Innervation and neurotransmitter localization in the lung of the Nile bichir Polypterus bichir bichir.
ABSTRACT Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P(2)X(2), and 5-hydroxytryptamine (5-HT)-immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5-HT and P(2)X(2) receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase-immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods.
- SourceAvailable from: Chandra LalClinica chimica acta; international journal of clinical chemistry 12/2010; 411(23-24):2112-3. · 2.54 Impact Factor
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ABSTRACT: How important are sexual hormones beyond their function in reproductive biology has yet to be understood. In this study, we analyzed the effects of sex steroids on the biology of the embryonic amphibian epidermis, which represents an easily amenable model of non-reproductive mucociliary epithelia (MCE). MCE are integrated systems formed by multiciliated (MC), mucus-secreting (MS) and mitochondrion-rich (MR) cell populations that are shaped by their microenvironment. Therefore, MCE could be considered as ecosystems at the cellular scale, found in a wide array of contexts from mussel gills to mammalian oviduct. We showed that the natural estrogen (estradiol, E2) and androgen (testosterone, T) as well as the synthetic estrogen (ethinyl-estradiol, EE2), all induced a significant enhancement of MC cell numbers. The effect of E2, T and EE2 extended to the MS and MR cell populations, to varying degrees. They also modified the expression profile of RNA MCE markers, and induced a range of "non-typical" cellular phenotypes, with mixed identities and aberrant morphologies, as revealed by imaging analysis through biomarker confocal detection and scanning electron microscopy. Finally, these hormones also affected tadpole pigmentation, revealing an effect on the entire cellular ecosystem of the Xenopus embryonic skin. This study reveals the impact in vivo, at the molecular, cellular, tissue and organism levels, of sex steroids on non-reproductive mucociliary epithelium biogenesis, and validates the use of Xenopus as a relevant model system in this field.Frontiers in Zoology 02/2014; 11(1):9. · 3.87 Impact Factor
Innervation and Neurotransmitter
Localization in the Lung of the Nile
bichir Polypterus bichir bichir
GIACOMO ZACCONE,* ANGELA MAUCERI, MARIA MAISANO,
ALESSIA GIANNETTO, VINCENZO PARRINO, AND SALVATORE FASULO
University of Messina, Department of Animal Biology and Marine Ecology, Faculty of
Science, Section of Comparative Neurobiology and Biomonitoring, Messina, Italy
Anatomical and functional studies of the autonomic innervation in
the lung of dipnoan fishes and the bichirs are lacking. The present immu-
nohistochemical studies demonstrated the presence of nerve fibers in the
muscle layers of the lung of the bichir, Polypterus bichir bichir, and iden-
tified the immunoreactive elements of this innervation. Tyrosine hydroxy-
lase, acetylcholinesterase, and peptide immunoreactivity was detected in
the intramural nerve fibers. Extensive innervation was present in the
submucosa where adenylatecyclase/activating polypeptide 38, substance
P, P2X2, and 5-hydroxytryptamine (5-HT)–immunoreactive nerve fibers
mainly supplied blood vessels. A collection of monopolar neurons located
in the submucosal and the muscular layers of the glottis expressed a vari-
ety of various transmitters. These neurons may be homologous to gan-
glion cells in the branchial and pharyngeal rami of the vagus in fishes.
Nerves containing 5-HT and P2X2receptor immunoreactivity projected to
the lung epithelium. Associated with neuroepithelial cells in mucociliated
epithelium, were neuronal nitric oxide synthase–immunopositive axons.
The physiological function of this innervation is not known. The present
study shows that the pattern of autonomic innervation of the bichir lung
may by similar in its elements to that in tetrapods.
? 2007 Wiley-Liss, Inc.
Anat Rec, 290:1166–
Key words: autonomic nerves; NECs; nerve cell bodies; lung;
muscle; epithelium; bichir
The bichirs (Polypterus spp) belong to the family Poly-
pteridae and are placed on an early evolutionary branch
within the Actinopterygians. Although they have been
assigned to higher level groups of bony fishes, the bichirs
are among one of the primitive forms of the jawed verte-
brates of Gnathostomes (Noack et al., 1996). The bichirs
possess richly vascularized air sacs and develop as
pouches of the pharynx and are more primitive than the
lungs of dipnoan fishes. They are dual breathers and
exchange O2and CO2in both aerial and aquatic environ-
ments. During oxygen lack, the gills are not sufficient for
gas exchange. Therefore, the bichirs must inhale air
through air-sacs in addition to branchial respiration (Gra-
ham, 1997). The dependence of teleost fishes on alterna-
tive air-breathing structures is strongly correlated with
their adaptative radiation and with the evolutionary ca-
nalization of gas bladder structure (Graham, 1997). New
structures derived from branchial chambers, which are
found in more advanced teleost fishes, differ anatomically
and embryologically from the archetypical polypterid
lungs. Studies of air-breathing fishes have contributed to
our comprehension of the many integrated changes
involved in water/air transitions. However, compared with
the lungs of dipnoan fishes (Holmgren et al., 1994), which
do not receive any innervation from spinal autonomic
nerves (Axelsson et al., 1989), the bichirs have developed
*Correspondence to: Giacomo Zaccone, University of Messina,
Department of Animal Biology and Marine Ecology, Faculty of
Science, Section of Comparative Neurobiology and Biomonitor-
ing, Via Salita Sperone 31, I-98166 Messina, Italy. Fax:
90-393409. E-mail: email@example.com
Received 20 February 2007; Accepted 1 June 2007
Published online in Wiley InterScience (www.interscience.wiley.
? 2007 WILEY-LISS, INC.
