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Air- breathing in : Air-breathing Organs and Control of Respiration. Nerves and Neurotransmitters in the air-breathing organs and the .

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Air- breathing in fish: Air- breathing organs and control of respiration Nerves and neurotransmitters in the air-breathing organs and the skin ⁎ Giacomo Zacconea, , Eugenia Rita Laurianob, Gioele Capillob, Michał Kucielc a Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, 98168, Messina, Italy b Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale F. Stagno d’Alcontres 31, 98166, Messina, Italy c Poison Information Centre, Department of Toxicology and Environmental Disease, Faculty of Medicine, Jagiellonian University, Kopernika 15, 30-501 Kraków, Poland

ARTICLE INFO ABSTRACT

Keywords: In fishes, exploitation of aerial has evolved independently many times, involving a variety of air- Neurotransmitters breathing organs. Indeed, air-breathing occurs in at least 49 known families of fish (Graham, 1997). Many Nerves amphibious , at some stage of their development are actually trimodal breathers that use various NECs combinations of respiratory surfaces to breath both water (skin and/or ) and air (skin and/or ). The Skin-gill-lung present review examines the evolutionary implications of air-breathing organs in fishes and the morphology of ARO the peripheral receptors and the neurotransmitter content of the cells involved in the control of air-breathing. Air-breathing fishes Control of breathing, whether gill ventilation or air-breathing, is influenced by feedback from peripheral and/or central nervous system receptors that respond to changes in PO2, PCO2 and/or pH. Although the specific chemoreceptors mediating the respiratory reflexes have not been conclusively identified, studies in water- breathing have implicated the neuroepithelial cells (NECs) existing in gill tissues as the O2 sensitive chemoreceptors that initiate the cardiorespiratory reflexes in aquatic vertebrates. Some of the air-breathing fishes, such as , Polypterus and Amia have been shown to have NECs in the and/or , although the role of these receptors and their innervation in the control of breathing is not known. NECs have been also reported in the specialized respiratory epithelia of accessory respiratory organs (ARO’s) of some catfish and in the gill and skin of the schlosseri. Unlike teleosts matching an O2-oriented ventilation to ambient O2 levels, lungfishes have central and peripheral H+/CO2 receptors that control the acid- base status of the blood.

1. Introduction The major shifts in the integration of systems have coincided with the evolutionary transitions from aquatic to aerial respiration and from Primitive fishes were the first vertebrates to exploit atmospheric aquatic to terrestrial life. In freshwater fish, respiration, ion and water respiratory gases, in addition to gases dissolved in their aquatic milieu, regulation and acid-balance reside mainly within the gills. By contrast, prior to the colonization of the terrestrial by (see in , gas exchange and respiratory acid-base regulation are for review Hedrick and Katz, 2016). The ability to extract di- lung functions whereas ion and water regulation, nitrogen excretion, rectly from the atmosphere enabled ancient fish to survive in hypoxic and metabolic acid-base regulation depend on the kidney. In larval environments. Extant air-breathing fish are now the subjects of many amphibians, excretion, osmoregulation, and respiration are branchially studies coming from diverse laboratories since they are considered mediated. However, the post-metamorphic amphibians show an inter- physiological models to study the evolutionary transition from gill to mediate position between fish and mammals in terms of kidney func- air-breathing ventilation. A consequence of this transition is the addi- tion, while auxiliary organs such as the skin and urinary bladder may be tion of accessory respiratory organs (ARO’s) that necessitate changes in involved in respiratory and osmotic functions (Graham, 1997). both the general circulatory system and the microcirculation of the The ancient fish lineages are viewed as the archetypes for the respiratory epithelia, thus providing indication of the asso- physiological adaptations to amphibious life (Hedrick and Katz, 2016). ciated with adaptation to the terrestrial habitats (Olsson et al., 1995). Consequently, much research is now addressed on the basic metabolic

⁎ Corresponding author at: University of Messina, Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Polo Universitario dell’Annunziata, 98168, Messina, Italy. E-mail addresses: [email protected] (G. Zaccone), [email protected] (E.R. Lauriano), [email protected] (M. Kuciel). https://doi.org/10.1016/j.acthis.2018.08.009

0065-1281/ © 2018 Published by Elsevier GmbH.

