, NECs and associated innervation in thespecies studied may be a common feature, owing totheir appearance in homologous structures found inthe autonomic nervous system early in vertebrateevolution. An understanding of the evolutionaryhistory of the sympathetic and cranial parasympa-thetic nervous system in non-teleost fishes awaitsinvestigation (Funakoshi and Nakano, 2007). Thesestudies may also help to characterize neural typesinnervating the chemosensory neuroepithelial cellsfor comparison with those found in the mammalianNEBs consisting of vagal and spinal afferentcomponents (Brouns, et al. 2007; Van Lommel,2007).

Acknowledgements

The assistance of Professor Alfons Van Lommel inreading the manuscript is gratefully appreciated.

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