THE ANATOMICAL RECORD 290:1166–1177 (2007)
a more complex autonomic pathway due to the presence
of an adrenergic component of intramural innervation
(Zaccone et al., 2006c). Occurrence of the ganglion cells is
another aspect of the autonomic nervous system of the
bichir (Zaccone et al., 2006a) that is similar to neurons of
the ganglionic plexuses present in the mammalian respi-
ratory tract. Autonomic nerves control vascular resistance
by the release of neuroactive substances such as cathecol-
amines and neuropeptides, which act on the smooth mus-
cle of blood vessels and play a key role in modulating
Gills, accessory respiratory organs, and lungs are
organized in series with the systemic vasculature and,
therefore, the entire cardiac output must pass through
the respiratory circulation (Olson, 1998). The innerva-
tion of the airways in mammals resembles that of the
upper gastrointestinal tract in that the vagus nerve is
the principal source of the parasympathetic input to the
lung and the sympathetic nerves arise from the sympa-
thetic trunk. Accumulated morphological, physiological,
and pharmacological data indicate that a parasympa-
thetic cholinergic innervation results in the contraction
of airway smooth muscle and a sympathetic adrenergic
output to muscle is sparse or absent (Black, 1997).
There is growing evidence that nitric oxide (NO) func-
tions as a primary nonadrenergic noncholinergic (NANC)
neurotransmitter in the relaxation of lung muscle and in-
hibitory responses are also mediated by vasoactive intes-
tinal polypeptide (VIP). The lung visceral muscle of Pro-
topterus is contracted by acetylcholine (Ach) and vagal
nerve stimulation (Abrahamsson et al., 1979), but there
is no evidence of NANC transmission, which is a com-
mon feature of lung visceral muscle in Tetrapods (Camp-
bell and McLean, 1994). In the respiratory tract of uro-
deles, NO is a bioactive substance involved in inhibitory
neurotransmission in the pulmonary nervous system,
where it may be colocalized with VIP (Adriaensen et al.,
Histochemical studies have localized NO to a discrete
subset of extra- and intramural neurons in primarily
parasympathetic as well as enteric ganglia in mamma-
lian vertebrates. Parasympathetic ganglia are found
along intrapulmonary airways. Neurons of these ganglia
contain Ach, as well as various peptides, including VIP,
galanin, and substance P (Undem and Myers, 1997). In
the respiratory tract of several mammalian species, ni-
tric oxide synthase (NOS) has been demonstrated in a
subpopulation of ganglion cells located in the posterior
wall of the trachea and the extrapulmonary bronchi
(Diaz de Rada et al., 1993). Also in these regions various
neuropeptides associated with sensory nerves including
substance P have been observed in nerve terminals of
autonomic ganglia (Kummer et al., 1992).
By using double-label immunofluorescence methods,
we have investigated the occurrence of peptide and non-
nerves, intrinsic neurons, and ganglionic structures
including the presence of neuroepithelial cells, in the
lung of the Nile bichir Polypterus bichir bichir.
with the autonomic
MATERIALS 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 in aquaria at 278C,
and fed amphibian larvae and goldfish. Maintenance
and killing of the fish used in this study followed the
guidelines of animal care and experimentation of Mes-
sina University. Adult specimens were anesthesized with
0.01% ethyl 3-aminobenzoate methanesulfonate (MS
222, Sigma). The ventral glottis with right and left lung
were perfused with 4% paraformaldehyde in 0.1 phos-
phate buffer (pH 7.4). Tissue was dissected, degassed
and immediately immersed in the same fixative for 2–4
hr. Thereafter, all specimens were dehydrated in an
ascending series of ethanol and routinely embedded in
Techniques for immunolabeling and immunoperoxi-
dase were previously reported for catfish gill and am-
phibian epidermal tissues (Zaccone et al., 2003, 2006b).
Deparaffinized and rehydrated sections were treated
with 0.1 M phosphate-buffered saline (PBS, pH 7.4), con-
taining 1% bovine serum albumin at room temperature
(RT) for 30 min to block endogenous peroxidase and con-
secutively incubated with the primary antisera (Table 1)
overnight at 48C in a moist chamber. The antigen-anti-
body complexes were visualized using goat anti-mouse
IgG (1:100; Chemicon, Temecula, CA) or goat anti-rabbit
Ig peroxidase conjugate (1:100; Sigma). Peroxidase activ-
ity was demonstrated by incubation for 50–120in a solu-
tion of 0.015% 3-30-diaminobenzidine in 0.01 M Tris
buffer (pH 7.6), that contained 0.005% H2O2.
After several rinses in PBS, sections were incubated
for double immunofluorescence labeling with antisera
TABLE 1. Primary antibodies used
AntigenAnimal source DistributorDilution
Tyrosine hydroxylase (TH)
neuronal nitric oxide synthase (nNOS)
Substance P (SP)
Vasoactive intestinal polypeptide (VIP)
Polypeptide 38 (PACAP)
Sigma, St. Louis (U.S.A.)
Biomol, Milan (Italy)
Sigma, St. Louis (USA)
Alomone Labs, Jerusalem, Israel
Sigma, St. Louis (USA)
Biomeda, Milan (Italy)
Peninsula Labs. (USA)
Sigma, St. Louis (USA)
Sigma, St. Louis (USA)
NEUROTRANSMITTERS IN LUNG OF Bichir P. bichir bichir
Diagram 1. A,B: Schematic illustration of the bichir lungs and the glot-
tis as viewed from the dorsal side (A) and ventral side (B), respectively.
A: Disposition of pulmonary branches of vagus rami intestinalis nerves
and anatomical location of pulmonary artery are shown. RL, large right
lung; LL, l small left lung; RV, right vagus; LV, left vagus; RP, right pul-
monary artery; LP, left pulmonary artery; RPN, right pulmonary nerve;
G, glottis; OE, esophagus; S, stomach, I, IntestineB. Opening (OP) of
the glottis into the right lung is shown. Adapted from Kerr (1907).