Please cite this article as: Zaccone, G., Acta Histochemica, https://doi.org/10.1016/j.acthis.2018.08.009 G. Zaccone et al. Acta Histochemica xxx (xxxx) xxx–xxx and physiological modifications that have occurred during the transi- located in the gills. These O2 sensors correspond to the NECs that have tion to air-breathing. The main aspect of this transition is the reduction been localized in the gill filaments of a wide variety of fishes, including of the gill blood flow associated with air-breathing since it compromises teleosts (Zaccone et al., 1997; Jonz and Nurse, 2003; Jonz and Zaccone, basic gill functions such as CO2 removal, osmotic regulation, acid-base 2009; Porteus et al., 2015) and non- (Zaccone et al., 1997; Jonz balance and nitrogen excretion. Another important aspect is the control et al., 2016) species. NECs share several morphological features of air-breathing. Air-breathing fishes must be able to sense and to re- with other peripheral O2 chemoreceptors such as the carotid body cells spond to changes in external and internal partial pressures of re- of mammals and the NEC-like cells found in the lung of lungfishes and spiratory gases (O2 and CO2) via chemoreceptors, as well as to sense (Zaccone et al., 2007, 2012). Although the specific O2-sensitive changes in the volume of the air-breathing organ via mechanoreceptors chemoreceptors and gill arch mechanoreceptors that mediate the gill (Hedrick and Katz, 2016). Some aspects related to nitrogen excretion in ventilatory and air-breathing responses to hypoxia have not been con- air-breathing fishes remain somewhat obscure since air-breathing in- clusively identified (Hedrick and Katz, 2016), the NECs of teleost gills terrupts or reduces branchial function. Data obtained in the amphibious are being considered to be the O2 sensitive chemoreceptors (Jonz and fishes, the , revealed that the gills, skin and urinary tracts Nurse, 2009; Porteus et al., 2013). were all involved in nitrogen excretion (Graham, 1997) and, that, We devote the bulk of this chapter to review the neurochemical during forced emersion, they switched from ammonotelism to ur- pro files of the NECs located in the gills and air-breathing organs of a eotelism. selected group of primitive fishes and advanced teleosts that have de- The two major clades of , the (ray- veloped air-breathing structures, probably as a plastic response to en- finned fishes) and (lobe-finned fishes), diverged some- vironmental modifications. times in the late Silurian (438 to 408 million years ago). It is generally accepted that air-breathing evolved in the two major lineages prior to 2. Phylogenetic origin of air-breathing the fish- transition in the Devonian (approximately, 385 to 360 million years ago) (Graham, 1997; Clack, 2012; Hedrick and Katz, As emphasized by Gilmour and Milsom (2009), the respiratory 2016). Among the extant actinopterygians, the most primitive forms passages of all the vertebrates have arisen from digestive passages and, include bichirs (Polypterus), (Lepisosteus) and the bowfin(Amia). that, with regard to CO2, and/or pH, the taste, smell and cardior- Among the sarcopterygians, the only remaining extant air-breathing espiratory chemoreception are arbitrary distinctions. It seems probable group corresponds to the Dipnoii (lungfishes: Protopterus, Lepidosiren that airway chemoreceptors aroused from digestive (olfactory, gusta- and Neoceratodus). Our primary focus in this chapter will be the pre- tory) chemoreceptors including, possibly, a diffuse system of chemor- sumptive peripheral respiratory chemoreceptors of air-breathing fishes eceptors that are found in the skin, gills and oropharyngeal surfaces of that were initially located in the gills of the teleosts (Zaccone et al., primary aquatic vertebrates. These chemoreceptors constitute the so- 2006; Jonz and Nurse, 2009; Jonz and Zaccone, 2009; Jonz et al., termed solitary chemosensory cell (SCCs) system (Sbarbati et al., 2009). 2016). Morphological and physiological studies of the peripheral O2 All the including vertebrates have a pharyngeal branchial sensing cells have been performed in a few number of air-breathing basket that is regarded to have respiratory function. This occurs in fishes, being compared to those of water-breathing fishes and mammals jawed fish (Hsia et al., 2013). In particular, a is a pe- to study the evolution of O2 chemoreception. This chapter also focuses culiar characteristic of vertebrates during their development (Cameron on the characteristics of the air-breathing that occurred in a group of et al., 2000). In primitive chordates, the first pharyngeal slits evolved teleosts that were secondarily adapted to aerial respiration. These in- into required for feeding. Subsequently, pharyngeal slits and clude the air-breathing organs (ABO’s) and the aerial respiratory sur- buccal pumps, that primarily evolved for feeding, gave rise to gills for faces of the higher euteleosts (ABO’s derived from the gills and the breathing, with water flow being driven by a buccal pump involving modified branchial chambers) that are present in the members of the muscles primarily innervated by the trigeminal and facial nerves Clariidae and Heteropneustidae families, grouped into the superfamily (Gilmour and Milsom, 2009). A recent study by Icardo et al. (2017) has Claroidea (Sullivan et al., 2006). The gills and the skin of the amphi- shown that the lungs in two polypterid fish, the , Polypterus se- bious fishes are also considered to be functional for aerial respiration. In negalus, and the reedfish, Erpetoichthys calabaricus, originate from the mudskippers, the sensory system required to switch the site of gas ex- ventral side of at about the level of oesophagic aperture. It change in emersed air-breathing species and in those having terrestrial demonstrates that the structures that function both as air-breathing and habits, is not well characterized. Extrabranchial sites of respiration in buoyancy organs aroused from the alimentary tract. The term 'lung' is the mudskippers include the cutaneous surfaces, where rapid circula- not consistently used to describe the air-breathing organs found in all tory adjustments increase blood flow and facilitate O2 transfer (see for the primitive fishes (Hedrick and Katz, 2016). Graham (1997) uses the review Wright and Turko, 2016). According to Wright and Turko term 'gas bladder' and suggests there are fundamental differences in (2016), the cutaneous surfaces of amphibious fishes such as the rivu- embryonic origin, location of the glottis and differences in pulmonary lines and the mudskippers are primed for aerial respiration, and several circulation. The lungfishes (Neoceratodus, Lepidosiren, Protopterus) and plastic traits associated with locomotion, gas exchange, nitrogen ex- the polypterids (Polypterus and Erpetoichthys) all possess lungs that arise cretion, ionoregulation and osmoregulation must be taken into account from a ventral glottis, a muscle-ridged slit. Respiratory gas bladders are when explaining ABO specialization. The skin surfaces are also the present both in Amia (Halecomorpha) and the gars (Lepisosteus and histological site for the occurrence of putative oxygen receptor cells Astractosteus, Ginglyostoma). (the neuroepithelial cells, NECs) that show peculiar neurotransmitter Air breathing first evolved in fishes and, over the 400 million year profiles (Zaccone et al., 2017). Unlike in teleosts, where the control of history of this group, this capacity has persisted in certain lineages and breathing is influenced by feedback from peripheral and/or central has been reacquired by others. Graham (1997) suggests that the air- nervous system by O2 sensing cells, the function of the NECs in the skin breathing is one of several adaptive responses utilized by fishes of amphibious fishes remains obscure. dwelling in habitats where O2 supplies may be severely depleted. Ra- NECs are present in the gill filaments of fish, appear strategically ther than having tendencies toward invading the land, most of these located at the interface between the respiratory water and the arterial fishes remain tied to an exclusively aquatic existence. Various theories blood flow, and exhibit morphofunctional characteristics that are ty- have been advanced concerning how the transition from bimodal to pical of the O2 chemoreceptors present in the lung of the air-breathing unimodal respiration could have affected the evolution of tetrapod gas vertebrates (reviewed by Bailly, 2009; Jonz and Nurse, 2009). Several exchange, to eliminate gill function. The ecological radiation has oc- responses to hypoxia such as hyperventilation, variation in gill vascular curred in groups such as anabantoids, clariids, and callichthyids in resistance arise, as stated above, from O2 peripheral chemoreceptors which bimodal breathing has been integrated with their natural history.

2 G. Zaccone et al. Acta Histochemica xxx (xxxx) xxx–xxx

Siluriformes breathe air using structures derived from their gills, Erpetoichthys a recoil aspiratory-like mechanism is described, but it is branchial chambers or both. In Clarias branched organs extend dorsally not related to the aspiratory mechanism for lung ventilation in tetra- from the branchial chambers. In Heteropneustes, the suprabranchial pods (Hedrick and Katz, 2016). chambers are not branched and penetrate into the musculature forming The ventilator control mechanisms in all the vertebrates involve long sacs homologous to the lungs in bichirs (Zaccone et al., 2002, three basic elements, namely, the peripheral sensory receptors (oxygen 2015). Despite the morphological specialization, the ray-finned fishes sensors and mechanoreceptors), the central nervous system and motor- did not evolve a separate venous return or blood separation within the neuron effectors. Unlike the unimodal control of respiration in fishes heart, that are characteristics of lungfishes and . The evolution and mammals having both a continuous ventilatory pattern, in the of air-breathing in these fishes probably occurred several times through ABO’s of the primitive actinopterygians, in view of their diversity, the a process of cladogenesis (Devaere et al., 2007; Ratmuangkhwang et al., coordination of the aquatic and aerial respiration played a critical role 2014). A recent comprehensive molecular study using complete mi- as well as the cardiorespiratory interactions. Ventilation is driven by tochondrial DNA sequences (Nakatani et al., 2001) confirmed the sister sensory receptors that also include taste and olfactory receptors. The relationship between Clariidae and Heteropneustidae. However, a dif- peripheral-receptor feedback occurs via the sensory afferent cranial ferent level of strategy suggested different origin of the air-breathing nerve branches (Milsom, 1990; Smatresk, 1994) that in fish connect the amongst the catfish families within the order of Siluriformes. The origin gill arches, the heart and the organs within the coelomic cavity. The of encapsulation of the swimbladder and air-breathing capacity could ventilator control in air-breathing fishes is dominated by the peripheral be interpreted as synapomorphy for a group of Siluriformes (South, chemoreceptors located in the gill arches, primarily in response to American, African, Asian) (De Pinna, 1998). The Siluriformes have been changes in PO2 in contrast to terrestrial vertebrates where ventilation is separated for at least 100 million years, probably enough time for the primarily driven by central chemoreceptors in response to changes in development of different air-breathing behaviors. PCO2/pH (Jones and Milsom, 1982; Hedrick and Katz, 2016). The lung is thought to have evolved during the Silurian The lungs of primitive air-breathing fishes are known to have me- period in fishes ancestral to the ray- and lobe-finned Osteichthyes. It chanoreceptors that sense changes in organ volume. These mechan- was in the Upper Devonian that terapods were derived from the lobe- oreceptors are generally characterized as Slowly-Adapting Receptors finned fishes. Air breathing evolved independently many times, as (SARs) or Rapidly-Adapting Receptors (RARs) based on their afferent suggested by Graham (1997). Air-breathing is considered to be sym- firing patterns in response to inflation or deflation of these organs. The plesiomorphic, that is, a shared primitive trait for all the bony fishes. afferent fi ring frequency of single unit neurons within the vagus nerve Molecular evidence is now demonstrating that the developmental pro- increases upon inflation and decreases upon deflation (Hedrick and gram for lung was established in the common ancestors of actinopter- Katz, 2016). Both SARs and RARs occur in Protopterus, Lepidosiren and ygians and sarcopterygians (Tatsumi et al., 2016). This symplesio- Lepisosteus (DeLaney et al., 1983; Smatresk and Azizi, 1987). The pri- morphy, based on the common presence of a lung and a lung-like gas mitive function of the ABO mechanoreceptors is to aid in buoyancy bladder for aerial respiration, extended from Sarcopterygii to the regulation (DeLaney et al., 1983), but future experiments are needed to and perhaps even to some of the . In this distinguish the respiratory versus buoyancy relevant inputs of ABO evolutionary scenario we must include the ABOs of the more advanced mechanoreception. teleosts that, in absence of a modifiable gas bladder, show new struc- In conclusion, the transition to air-breathing is accompanied by a tures that were recruited by diverse organs such as the pharyngeal, transition to a primarily CO2/pH keyed ventilatory drive and the ap- branchial and opercular chambers, the oesophagus, stomach and in- pearance of central CO2/pH chemosensitivity. In bimodal breathers, testine (Graham, 1997). Additionally, the skin is an auxiliary aerial the available evidence supports the existence of both water-sensing respiratory organ in many fish species thus suggesting that respiration CO2/pH chemoreceptors, presumably located in the gills or the oro- was primarily cutaneous. In fact, the walls of the slits in primitive branchial cavity, and of CO2-sensitive pulmonary stretch receptors chordates were associated with mucus-bearing cilia that served to trap (Gilmour and Milsom, 2009). Also, in the ARO (air sac) of the bimodal suspended particles (Graham, 1997; Gilmour and Milsom, 2009). The breathers, such as the air-breathing catfish, Heteropneustes fossilis, there complexity of the ABO structures was a consequence of the independent are no available data regarding the importance of mechanoreceptor evolution of air breathing and the diversity of ABO’s in several ostar- input (probably located in vagal afferent of the cucullar muscle of the iophysans and higher teleosts. This suggests a noteworthy convergence air sac) for the coordination of motor control over the respiratory of the air-breathing structures due to the evolutionary constraints as- muscle and structures along the aerial respiratory pathway (the gill sociated with air capture and gas exchange surfaces. That is, these arches). structures and their connecting respiratory passages also arose from various parts of the alimentary canal (Gilmour and Milsom, 2009). 4. Innervation of the air-breathing organs

3. Mechanisms of air-breathing in primitive fishes and control of Stimuli for air-breathing in fishes include hypoxia and hypercapnia, respiration both modulated by increased temperature and exercise, which increase oxygen demand and CO2 production. Air-breathing fishes utilize well- The majority of air-breathing fishes employ a buccal force pumping vascularized ABO’s, and there are, to date, very few studies regarding mechanism to ventilate their lungs. The lungfish first aspirate air into the distribution of receptors stimulating air-breathing in fishes that use the buccal cavity with the glottis closed preceding expiration from the various types of ABO’s. The sites and afferent innervation of oxygen- lung. With expiration there is mixing of fresh air and lung gas in the sensitive chemoreceptors that stimulate air-breathing and gill ventila- buccal cavity. Subsequently, this mixed gas is pumped into the lung by tion have been studied in various species of air-breathing fishes contraction of the buccal musculature. The air-breathing activity in (especially gars and bowfin). They are found diffusely distributed in the these fishes is affected by mechanoreceptors sensing the changes in gills and innervated by branchial nerves VII, IX, and X pressure or wall tension. These receptors play a role in inhibiting in- (Taylor et al., 1999). It has been suggested that the integration of the flation or promoting deflation (Hedrick and Katz, 2016). The mechan- input occurs via internally and externally oriented receptors that are ical act of breathing has changed from buccal force pump in air- responsible to set the level of hypoxic drive and the balance between breathing fishes and amphibians to sectional breathing based on con- air-breathing and gill ventilation, respectively. Application of horse- traction of the diaphragm and chest wall musculature. It is also likely radish peroxidase to nerves supplying the glottis and bladder in Amia that there are pacemaker cells or inhibitory interaction in pulmonary revealed the occurrence of a group of nerve cell bodies in a ventral ventilatory control. In the bichir Polypterus, and the reedfish location in the brain stem and the ventral horn of the anterior spinal