ZACCONE ET AL.
against nNOS/AchE, nNOS/5-HT, TH/nNOS, SP/AchE,
PACAP/AchE, VIP/AchE, AchE/S100 protein, P2X2/5-HT,
5-HT/5-HT3 (concentrations and suppliers as indicated
in Table 1). The distribution of neuronal cell bodies was
evaluated after colabeling with antibodies to both pep-
tide and nonpeptide transmitters and the general neuro-
nal marker S100. After four rinses in PBS, binding sites
of primary antibodies were visualized by corresponding
fluorescein isothiocyanate (FITC)-conjugated goat anti-
mouse IgG (Sigma) and tetramethylrodamine isothiocya-
nate (TRITC)-goat anti-rabbit IgG (Sigma), diluted 1:100
for 2 hr at RT.
To document immunofluorescence double labeling, a
Zeiss Axio Imager Z1 microscope was used. It is inte-
grated with AxioVision 4.5 and an AxioCam digital cam-
era (Zeiss) was used for image processing. Sections were
imaged using the appropriate filter settings for the exci-
tation of FITC (480–525 nm; channel 1) and TRITC
(515–590; channel 2). Channel 1 and 2 were coded green
and red, respectively. The colocalization of two markers
in the same structure, or an overlap of closely apposed
structures in the same focal plane, resulted in a yellow
mixed color. Sections were then taken at the same plane
and the two channels were merged. Image processing
including merging was carried out using AxioVision 4.5
Negative controls for all immunohistochemical label-
ing were performed by substitution of nonimmune sera
for the primary or secondary antisera. Specificity of the
labeling of some peptides was verified by incubating sec-
tions with antiserum preabsorbed with the respective
antigen (10–100 mg/ml). The preabsorption procedures
were carried out overnight at 48C. The purified antigens
were obtained from Bachem (VIP, PACAP), Biomol-DBA
(nNOS), and Sigma (5-HT, SP).
The paired lungs of Polypterus bichir bichir extend as
long hollow unchambered sacs. The larger right lung
runs along the whole length of the body cavity, whereas
the smaller left lung extends to the stomach (Diagram
1). The two lungs arise from a ventral glottis which
opens into the right lung (Fig. 1A,B); the left lung being
connected to the right by a separate opening. A muscle-
ridged slit arranged as a sphincter is observed around
the glottis (Diagram 1). Each lung is innervated by a
branch of the ramus intestinalis from the vagus nerve.
These pulmonary branches reach the lungs on the dorsal
surface and run to the distal end of each lung (Diagram
1). The right lung is innervated by the pulmonary
branch of the left ramus intestinalis, whereas the left
lung receives an innervation from a branch of the right
ramus intestinalis (Lechleuthner et al., 1989). Pulmo-
nary arteries and veins run along the length of the wall
of the lungs and branch perpendicular to the axis.
A description of sympathetic chains and postgan-
glionic sympathetic fibers connecting with the outflow of
vagus nerve has not been reported in a bichir species.
The following account provides evidence for a sympa-
thetic adrenergic innervation (see below) by immunohis-
The glottis is lined by ciliated, goblet cells and neuroe-
pithelial cells (NECs), including areas adjacent to the
lung opening, whereas the lung itself is characterized
only by small patches of ciliated cells (see below). The
row. A: The glottis-a muscle-ridged slit consisting of smooth muscle
cells (SM) is seen opening into the right lung. B: A furrow in the lung
A,B: A view of the glottis muscle and the lung ciliated fur-
epithelium showing presence of mucous goblet cells (g) and ciliated
cells. Flat cells of the respiratory epithelium (Re) are richly vascular-
ized. Scale bar 5 20 mm.
NEUROTRANSMITTERS IN LUNG OF Bichir P. bichir bichir
internal surface of the lungs is smooth except for a pat-
tern of longitudinal striations, called furrows (Fig. 1B).
The furrowed epithelium contains a rich concentration
of ciliated cells, Type II pneumocytes (Zaccone et al.,
1989), mucous cells, and NECs. Type II pneumocytes
overlie the surface capillary bed and, together with the
endothelial cells, form the blood–air barrier. Two layers
of striated muscle line the lung walls.
Paraganglia within the trunk of the pulmonary vagus
nerves are present. Local ganglion cells are sometimes
found next to large vessels. Nerve cell bodies and nerve
fibers are also found in submucous localizations in the
glottic region and form ganglionic plexuses.
AchE and S100 Protein
Blood vessels in the submucosa were supplied by
dense plexuses of AchE- and S100-positive nerve fibers.
Nearly all nerve fibers running within the layers of stri-
ated muscle lining the lung wall displayed strong AchE
immunoreactivity. Most of these fibers did not co-label
with S100 (Fig. 2A,B). Many AchE-positive nerve fibers
were seen penetrating the muscle layers of the glottis.
Neurons immunoreactive for both AchE and S100 were
also found in nerve fiber bundles within the glottis mus-
cle layer (Fig. 2C). Associated with the soma of some
neurons were AchE- and S100-positive nerve fibers.
AchE and nNOS
The AchE-nNOS double labeling method does not
demonstrate different nerve fiber populations. Details of
the double label method revealed that a population of
nerve fibers that approached paraganglion cells showed
nNOS could not be demonstrated. Figure 2D shows that
intravagal paraganglionic cells were surrounded by a
basket of AchE-immunopositive nerve fibers. A small
number of cells were reactive to AchE and nNOS, and
occasionally found within the outer part of the muscle
coat. Thick nerve trunks were located on the external
surface of the muscle coat exhibiting double immunolab-
eling of nNOS and AchE, thus showing that these nerve
fibers belong to the same population (Fig. 2E). NECs
were localized with antibodies against AchE and nNOS
(Fig. 2F,I) within the epithelial lining (mucociliated epi-
thelium) of the lung where they formed a thin apical
process that extended to the airway lumen. AchE-nNOS
merged images show an association of nNOS nerve ter-
minals running between the NECs toward their apical
part (Fig. 2G). There were sparse submucosal nerve
fibers displaying immunoreactivity for AchE. Some of
these fibers were in contact with blood vessels (Fig. 2H).