3 G. Zaccone et al. Acta Histochemica xxx (xxxx) xxx–xxx cord. This suggests that hypobranchial nerves are distributed to the supported by thick and thin septa containing variable amounts of stri- glottis and bladder and innervate the muscular elements associated ated and smooth muscle (Icardo et al., 2015). Tyrosine-hydroxylase with feeding movements. The hypobranchial innervation of the glottis (TH), choline acetyltransferase (ChAT), PACAP, 5 H T and SP neuro- and bladder ensures control of the glottal opening and effective venti- peptide immunoreactivities were detected in the intramural nerve fi- lation of the swimbladder (Taylor et al., 1999). This is in agreement bers coursing in the musculature (Zaccone et al., 2012). with the immunolocalization of intrinsic nerve cell bodies within the muscle and the submucosa in the glottis of the Polypterus bichir bichir 6. Chemoreceptors (Zaccone et al., 2007). Most of air-breathing fishes supply their various types of ABO’s by a systemic circulation, whereas lungfishes and all the Oxygen-sensitive chemoreceptors exerting a dominant control over tetrapods have distinct pulmonary arteries and veins in association with cardiorespiratory reflexes in fishes show a typical response to ambient true lungs. hypoxia by producing bradycardia and increasing the ventilatory efforts (Butler et al., 1977; Burleson and Milsom, 2003). Many physiological 5. Neurotransmitter substances in lung muscle and immunohistochemical studies carried out in several fish species have supported the existence of these chemoreceptors in or near the Actinopterygians and sarcopterygians have swimbladders or lungs gills (orobranchial and parabranchial cavities). However, the precise adapted for air-breathing. On the actinopterygian branch, Polypterus, anatomical sites and functional properties of these cells have not been Amia, Lepisosteus and several teleost species have a lung that is used for clarified. air-breathing. A true lung with separate pulmonary vein that empties Peripheral oxygen-sensing chemoreceptors were located primarily into the atrium of the heart, is present only in lungfishes. The lungfish in the teleost gills and have been identified as neuroepithelial cells lung contains both visceral and vascular muscle but, in comparison to (Zaccone et al., 1992, 1995, 1997; Jonz et al., 2004; Jonz and Nurse, tetrapods, there is little information on the control of these muscles by 2009; Porteus et al., 2015; Jonz et al., 2016). The NECs of fish gills the autonomic nervous system. The lungs are innervated by vagal containing a broader spectrum of neurotransmitter substances,are cranial autonomic nerves, and intrinsic nerve cell bodies are found neurosecretory and associated with nerve fibers. O2 sensing mechan- within the vagus and the pulmonary artery of Lepidosiren, Protopterus, isms have been only examined in the gills of a few fish species but these Neoceratodus forsteri and Polypterus bichir (Campbell and McLean, 1994; studies (Jonz and Nurse, 2009) have demonstrated that the NECs in- Zaccone et al., 2007). In Protopterus, a cholinergic excitatory vagal in- itiate cardiorespiratory reflexes in aquatic vertebrates. Physiological nervation of the smooth muscle in the lung wall occurs during expira- evidence of gill O2 chemoreceptors has been mainly reported in zeb- tion. In fact, the lung visceral muscle is contracted by Ach and vagal rafish and has not described in air-breathing fish species. nerve stimulation. However, there is not information on the presence of a non adrenergic-non cholinergic (NANC) innervation that is a feature 7. NECs in the air-breathing fishes of the visceral muscle in the tetrapod lung (see for review Donald, 1998). Immunohistochemical investigation on the lung of N. forsteri 7.1. Gill revealed a rich supply of nerve fibers containing immunoreactivities for galanin (GAL), somatostatin (SOM), substance P (SP), and vasoactive In the gills of one facultative air-breather, the bowfin Amia calva, intestinal polypeptide (VIP) in the pneumatic duct, the lung wall and Porteus et al. (2014) found the presence of multiple 5 H T- im- lung trabeculae. GAL-IR cell bodies are present in the pneumatic munoreactive NEC populations. Some of these cells (Type II, III) are sphincter. In contrast to N. forsteri, only met-ENK –IR and VIP-IR occur thought to be putative oxygen chemoreceptor cells since they monitor in the lung of Lepidosiren and Protopterus. In the Lepisosteus pla- hypoxia and hypoxaemia and increase in size upon exposure to sus- tyrhincus, the lung is innervated by the vagi, which carry excitatory tained hypoxia (Fig. 1a–d). These cells are also similar in function to the cholinergic nerve fibers to the musculature of the lung wall and tra- NECs identified in zebrafish and the , Kryptolebias beculae. A dense adrenergic innervation has been noticed in the blood marmoratus gills, that also increase in size during exposure to sustained vessels of the lung wall whereas only sparse innervation of adrenergic hypoxia (Jonz et al., 2004; Regan et al., 2011). It is interesting that nerve fibers could be demonstrated in the smooth muscle bundles. The VAChT and 5 H T immunopositive Type II cells (also regarded as in- function of adrenergic innervation of the vasculature and muscles of the trinsic neurons) in the bowfin are also very similar to the chain neurons lung wall is not known (Campbell and McLean, 1994). In lungfishes, in the gills of the amphibious giant mudskipper, Periophthalmodon such as in Protopterus, there is no conclusive evidence for adrenergic schlosseri where they express the same neurochemicals (Zaccone et al., innervation of the lung (Abrahamson et al., 1979). 2017). In the lung of the bichir Polypterus bichir bichir, Zaccone et al. (2007) Three types of NECs are recognizable in the gills of the Indian cat- described the presence of paraganglia within the trunk of the pul- fish, Heteropneustes fossilis (Zaccone et al., 2002) using antibodies to monary vagi, and intrinsic nerve cell bodies within the muscle and in neuropeptides (Leu- and met-enkephalin), nNOS and VIP. However, no submucous localizations in the glottis. A dense adrenergic innervation co-localization procedures are available yet. The presence of a dense was noticed in the pulmonary artery and the striated muscle of the lung neuropeptide (Met- and leu-enkephalin) innervation is found along the wall. In both longitudinal and circular muscle of the lung wall, a sparse anterior and posterior filament nerves, branchial arches, efferent innervation of cholinergic, serotonergic and nitrergic (nNOS positive) branchial arteries, efferent filament arteries, efferent lamellar arterioles nerve fibers was found. A dense innervation of cholinergic nerve fibers and branchial muscle. In the interfilament epithelium, NECs containing is also present in the glottis muscle, the fibers intermingling with the immunoreactivities for neuropeptides (met-and leu-enkephalin) have cell bodies. In the lung wall of the gray bichir Polypterus senegalus, been demonstrated. A similar innervation pattern is seen using nNOS antibodies against the neurotransmitter 5 H T and the calbindin D 28 K immunohistochemistry. A dense nNOS positive innervation is found in revealed nerve bundles and varicose nerve fibers in the striated muscle. both anterior and posterior gill filament nerves branching along the Double immunolabeling with antibodies directed against ChAT and efferent filament arteries. nNOS-immunopositive nerve fibers were also nNOS demonstrated the coexistence of these neurochemicals in varicose traced along the afferent and efferent branchial arteries.Experiments nerve bundles in lung muscle. Thick nerve fibers and perivascular using a combination of nNOS and TH antibodies revealed a dense TH nerves in the muscle are found containing 5 H T and nNOS (Zaccone perivascular innervation of the efferent filament arteries (Fig. 2a). et al., 2015). nNOS immunoreactive NECs are seen mainly in close association with The gas bladder in the spotted gar Lepisosteus oculatus and in the efferent filaments and lamellar arteries. NECs were also located in the longnose gar L. osseus is a membranous sac. In L. oculatus, the bladder is vicinity of nNOS positive nerve fibers. NADPH-d histochemistry is also

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Fig. 1. A–D. Labelling of neuroepithelial cells (NECs) in the bowfin, Amia calva. A) Type I cells labelled with serotonin (5-HT, green) antibody are found in close proximity to peripheral nerves positive to the neuronal marker, zn-12 (magenta). B) Type I cells labelled with the 5-HT (magenta) antibody show the coexistence of vesicular acetylcholine transporter (VAChT) (arrows), likely in nearby synapses. C) Type II (arrow) and type III (arrowheads) cells coexpressed 5-HT (green) and zn- 12. D) Type II cells coexpressed 5-HT (green) and VAChT. The cell nuclei (blue) are labelled with DAPI. Scale bars, 10 μm. Images kindly provided by C. Porteus (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). used showing a distribution pattern that was very similar to nNOS filament arteries. Numerous VIP-immunoreactive NECs were seen dis- immunohistochemistry. A large number of NADPH-d positive NECs was persed in the interfilament and lamellar epithelium, and are located seen in close association with efferent lamellar arteries and efferent along the side of the efferent filament arteries and efferent lamellar filament arteries. Recent investigations (Zaccone et al., 2018) have arteries. shown that two cell populations are found in the distal regions of the In the gills of the mangrove rivulus, Kryptolebias marmoratus, Regan gill filaments using a combination of antibodies to 5-HT and VAChT et al. (2011) found two NEC populations immunopositive to serotonin (Fig. 2d). In the subepithelial areas of the distal quarters and the and acetylcholine using immunohistochemistry. Acute exposure to hy- proximal part of the gill filament, a collection of VAChT positive in- poxia and the neurochemicals (serotonin and acetylcholine) resulted in trinsic neurons was noticed. They projected to filament arteries the fish emersing thus indicating that these neurotransmitters mediate (Fig. 2c). the oxygen sensing function of NECs. NECs are confined to efferent aspects of the filaments in the catfish In the gills of the giant mudskipper, Periophthalmodon schlosseri, Clarias gariepinus (Zaccone, Maina et al., unpublished). Most of the Zaccone et al. (2017) identified the presence of NECs inmmunoreactive NECs in this species are exposed directly to external environments. to 5 H T. These serotonergic cells are seen dispersed along the length of Confocal images from double immunolabeling with antibodies to TH the gill filaments but also occurred in cell clusters in the distal halves of and nNOS have shown the co-localization of these neuronal markers the filaments, in the lamellar epithelium and in the gill arches. A col- (Fig. 2b). NECs are absent on the afferent side of the filaments. Sub- lection of intrinsic neurons was noticed at the base of the gill filament epithelial TH immunopositive nerve fiber plexuses are present in the arteries (Fig. 4 a–i). The serotonergic cells colabelled with VAChT, distal half of the filament, probably contributing to the innervation of nNOS and TH, thus indicating the presence of multiple cell populations the efferent arteries (Fig. 2b). containing a wide array of transmitters that act in hypoxic reflexes in VIP-immunopositive varicose nerve fibers were mainly found fish gill. Immunolabeling with TH antibodies revealed that NECs in the around the vascular walls of efferent lamellar arteries and efferent gill are innervated by catecholaminergic nerves thus suggesting that

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Fig. 2. A–D. Labelling of nerve fibers, NECs and gill neurons in Heteropneustes fossilis and Clarias gariepinus. A) Nerve fibers innervating the efferent artery of the gill filament (eFA) of H. fossilis are labelled with the tyrosine hydroxylase (TH, green) antibody. B) NECs (arrows) in the distal half of the gill filament of C. gariepinus are labelled with antibodies to TH and neuronal nitric oxide synthase (nNOS). Subepithelial nerve plexuses (arrows) are positive to the TH antibody. C) Neurons (arrows) running in close proximity to the filament artery of H. fossilis are positive to VAChT. D) NECs (arrows) are labelled with the 5-HT antibody (green) in the distal half of the gill filament of H. fossilis. Scale bars, 20 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). these cells are involved in the control of branchial functions through receptor, 5 H T, and PACAP are seen in submucous localization and relationships with the sympathetic branchial nervous system. intermingled with muscle layers of the glottis. Very recently, the co- existence of 5 H T with calbindin D28 K, of 5 H T with nNOS and of ChAT with nNOS has been found in the NECs of the mucociliated epi- 7.2. Lung thelium in the lung of the Senegal bichir, Polypterus senegalus. A poly- morphic population of lung cells, the so-called polymorphous granular Combined techniques of confocal microscope and immuno- cells (PGCs) is also present in the same epithelium showing a similar fluorescence have allowed visualization of NEC populations in the lung neurochemical profile to that of NECs (Fig. 3c–f). Much remains to epithelium of the bichirs (Zaccone et al., 2007, 2008, 2015). In the ascertain if the NECs respond to changes in O2 and/or CO2 whereas Polypterus bichir bichir, a variety of neurotransmitters is located in the PGCs, that show a characteristic morphology and distribute in both NECs of the mucociliated furrowed epithelium. These NECs are positive epithelium and subepithelial areas, are likely resident cells involved in to 5 H T, AchE, TH and nNOS. NECs are also found in the epithelium local immune responses. lining the glottis. All the NECs are easily recognizable by the slender The expression of a wide variety of transmitter substances in the apical process running in between the mucous cells, and reaching the bichir lungs, including the presence of acetylcholine and catechola- apical surface (Fig. 3a–b). Double immunolabeling for nNOS with AchE mines, are a peculiar characteristic of parasympathetic and sympathetic and nNOS with TH revealed a close association of varicose nerve fibers nervous systems from the early stages in the evolution of the verte- with NECs. nNOS nerve fibers came into contact with NECs, and several brates. The lung is an important organ for air breathing and originates nerves converged on a single cell. A collection of nerve cell bodies well before the terrestrialization of vertebrates as recently evidenced by displaying immunoreactivities for AchE, P2 × 2 receptor, 5 H T3