A rich supply of AchE-immunoreactive nerve fibers
formed a dense network with a circular orientation
along the long axis of the pulmonary artery. These fibers
followed a conspicuously similar course as the nNOS-
immunoreactive nerve fibers, as demonstrated by the
combination of the two channels.
nNOS and 5-HT
Single or small groups of NECs showing nNOS and 5-
HT immunoreactivity (Fig. 2I,J) were present in the
mucociliated epithelium of the lung. NECs were associ-
ated with a dense network of nNOS-immunopositive var-
icose nerve fibers running in an apical direction. Double
labeling with antibodies against nNOS and 5-HT charac-
terized the presence of ganglionic plexuses showing
nerve cell bodies both in the submucosal and muscle
layers of the glottis. The immunohistochemical analysis
revealed the presence of 5-HT in these cells. Paragan-
glionic cells were nNOS-immunopositive and surrounded
by nerve fibers with a vesicular morphology showing 5-
HT immunoreactivity (Fig. 2K). Combinations of both
channels revealed that few nNOS-immunopositive nerve
fibers ran in proximity to 5-HT–positive fibers, thus
belonging to separate populations. Thick nerve trunks
were located on the external surface of lung muscle coat.
They contain nerve fibers as evidenced by 5-HT immu-
noreactivity. 5-HT–immunoreactive nerve fibers were
found in the longitudinal and circular muscle of the lung
wall. Numerous 5-HT–immunoreactive nerve fibers were
present in the adventitia of pulmonary artery wall that
formed dense nerve plexuses.
nNOS and TH
In double-label experiments with TH and nNOS, com-
plex neural plexuses in striated muscle are shown in the
external surface of lung muscle coat. All the immunore-
active nerve terminals in between muscle cells label for
TH (Fig. 2L). Small paraganglionic cells are seen within
vagal nerves. These cells stain sometimes for TH (Fig.
2M,N) and are surrounded by TH-immunoreactive nerve
fibers which also supply blood vessels associated with
the paraganglia. Thicker nerves showing only immunor-
eactivity for TH are found in the muscular layers of the
glottis (Fig. 2O) and the submucosal vasculature of the
Double-label immunohistochemistry revealed that NECs
in the mucociliated lung epithelium coexpressed TH and
nNOS. Most single NECs formed a thin apical process that
gave a positive immunoreaction for TH and nNOS (Fig.
3B). A dense TH-positive perivascular plexus was found
that surrounded the pulmonary artery (Fig. 3A).
AchE and SP
Both green and red channels revealed that bundles of
nerve fibers both in the outer and inner layers of the mus-
cle coat in the lung wall contain SP- and AchE-immuno-
positive nerve terminals (Fig. 3C,D). Merged images
showed that only few nerve fibers were labeled only with
AchE antibodies. Immunocytochemical double labeling
demonstrated plexus of AchE- and SP-immunopositive
nerve fibers at the wall of the pulmonary artery. Several
endothelial cells were also found to express SP. NECs in
the furrowed epithelium showed SP immunoreactivity.
AchE and PACAP
Combination of both channels clearly demonstrates
that PACAP and AchE immunoreactivity is in the sub-
mucous neurons in the glottic region (Fig. 3E). Globular
or ellipsoid-shaped neurons in a nerve fiber bundle in
the smooth muscle layer of the glottis were immunoreac-
tive for AchE (Fig. 3G) and PACAP (Fig. 3F). The red
channel (PACAP) shows a perivascular plexus around
submucosal blood vessels and pulmonary artery in the
lung and the glottis, but combination of the two channels
revealed no colocalization with AchE. Intraepithelial
ZACCONE ET AL.
terase (AchE) and S-100 protein in the glottis and lung muscle. A,B: Combi-
nation of both channels showing the immunostained cholinergic innervation
inthe striated lung muscle (arrowheads). C: Combinationof the two channels
reveal that nerve terminals in glottic muscle are AChE-immunopositive
(arrows) and a group of neurons aligned along the nerve fibers in the muscle
layer show the overlap signal (yellow orange). D–I: Immunohistochemical
double staining for AchE and neuronal nitric oxide synthase (nNOS) in the
lung muscle submucosa, neuroepithelial cells (NECs), and paraganglia. D:
Paraganglion cells are surrounded by dense networks of AchE-immunoreac-
tive axons (arrowhead). Only a few nitrergic nerve terminals running along
the cells are seen (arrow). E: Overview of a large nerve trunk (arrows) running
along outer wall of the lung. A delicate neural network (arrow) is visible show-
ing colocalization of AchE and nNOS in the nerves. F: AchEimmunoreactivity
of a NEC in the mucociliated epithelium with a thin apical process (arrow)
facing the airway lumen. G: Combination of green and red channel showing
the nerve fibers running between two NECs in an apical direction. These
fibers are nNOS-immunoreactive (arrows). The asterisk indicates the slender
process of a NEC touching the lumen (lu). H: Green channel showing a sub-
A–C: Immunohistochemical double staining for acetylcholines-
mucosal blood vessel (bv) contacted by a nerve bundle (arrow) composed of
AchE-immunopositive nerve fibers. I: nNOS immunopositivity of a NEC in
the furrowed lung epithelium with an apical process reaching the airway
lumen (lu). J,K: Immunohistochemical double staining for nNOS and 5-HT in
the lung epithelium and paraganglia. J: A 5-HT–immunopositive NEC with a
along process (arrow) facing the airway lumen and 5-HT–positive nerve
fibers (arrowhead) at the base of the epithelium. K: Combination of the green
and red channel showing the nNOS immunoreactivity of the paraganglion
cells (arrowhead), which are surrounded by a basket of 5-HT–immunoposi-
tive axons. L–N: Immunohistochemical double staining for nNOS and tyro-
sine hydroxylase (TH) in lung and glottis muscle and paraganglia. L: Green
channel showing a more extensive TH-immunopositive neural plexuses
(arrowheads) surrounding the striated muscle (m). No nNOS immunoreactiv-
ity is found in these plexuses. M: Green and red channel showing TH-immu-
nopositive nerve fibers (arrow) surrounding paraganglion cells (arrowhead)
showing nNOS immunoreactivity. N: Green channel of same structures
showing presence of TH-immunopositive nerve fibers (arrow) around the
paraganglion cells. O: A rich network of TH-immunopositive nerve plexuses
(arrowheads) in the muscle layers of the glottis. Scale bars 5 20 mm.