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Fig. 3. A–G. NECs and polymorhous granular cells (PGCs) in the lungs of the bichirs Polypterus bichir bichir and P. senegalus, and in the respiratory bladder of Lepisosteus oculatus. A) NECs labelled with TH (green) and nNOS (red) antibodies are found in the mucociliated epithelium (MC). Note the slender apical cell processes (arrow) facing the airway lumen (AL). A and B are reproduced from Zaccone et al. (2008) with permission. C–D) PGCs in the mucociliated epithelium of P. senegalus labelled by antibodies to calbindin (red) and 5-HT (green). E) Merged image. F) A NEC (arrow) labelled with calbindin and 5-HT antibodies. G) Double immunostaining revealed that calbindin and 5-HT are colocalized in a NEC in the mucociliated epithelium of the respiratory bladder of L. oculatus.C–F are reproduced from Zaccone et al., 2017 with permission. G is reproduced from Jonz et al., 2016 with permission. Scale bars, 20 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). the presence of tetrapod genes such as Fgf10, tbx4, tbx5 already present cutaneous blood vessels. Compared to other gas exchanging surfaces, in the developing lung of the bichirs (Tatsumi et al., 2016). the skin of fishes is relatively thick and not particularly ventilated and The mucociliated epithelium covering the apical surface of the perfused. Cutaneous air breathing has been studied in various species of thickest septa in the respiratory bladder of the spotted gar, Lepisosteus air-breathing fishes, but it likely occurs in all the amphibious species. oculatus (Icardo et al., 2015) harbours NECs that are seen sparse or in Both diffusion distances and skin densities affect cutaneous clusters. They are colabelled with 5 H T and calbindin DK28 (Fig. 3g), respiration (Graham, 1997). In mudskippers, a large number of capil- and are also immunopositive to VIP (Zaccone et al., 2012). laries and vessels near the surface of the epidermis were reported (Zhang et al., 2000). Epidermal vessels are known to occur in rivulus (Kryptolebias marmoratus) and mudskippers, and epithelial thickness 8. The skin of air-breathing fishes was found to be modified in some skin regions to favor cutaneous re- spiration. Estimation of morphological diffusing capacity for gas ex- The fish skin is comprised by both the epidermis and dermis. The changing was reported in various regions of the body of some muds- epidermis consists of metabolically active epithelial cell layers, various kipper species (Park, 2002; Park et al., 2003). In air-breathing fishes, type of secretory (mucous and serous) cells, sensory cells and ionocytes including the mudskippers, the middle layer of the epidermis contained (see for review (Zaccone et al., 2001). The role of fish skin as gas ex- several kinds of epidermal gland cells (the swollen cells) that function changer is auxiliary. Both the epidermis and dermis are the location of

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Fig. 4. A–M. Confocal images of the gills and skin of Periophthalmus schlosseri.A–C) Double immunostaining with antibodies directed against VAChT (red) and 5-HT (green) reveal that 5-HT(green) is found in some NECs(serotonergic) alone and other cells also contain VAChT (red) in the distal halves of the gill filament and the tip of gill lamellae. D–F) Labelling of the superficial and deep filament chain neurons (ChN) with antibodies directed against VAChT and 5-HT. Arrows in E indicate 5-HT intrinsic nerve bundles in branchial muscle, probably originating from serotonergic neurons. G–H) NECs in the distal filament regions labelled with antibodies to TH (green) and VAChT (red). Note the location of NECs with nearby nerve bundle. I) Merged image. j–m) NECs in the skin (branchiostegal membrane) labelled with antibodies to 5-HT (green) and nNOS (red) are scattered throughout the epidermal layers. Some cells (arrows in K, M) are exposed to the exterior surface. SC, swollen cells. Scale bars, 20 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). in oxygen uptake (Whitear, 1986; Park, 2002). a separate population of cells from the serotonin and SV2 positive NECs. To our knowledge, NECs were never reported in the skin of adult The authors concluded that the emersion of K. marmoratus is regulated teleosts except for those in the eye, tail and yolk sac in the zebrafish by an oxygen sensing mechanism and could be considered a reflex. A larvae (Porteus et al.,2015). NECs in the yolk sac were considered to recent study carried out by Zaccone et al. (2017) used confocal mi- function as gas exchangers in zebrafish larvae (Pelster and Bagatto, croscopy and double labelling procedures with antibodies to 5 H T, 2010; Porteus et al., 2015; Jonz et al., 2016). The presence of NECs was nNOS, TH and VAChT. These procedures have shown that NECs were not ascertained in the of the air-breathing fishes except in rivulus scattered throughout the skin epithelium and were classified of both (K. marmoratus) and the giant mudskipper (Periophthalmus schlosseri) closed and open type (Fig. 4j–m). The closed type of cells are located in (Regan et al., 2011; Zaccone et al., 2017). the basal, suprabasal and middle layers of the epidermis and do not Regan et al. (2011) described two separate NEC populations in the contact the epidermal surface. The open type of cells show a slender skin of Kryptolebias marmoratus. Immunoreactivity for serotonin was process reaching the outermost layer of the skin epithelium. NECs found in NECs distributed across the entire cutaneous surface. Ser- containing 5 H T colabelled with TH, nNOS, but never with VAChT. otonin immunopositive cells are also labelled by SV2 antibodies and Using double labelling with antibodies to 5 H T and nNOS, colocaliza- located amongst a network of zn-12-immunpositive nerve fibers. Cells tion showed that, at least, three NEC subpopulations occurred: a ser- labelled with antibodies to VAChT were also located in the skin and are otonergic and a nitrergic (nNOS positive) subpopulations with another