synthase (nNOS) and tyrosine hydroxylase (TH) in lung epithelium and
pulmonary artery. A: Cross-section of pulmonary artery stained with
the double-labeling method nNOS-TH. The TH-immunopositive nerve
fascicles and fibers form a dense network in the wall of the artery. B:
Combination of both channels revealing that a neuroepithelial cell
(NEC) in the mucociliated epithelium expresses both nNOS and TH,
although the staining intensity varies along the cell cytoplasm; g, mu-
cous goblet cells. The arrow indicates its apical process. C,D: Double
labeling for Acetylcholinesterase (AchE)/substance P (SP) in lung mus-
cle. C,D: Green and red channel showing the outer muscle layer con-
tacted by AchE (arrowhead) and AchE/SP-expressing nerve fibers. E–
H: Double labeling for AchE-adenylatecyclase/activating polypeptide
38 (PACAP38) in the nerve cell bodies of the glottis. E: The overlap
signal (yellow orange) reflects colocalization of AchE and PACAP38 in
the nerve cell bodies on the submucosal aspect. ep, epithelium. F:
A,B: Immunohistochemical staining for neuronal nitric oxide
PACAP38 immunoreactivity of the same neurons. G: AchE immunor-
eactivity of the neurons on the submucosal aspect. ep, epithelium.
H: Immunoreactivity for AchE showing nerve cell bodies in a nerve
fiber bundle that is entering the smooth muscle (sm) layer of the glot-
tis. I–K: Double labeling for AchE-vasoactive intestinal polypeptide
(VIP) in outer muscle and lung submucosa. I: Overview of the lung
with more extensive neural plexuses surrounding outer muscle layer
(m), immunostained with antibodies to AchE and VIP. Combination of
both channels revealing that AchE and VIP immunoreactivities are
expressed by the same nerve fiber populations (arrow). J,K: The green
channel and the red channels show AchE- and VIP-immunoreactive
nerve fibers are observed in contact with submucosal vasculature
(bv). L: Immunostaining for ATP receptor P2X2in the lung epithelium.
Red channel reveals P2X2-immunoreactive nerve fibers entering the
lung epithelium (arrows) to form intraepithelial terminals facing the air-
way lumen (a). Scale bars 5 20 mm.
ZACCONE ET AL.
complexes of PACAP-immunopositive nerve fibers were
AchE and VIP
VIP immunoreactivity was found in the external mus-
cle coat (Fig. 3I), longitudinal and circular muscle
layers, and submucosal vasculature. In the striated mus-
cle layer, the VIP innervation was pronounced on the
external surface where nerve trunks were associated
with lung muscle. Combination of the red and green
channel showed a direct relationship between the VIP
and AchE innervation of this muscle region. AchE- and
VIP-immunoreactivity of nerve fibers associated with
submucosal vessels are shown in Figure 3J,K.
5-HT and P2X2
Labeling of sections processed for P2X2receptor local-
ization with antibodies to 5-HT showed the coexistence
of immunopositive nerve fibers expressing P2X2purinor-
eceptors and 5-HT. These fibers approach the lung epi-
thelium, protrude between the epithelial cells, and form
intraepithelial terminals (Fig. 3L). Many terminals were
seen close to the epithelial surface. P2X2 and 5-HT
nerves and in nerve fibers in contact with blood vessels.
Immunoreactivity against P2X2and 5-HT was found in
the NECs (Fig. 4A) in the mucociliated lung epithelium,
in several monopolar neurons and nerve fibers occurring
in the submucosal layers of the glottis (Fig. 4B). On
occasion, cell bodies located along nerve strands were
seen in these layers (Fig. 4D–F). A high number of neu-
rons containing immunoreactivity to P2X2and 5-HT was
also present between nerve bundles that enter the
smooth muscle layer of the glottis, and the submucosal
plexus but ganglia were usually absent. Serotonergic
neurons were seen surrounding pulmonary artery (Fig.
4C), but were not shown by P2X2 staining. The 5-HT
plexus around the pulmonary artery.
5-HT and 5-HT3
After double labeling with antibodies against the 5-
HT3receptor and 5-HT, immunoreactivity was observed
in the monopolar neurons in glottic submucosal layers
tryptamine (5-HT) and P2X2 receptor in the lung epithelium and the
nerve cell bodies in the glottis. A: The green channel reveals a 5-HT–
immunopositive NEC with a long process reaching the airway lumen.
The red channel showing the P2X2receptor immunoreactivity is uni-
form throughout the cell cytoplasm. B: Green and red channels show-
ing the presence of two 5-HT and P2X2receptor-immunopositive glot-
tic monopolar neurons in the submucosal layer. C: Neurons (arrows)
containing immunoreactivity to 5-HT projecting to pulmonary artery
A–F: Immunohistochemical double staining for 5-hydroxy-
(a). D,E: Green and red channel demonstrating the localization of 5-HT
and P2X2receptor respectively in some nerve cell bodies in internodal
strands. F: Overlap signal showing 5-HT/P2X2receptor colocalization.