8 G. Zaccone et al. Acta Histochemica xxx (xxxx) xxx–xxx subpopulation expressing both 5 H T and nNOS. These cells are in- suprabranchial chamber forms above the gills and next to the cranium, timately associated with nerve fibers immunolabelled with antibodies and gill fans form at the top of each . The walls of the to 5 H T and TH. These results suggest that this source of innervation is posterior suprabranchial chamber and the air sacs are ensheathed by either intrinsic to the gill, or extrinsic to it and located within the gill the cucullaris muscle that functions in ABO ventilation. The cucullaris filament (Bailly, 2009; Jonz and Nurse, 2009).This is in agreement with muscle is innervated by a lateral branch of the occipito-spinal nerve the experiments designed to characterize this source of innervation originating from the posterior region of the auditory capsule, and en- revealing that the NECs of the gill filaments received innervation both circles the entire respiratory sac (Munshi et al., 1986). The respiratory from nerve fibers with corresponding nerve cell bodies that are extrinsic epithelium of the air sac is characterized by the alternating presence of to the gill, as well as from nerve fibers with cell bodies that are intrinsic highly vascularized respiratory islets and lanes of flattened cells. The and located within the gill filaments (Jonz and Nurse,2009). With TH- respiratory islets originate from the gill lamellae and are smaller than nNOS immunolabeling, a basket of TH immunopositive nerve fibers those in the gills (Graham, 1997). The islets are essentially blood were identified surrounding both the in the outermost sur- channels that are maintained opened by the pilaster cells. Mucus se- face of the epidermis and the swollen cells in the deeper epidermal creting cells are freely distributed across the ARO respiratory surface. layers. These findings suggest that the capillary networks may be There are striking similarities in circulatory specialization and AROs regulated by alpha-adrenoreceptors (Cooper et al., 2012) and speak in anatomy. in Clarias and Heteropneustes. These catfish appeared in a favor of a motor control exerted by the sympathetic nervous system in common silurid ancestor and were later modified into an everted ar- the evolutionary step of air-breathing (Taylor et al., 1999). This is also borescent organ or an inverted air sac, respectively (Olson et al., 1995) consistent with the capability and utility of cutaneous O2 uptake and Nerve studies on the air-sac of H. fossilis were previously reported by release that likely occurs in all amphibious fishes, and anticipates the Zaccone et al., (2002). Nerve supply of the air-sac is paralleled with that evolution of the mechanism for acid-base regulation and elimination of of the gill since the afferent branchial artery that runs into the main branchial functions. ridge of the air-sac produces a series of lateral blood vessels that supply blood to the lamellae of the respiratory epithelium, that is collected by the main efferent sac vessels. Innervation patterns have been identified 9. The accessory respiratory organs (ARO’s) of the branchial by NADPH-d enzyme histochemistry and immunohistochemistry by the region use of antibodies to neuropeptide met-enkephalin, nNOS, VIP and tyr- osine-hydroxylase. 9.1. Catfishes Antibodies to met-enkephalin label nerves in the walls of the main efferent vessels of the respiratory air-sac, efferent lateral islet vessels Fishes in the families Clariidae and Heteropneustidae include those and islet capillaries. A dense plexus of nitregic (nNOS positive) nerves is with air-breathing organs derived from modified gills and branchial located in the submucosal layer, in the walls of the afferent and efferent chambers. The anatomical description of the ARO in Clarias gariepinus is arteries of the islets and in the cucullaris muscle. Numerous mucous made by Maina (this issue). Briefly, in Clariidae the ARO’s are su- cells display nNOS immunoreactivity, and a fine nerve plexus of nNOS prabranchial organs having a complex structural organization. They are positive nerve fibers is seen in close association with these cells. VIP comprised of a dendritic organ, a pair of highly vascularized suprab- antisera label nerve fibers in the connective , and a perivascular ranchial chambers within which the respiratory trees are contained and nerve plexuses is observed in the vascular walls of afferent and efferent fan-like structures guarding the entrance of the supra branchial cham- lateral vessels of the islets. VIP immunopositive nerve fibers are noticed bers that help to take air. The suprabranchial organs, like the gills, are in the musculature of the air-sac. Adrenergic innervation is very sparse. lined by thin outer epithelial layers with intercellular spaces separated It appears associated with the main efferent vessels but not with the by pilaster cells. The organs and the suprabranchial chambers are musculature of the air-sac. VAChT positive nerve varicosities are dis- supplied by afferent and efferent blood vessels from the gill arches tributed throughout the muscle. Unlike in the gills (see above), 5-HT (Munshi, 1961; Maina and Maloiy, 1986). positive NECs were found very rarely in the respiratory epithelium, The Heteropneustidae family contains only two species: appearing in clusters of 2–3 cells (Fig.5A–C). These cells, acting as Heteropneustes fossilis and H. microps (Graham, 1997). In H. fossilis,a potential external chemoreceptors, were regarded as externally-or- pair of tubular pneumatic air sacs (one on each side of the body) act as iented chemoreceptors to differentiate them from the internally-or- ARO. These long tubular sacs arise as outgrowths from the branchial iented chemoreceptors responding to hypoxia (Smatresk, 1986). VAChT chamber and extend almost down to the tail between the body mus- antibodies labelled numerous mucous cells sparsely distributed across culature, near the vertebral column. As in Clarias, a paired

Fig. 5. A–C. Confocal images of the air sac of the catfish Heteropneustes fossilis. A) Cholinergic nerves scattered throughout the cucullaris muscle of the air sac are labelled with the antibody to VaChT. B) Mucous cells expressing VAChT are innervated by VAChT-positive nerve fibers (arrow). C) NECs and mucous cells in the respiratory epithelium are labelled with antibodies to 5-HT (green) and VAChT (red). Note the location of 5-HT positive NECs (green). Scale bars, 20 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

9 G. Zaccone et al. Acta Histochemica xxx (xxxx) xxx–xxx the ABO surface. A fine perivascular plexus of fine VAChT positive air-breathing species, as well as in lungfishes, there is no evidence for nerve fibers was also found in close association with these cells the presence of CO2 /H + receptors. In the African lungfish Protopterus, (Fig. 5b). Goblet cell secretion is stimulated by parasympathetic and peripheral O2 and CO2-sensitive chemoreceptors are likely to occur on sympathetic nerves (Rogers, 2001; Dartt et al., 2006). Parasympathetic the gill arches. Slowly adapting pulmonary stretch receptors in the lung nerves release the neurotransmitters acetylcholine and VIP, that sti- of Protopterus and the South American lungfish Lepidosiren are inhibited mulate mucous secretion. Acetylcholine interacts with muscarinic re- by CO2 (Delaney et al., 1983). This is also reported in the spotted gar ceptors located in the mucous cell membrane. VAChT or NO released Lepisosteus oculatus (Smatresk and Azizi, 1987). O2 and CO2 sensitive from mucous cells in the air sac could be a neutrally mediated pathway chemoreceptors are thought to occur on the gill arches in this fish functioning with parasympathetic transmitters (VAChT and NO), or a (Porteus et al., 2014; Hedrick and Katz, 2016). paracrine or autocrine pathway in the ARO of Heteropneustes under A fundamental problem remains.It is that a functional conflict be- hypoxic conditions. However, the role of VAChT in the control of mucus tween a gas exchanger and a buoyancy(hydrostatic) organ in all the secretion and as sensors of hypoxic stress is unknown. A dense plexus of primitive fishes (Hedrick and Katz,2016). cholinergic nerve fibers is located in the musculature of the air sac but it Fishes and amphibians utilize a suction/force pump to ventilate gills never appears associated with the vasculature. or lungs with respiratory muscles innervated by cranial nerves. Fishes Recent studies by Zaccone and Maina (unpublished observations) can recruit a hypobranchial pump for active occlusion during hy- reported the presence of NECs in the gill fans and in the suprabranchial poxia, using feeding muscles innervated by anterior spinal nerves chamber, in areas of the filament-lamella structures, of the African (Taylor et al., 1999). Spinal innervation encircling the respiratory air catfish, Clarias gariepinus. The TH-nNOS immunopositive NECs were sac of the catfish H. fossilis has also been reported by Munshi et al. found touching the external medium, but others are seen at the base of (1986). the epithelium. Double immunolabeling with antibodies to VAChT and 5-HT revealed a population of 5-HT positive NECs in close association with VAChT positive nerve bundles (Fig. 6a–d, g). NECs are absent in 11. Conclusions and unanswered questions the respiratory membrane covering the dendritic organ. The respiratory surfaces of the dendritic organ have a large surface area and contain Much research remains to be accomplished regarding the cellular pillar cells that are the structural entities of the vascularized membrane. identification, distribution and neurophysiological characterization of A dense VAChT innervation is seen running along the basement mem- the peripheral chemoreceptors and their broader spectra of neuro- brane that surrounds the vascular channels. They also bifurcate near the transmitters that enabled to identify multiple populations of NECs. TH epithelial surface (Fig. 6e). VAChT perivascular nerve fibers are also is now added as a neuronal marker for NECs in the gill and skin of the found around the afferent and efferent arteries in the underlying con- mudskipper, P. schlosseri, including their sympathetic innervation nective tissue. Numerous VaChT positive neurons showed a sub- (Zaccone et al., 2017). The role of afferent and efferent complex in- epithelial localization in gill fans and their occurrence in the exterior nervation and hypoxia sensitivity of the NECs, and the interaction of the part of the cartilaginous core in the dendritic organ (Fig. 6a, f). Given chemo- and mechanoreceptors in the regulation of both aquatic and the available evidence in air-breathing fish (Regan et al., 2011), it ap- aerial gas exchange in air-breathing fishes, is still incomplete. NECs are pears that Ach may be important in mediating the hyperventilation distributed throughout the gill filaments and lamellae of all the gill response to hypoxic stimuli. VAChT extrabranchial innervation of the arches in several teleost species, the air-breathing organs of primitive gill fans and dendritic organ may be correlated with the modulation of fishes and the gill fans and the suprabranchial chamber in the AROs of response to hypoxia. The importance of gill fans in aerial respiration the air-breathing catfish. We would like also to include in this scenario derives from their well-developed gas-exchanging surface, ventilation the role of cutaneous chemoreceptors. We also think that the NECs of and air retention (Graham, 1997). the skin of air-breathing fishes await investigation based on the current knowledge regarding the origin of the fish gill NECs and the 10. Receptor responses in bimodal breathers pulmonary neuroendocrine cells (PNECs), both differentiating in situ within airway epithelia (Hockmann et al., 2017). The data collected from the immunohistochemical analysis of the Finally, many neurotransmitter substances (serotonin, neuropep- air-sac in H. fossilis may suggest a main inhibitory regulatory role of the tides, acetylcholine, nitric oxide, catecholamines) were found in the vasculature by a NANC component of innervation possibly arising from NECs of teleost and air-breathing fishes (Zaccone et al., 1997, 2002, nearby cell bodies. In addition, baroreceptors have been shown to be 2006, 2017; Jonz and Zaccone, 2009; Porteus et al., 2015; Jonz et al., free nerve endings and such as the extensive nitrergic perivascular 2016). The neurotransmitters could act as transducing substances that nerves terminating to the efferent air-sac vessels, as reported by are released after proper stimuli and produce a specific post-synaptic Funakoshi et al. (1999) for the efferent filament arteries of the gill. The effect. The cells have to convey stimuli to the sensory nerve endings existence of mechanoreceptor fibers should also be recognized in vagi they are associated with, and the associated nerves must carry the ap- providing a cholinergic constrictor innervation to the cucullaris muscle propriate membrane receptors. In the NECs of fish, the picture is still far as evidenced by a plexus of VAChT positive nerve fibers terminating in from complete. It is to be expected that future research will focus on the the musculature. Graham (1997) reported a functional role of the cu- knowledge of membrane receptors tuned to the different neuro- cullaris muscle in the regulation of ventilation and air breaths (in- transmitter types for a better understanding of the stimulatory and in- spiration and expiration) that are served by a single notch between fans hibitory circuits active in the chemosensory cell-nerve ending unit. 2 and 3. The respiratory muscles in fish contain length and tension The basic aspects of O2 chemoreception in the gills and air- receptors in common with other vertebrate muscles. The gill arches breathing organs of fish may involve multiple populations of NECs, bear a number of mechanoreceptors with various functional char- multiple neurotransmitters and neuropeptides.The extrinsic(sympa- acteristics. However, very little is known on the respiratory function thetic) innervation of the NECs is not well appreciated.Synaptic con- and innervation of the cucullaris muscle including receptor localization. tacts between the NECs and nerve endings displayed the features of In bimodal breathers, the available evidence supports the existence afferent synapses (Bailly, 2009).This suggests that oxygen chemor- of both water-sensing CO2/pH chemoreceptors on the gills and CO2- eception may depend on a centrally mediated reflex triggered by hy- sensitive pulmonary stretch receptors (Gilmour and Milsom, 2009). poxia and mediated by NECs through the afferent-like synapses which These receptors are located within the lung of various vertebrate spe- send information to the central nervous system such as the medulla that cies, are innervated by the vagus nerve and have evolved after mam- integrates oxygen-receptor information (Sundin et al., 2003; Bailly, malian split from the reptilian stock (Gilmour and Milsom, 2009). In 2009).

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Fig. 6. A–G. Confocal images of the accessory respiratory organ (ARO) of the catfish Clarias gariepinus.A–C) NECs (arrows) labelled with 5-HT (green) antibody are found in the epithelium of the gill fans in close proximity (B) to VAChT-positive nerve bundles (red) and mucous cells (mc). VAChT-positive (red) intrinsic neurons (n) are found in the connective tissue layers (A). Cholinergic nerves also run around a large blood vessel. D) Double immunostaining for TH (green) and nNOS (magenta) showing NECs in the gill fans epithelium (arrows) in close contact with mucous cells (mc). E–F) Double immunostaining for 5-HT (green) and VAChT (red) showing VAChT-positive nerves running along the basement membrane of blood channels, in the respiratory membrane of the labyrinthine organ. VAChT (red) positive neurons are seen in subepithelial localization. G) Double immunolabelling for TH (green) and nNOS (red) reveals the localization of NECs in the suprab- ranchial chamber epithelium in close proximity to mucous cells (mc). Peripheral nerves labelled with TH (green) antibody are seen in the subepithelium. Scale bars, 20 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

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