G–I: Immunohistochemical staining for 5-HT and 5-HT3receptor in the
nerve cell bodies of the glottis. G: Monopolar neurons imaged by im-
munostaining for 5-HT3 receptor in submucosal plexus adjacent to
smooth muscle. E, epithelium. H,I: Green and red channel demonstrat-
ing the presence of 5-HT and 5-HT3receptor respectively in ellipsoid-
shaped neurons underneath the glottic epithelium. Scale bars 5 20 mm.
NEUROTRANSMITTERS IN LUNG OF Bichir P. bichir bichir
and at junctions between fiber bundles in the smooth
neurons also occurred underneath ciliated mucous epi-
thelium of the glottis (Fig. 4H,I). NECs in the lining
mucosa were 5-HT positive, but did not express 5-HT3
A general account of the fish autonomic nervous sys-
tem (ANS) has been given by Campbell (1970). However,
have been no structural or functional studies of the
innervation of visceral and vascular muscles in the lung
of the bichirs, which comprise both Polypterus and Erpe-
toichthys extant forms. These fishes are non-teleostean
fishes that are among a group of brachiopterygians and
are considered to be more primitive than teleosts. How-
ever, their systematic position has been questioned, lead-
ing to a proposal to include these fishes in a group of tel-
eosts of its own (Bjerring, 1985). The present study pro-
vides, for the first time, a framework for interpreting
the structure of the ANS in the lung of the Nile bichir,
Polypterus bichir bichir. In comparison to the lungs of
dipnoan fishes, there appears to be a complexity of the
ANS of lung tissue in bichirs associated with a highly
developed sympathetic adrenergic system. A vagal inner-
vation of the lungs and gill arches in bichirs has only
been inferred from anatomical studies (Lechleuthner
et al., 1989; Piotrowski and Northcutt, 1996), and there
is no information on the spinal autonomic system or
functional studies on the role of innervation in polypter-
ids. The lungs of the bichirs receive output from both
cranial and spinal autonomic sources, but the location of
these nerves and their functional significance await
were seen running in the striated muscle, whereas the
outer muscle was innervated by vagal nerve trunks. A
submucosal plexus was present and often associated
with the vasculature, but lacked nerve cell bodies in the
lung region. By contrast, numerous globular or ellipsoid
monopolar neurons were present in the submucous and
muscle regions limited to the glottis and pulmonary
artery. A diffuse neuroepithelial cell system was found
in mucociliated epithelium of the furrows in the lung
Despite physiological studies showing the locations
and effects of neurotransmitters in some fish tissues
(Donald, 1998), the role of the ANS in lung tissue is
more difficult to elucidate. Based on immunohistochemi-
cal evidence, cholinergic and adrenergic components
characterized the lung and glottis intramural innerva-
tion and primary systemic vasculature. The pulmonary
nitrergic and cholinergic nerves. PACAP-, VIP-, 5-HT-,
and P2X2-containing nerves were also present near ves-
sels in the submucosa. These observations are consistent
with previous studies which have shown in fish vascula-
ture the presence of peptidergic nerves (Nilsson and
Holmgren, 1992a; Donald, 1998; Zaccone et al., 2006a)
and purinoreceptors (Burnstock, 1996). Fish gill vascula-
ture is innervated by axons of spinal autonomic origin
facilitating adrenergic regulation (Wahlqvist and Nilsson,
1981). Furthermore, catecholamine-containing nerves are
present on the gill afferent filament arteries and venous
sinus (Donald, 1998). These nerves are not found on the
pulmonary artery of the lung of dipnoan fishes, but adre-
nergic regulation may occur by means of circulating cate-
cholamines (Axelsson et al., 1989) released from the chro-
maffin tissues in the wall of intercostal arteries and the
posterior cardinal veins and the atrium of the heart
(Nilsson and Holmgren, 1992b). The dipnoan lung does
not receive any innervation from spinal autonomic fibers
(Parker, 1892; Holmgren et al., 1994). Sympathetic nerves
run to the posterior cardinal veins, while the parasympa-
thetic nervous system is limited to the vagal nerves
innervating the heart, gut and lung (Nilsson and Holmg-
There is no evidence for cholinergic innervation of the
fish systemic vasculature. The lung visceral muscle of
dipnoans is contracted by Ach and vagal nerve stimula-
tion (Abrahamsson et al., 1979) and is innervated by
cholinergic nerves. A vagal cholinergic, sympathetic, ad-
renergic and NANC component, including the serotoner-
gic neurons, may be used by pulmonary arteries to con-
trol lung blood flow in our bichir species. Future work is
needed to clarify the physiological control of the sympa-
thetic and neuropeptide innervation of the pulmonary
artery and changes associated with physiological events
due to air breathing. It is possible that the cholinergic
innervation arose in this ancient air-breathing fish when
initial selective forces for buoyancy, respiration, or both,
led to the appearance of the primitive lung-like organ
(Graham, 1997), a beautiful example of the plasticity of
the nervous system (Campbell and McLean, 1994).
In a few teleosts and holosteans, including the bichirs
(Lechleuthner et al., 1989), the swimbladder is primarily
a respiratory organ analogous to the lung. In most acti-
nopterygians with a swimbladder its function is to regu-
No information exists on the control of visceral muscle
of the lung of bichirs in comparison to that in dipnoan
fishes. The lung of these fishes is innervated by vagal
autonomic nerves, and intrinsic nerve cell bodies are
recognizable within the vagus and on the pulmonary ar-
tery of some species.
In particular, the lung wall showed a parasympathetic
and sensorimotor innervation (Holmgren et al., 1994). A
NANC and cholinergic innervation was not studied,
although there is information about the contraction of
lung visceral muscle of Protopterus by Ach and vagal
nerve stimulation (Nilsson and Holmgren, 1992b). In
this study, it was found that the lung musculature was
innervated by cholinergic, NANC, and sensorimotor
nerve fibers. There is, however, no physiological evi-
dence to verify these innervation patterns and to deter-
mine whether parasympathetic and sympathetic nerve
supplies follow separate paths to the lung. The origin of
adrenergic innervation of lung muscle awaits further
investigation. Further experiments will clarify if the
arrangement of sympathetic chains in the bichirs resem-
bles that of teleost and ganoid fishes, and if there are
substantial contributions of spinal autonomic neurons to
the vagi, as reported in the tetrapods. Interestingly,
lungfishes lack vagosympathetic nerve trunks, and it is
surprising that non-teleostean fishes (for instance, the
bichirs) which comprise an extremely heterogeneous
group and are considered primitive, show relationships
to the tetrapods. As explained above, we are not sure
that the innervation of the bichir lung muscle is homolo-
gous with the smooth muscle of fish swimbladder,
ZACCONE ET AL.
although both are primarily concerned with adrenergic
and cholinergic nerves (McLean and Nilsson, 1981;
Zaccone et al., 2006c) and also show a peptidergic inner-
vation (Lundin and Holmgren, 1989; Zaccone et al.,
2006a). This finding may be related to the dependence of
teleosts on alternative air-breathing structures, which
are strongly correlated with their adaptative radiation
and with evolutionary canalization of gas bladder struc-
ture. Therefore, some fish species have respiratory gas
bladders differing in complexity or retaining a primitive
organ trait to support buoyancy (Graham, 1997).
Although the ANS in teleosts is more sophisticated
than in elasmobranchs and cyclostomes, there is an
increased level of organization of the branchiomeric
pathways in mammals compared with fish, and the neu-
rochemistry of ganglion cells within the cranial nerves
of lower vertebrate species has been overlooked (Gibbins,
1994). The present study demonstrates the presence of
paraganglionic cells in the vagal nerve trunks or within
the lung wall, and sympathetic cholinergic postgan-
glionic fibers terminating on the lung muscle. Paragan-
glionic cells are also surrounded by a basket of both cho-
linergic and adrenergic nerve terminals, suggesting a
possible extent of a cephalic sympathetic chain and a
contribution of spinal autonomic neurons to the vagus
nerve. These cells contain acetylcholine, tyrosine hydrox-
ylase as well as substance P, nNOS, and PACAP. It is
possible that adrenergic components in the lung muscle
use NO, 5-HT, or PACAP as possible neuromodulators.
These compounds may be, in turn, modulated in their
release by the cholinergic innervation whose presence is
histochemically demonstrated in relation with the neuro-
peptide-immunoreactive neurons showing a different an-
atomical distribution. On the other hand, a further rela-
tionship is suggested between NO and the adrenergic
and cholinergic components of the intramural innerva-
tion because nNOS is shown in those submucous neu-
rons of the glottis and paraganglion cells within the
trunk of vagus nerve in which AchE and TH immunor-
eactivity is present. It is surprising that there is no evi-
dence for adrenergic innervation of the dipnoan lungs
(Nilsson and Holmgren, 1992b). Dipnoi may be closely
related to ancestors of amphibians (Glass, 1992), which
show adrenergic nerves in lung muscle (Campbell and
A prominent feature of the autonomic nervous system
division of the lung in bichir is the large collection of
monopolar neurons located in the submucosa and mus-
cular layers of the glottis. These neurons express AchE,
5-HT, VIP, PACAP 38, SP and 5-HT3and P2X2receptors.
The coexistence of many peptide and nonpeptide trans-
mitters, including receptors, may be species-specific, but
the more complex functional consequence of the combi-
nations of these transmitters is not known. Nerve cell
bodies are also encountered in the pneumatic duct in
the swimbladder and the gill arch of some teleost species
(Lundin and Holmgren, 1989; Bruning et al., 1996; De
Girolamo et al., 1998; Finney et al., 2006), including
lungfishes (see Holmgren et al., 1994). But the precise
arrangement and function of the above neurons of vari-
ous shapes and sizes, varies within a single species, and
in the same region in different species (Gibbins, 1994),
thus indicating their involvement in the regulation of
several functions, i.e., smooth muscle motility, secretion,
and ciliary activity in the lung epithelium and local
blood flow. It is of interest to note that there exists a
homology between vagal innervation of the tetrapod
lungs and the various branches of the vagus in fish.
Based on the ventral origin of the lungs from the bran-
chial arch region of the pharynx, it is suggested that the
ganglia in the lung are likely to be homologous with the
small ganglia in the branchial and pharyngeal rami on
the vagus in fish (see for review, Gibbins, 1994). The
ganglion cells in the pulmonary rami of the vagus in
anurans are monopolar and morphologically identical to
other cranial postganglionic neurons. Ganglion cells are
usually scattered in the pulmonary rami of the vagus in
mammals. Therefore, both vagal paraganglion cells and
glottic neurons found in Polypterus may bear a morpho-
logical resemblance to genuine postganglionic vagal neu-
rons, some of which become incorporated into the enteric
plexuses of the foregut (Gibbins, 1994). Glottic neurons,
both in submucous and muscle localizations, do not show
any association with PACAP-, SP-, and 5-HT-immuno-
reactive axons and lie in the nerve branches in the glottic
region. Some AchE/S100-positive nerve cell bodies show
an array of AchE/S100-imunoreactive axons arranged
around them. Submucousal neurons express P2X2and 5-
HT3receptors. This may suggest a possible interrelation-
ship between the serotonin secretion and purinergic
mechanisms in chemosensory signalling in the foregut of
In addition to the lung muscles of bichir, extensive
innervation of the submucosa is present, and several
nerves contain a variety of neurotransmitters and pro-
ject to an epithelium that is endowed with specialized
NECs. PACAP-, SP-, P2X2-, and 5-HT-immunopositive
fibers are in contact with submucosal arteries. The
NECs are regarded as O2-sensitive cells that have been
described in the mammalian carotid body and pulmo-
nary epithelium, and in the fish gill (Zaccone et al.,
2003, 2006a; Jonz and Nurse, 2006; Saltys et al., 2006).
Associated with the apical cytoplasm of NECs are axons
that form varicosities. The majority contains nNOS
alone or in combination with TH or ACh. The origin of
these distinct populations of axons is unknown. A simi-
lar innervation pattern of NECs in the zebrafish gill was
observed by Jonz and Nurse (2003) and by Saltys et al.
(2006) in the NECs of gill tufts of Xenopus larvae, using
other antibodies. Several endocrine systems and epithe-
lial tissues are regulated by the activity of ANS in fishes
(Donald, 1998), but we do not know the functional role
of NO and adrenalin found in the nerves associated with
NECs. The results of the present study may be paral-
leled with those from the previous descriptions of NEBs
and nitrergic innervation of the rat lung (Brouns et al.,
2002). These authors suggest that NO, released from the
nerve terminals, might result in a direct inhibition of
the sensory discharge of NEBs in response to hypoxia.
Clearly, further investigations of neural mechanisms
controlling the bichir lung, and oxygen sensing in the
lung of bichirs, are warranted. However, we demon-
strated for the first time, intraepithelial nitrergic termi-
nals selectively related to NECs in the lung of bichir,
including the expression of nNOS, SP, P2X2purinorecep-
tors, and nonpeptide transmitters (AchE and TH) by
these cells. These findings may be consistent with the
hypothesis of NO, Ach, and adrenalin being used for
communication between NECs and afferent nerve end-
ings. A conspicuous feature is the terminal arborization
NEUROTRANSMITTERS IN LUNG OF Bichir P. bichir bichir
of P2X2receptor-positive nerve fibers in the lung epithe-
lial layer. It was, however, seen that these fibers did not
approach NECs. Rat pulmonary NEBs receive a supply
of P2X3 receptor-immunopositive nerve fibers and sen-
sory fibers showing a different origin (Brouns et al.,
2000). The expression of P2X2receptors on the lung epi-
thelial nerves and NECs in bichir may be intimately cor-
related with the vagal afferent ATP-mediated mecha-
nisms that have been postulated by Burnstock (1999)
and Brouns et al. (2000), indicating NECs as source of
secretable ATP with local effects on the lung function.
We may say that the structural morphology of the
lung tissue of the primitive bichir is very similar to that
of the lungs in tetrapod vertebrates. The saclike lungs,
however, do not contain orders of septa like in amphibia
(Goniakowska-Witalinska, 2001; Zaccone et al., 2004).
Lungs in these fishes play a hydrostatic role in addition
to a respiratory function. Bichirs also have the capacity
to breathe air using lung wall muscles that actively
force expiration, which is then followed by passive lung
aspiration (Graham, 1997). Both lungs and swimblad-
ders have a common evolutionary origin. It is generally
agreed that a respiratory lung, perhaps appearing first
in early jawed vertebrates, was the ancestral organ
(Liem, 1988), and Denison (1941) described in the placo-
derm Bothriolepsis. The lung was thus regarded as a
primitive fish characteristic present in both sarcoptery-
gians and actinopterygians (Liem, 1988). Another theory
holds the evolutionary transition of lung from gas blad-
ders (Kerr, 1919) showing a more complex structure due
to the reacquisition of the aerial respiration by these
organs (Graham, 1997). It seems that the evolutionary
history of the actinopterygian lung followed a different
course by its gradual transformation from a respiratory
glass bladder into a nonrespiratory physostomous gas
bladder, and finally to a physoclistous gas bladder pri-
marily specialized for sound reception, buoyancy control,
or both (Graham, 1997). In the bichir lung, the gas-
exchange area is located on very flat folds containing
the respiratory cells (pneumocytes I), which constitutes
the very thin blood–air barrier (Lechleuthner et al.,
1989). The airway cells in the mucociliated epithelium of
the furrowed epithelium are both ciliated cells, pneumo-
cytes II (Lechleuthner et al., 1989), and NECs. All of
these epithelial cell types are encountered in the lung of
tetrapods, but rare in the lung of the polypterids. Lung-
fishes contain an elaborate network of septa in their
lungs and have innervated NECs limited to the muco-
(Adriaensen et al., 1990). The physiological role of the
innervation patterns of the bichir lungs remains obscure
to us, as well as the function of neuroepithelial O2-sensi-
tive chemoreceptor cells and regulation of respiration.
The bichirs rely predominantly on gill ventilation, but in
anoxic water they depend exclusively on pulmonary ven-
tilation, unlike dipnoan fishes, which have proper lungs.
The origin of respiratory control is in these fishes not
known, but is driven primarily by changes in O2in tele-
ost fishes. Respiratory control in bichirs also raises the
question of the importance of the modulation of bicar-
bonate levels. Recent studies have confirmed common
features of respiratory control in lungfishes to that of
tetrapods (Sanchez et al., 2006).
We conclude that the different patterns of innervation
and populations of neurons including paraganglia in the
bichir lung are unique features among autonomic path-
ways in air-breathing organs of fishes. In contrast to
dipnoan fishes, several neurotransmitters are involved.
The arrangement of the neurons, and their distinction
by their contents of transmitters, may be a rule in the
autonomic neurotransmission in fish tissues, although
autonomic control of the regulation of circulation and
respiration is not fully investigated. In the transition
from gill breathing to predominantly air breathing tetra-
pods, the neuroepithelial oxygen-sensitive chemoreceptor
pathways have undergone extensive changes. It is prob-
ably due to the considerable fluctuations in atmospheric
oxygen during the geological eras leading to the develop-
ment of the lungs of sarcopterygian lungfishes and the
bichirs, although a uniform evolutionary pattern could
be not expected due to their phylogenetic diversity. It
seems, however, that the diversification of bioactive com-
pounds and nerve control of the lungs are far less vari-
able than the distribution of respiratory chemoreceptors
in air-breathing fishes and tetrapods.
The authors thank Professor Geoffrey Burnstock for
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