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Nervous control of the gills
ARTICLE IN PRESS Acta histochemica 111 (2009) 207—216
www.elsevier.de/acthis
Nervous control of the gills Michael G. Jonza,, Giacomo Zacconeb a
Department of Biology, University of Ottawa, 30 Marie Curie, P.O. Box 450, Station A, Ottawa, ON, Canada K1N 6N5 Department of Animal Biology and Marine Ecology, Section of Comparative Neurobiology and Biomonitoring, University of Messina, Via Salita Sperone 31, I-98166 Messina, Italy
b
KEYWORDS Review; Fish; Gill; Autonomic; Innervation; Sympathetic; Parasympathetic
Summary The fish gill is a highly complex organ that performs a wide variety of physiological processes and receives extensive nervous innervation from both afferent (sensory) and efferent (motor) fibres. Innervation from the latter source includes autonomic nerve fibres of spinal (sympathetic) and cranial (parasympathetic) origin whose primary role is to induce vasomotor changes within the respiratory or nonrespiratory pathways of the gill vasculature. Autonomic control of the gill occurs by nerve fibres identified as adrenergic, cholinergic, and more recent evidence indicates that nonadrenergic–noncholinergic (NANC) nerve fibres, such as those that express amines, peptides, or nitric oxide, may also play an important role. The distribution and physiological function of NANC nerve fibres, however, is less clear. This review primarily discusses histochemical studies that have characterized the nervous innervation and autonomic control of the gill vasculature. In addition, supporting evidence from recent studies for the efferent control, or modulation, of other homeostatic processes in the gill is examined. & 2008 Elsevier GmbH. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General organization of the gills and associated vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . Autonomic innervation of the gill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cranial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spinal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catecholaminergic nerves and vascular control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cholinergic and aminergic nerves and vascular control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitrergic and peptidergic nerves and vascular control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other putative roles for efferent innervation in the gill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Innervation of the respiratory lamellae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corresponding author. Tel.: +1 613 562 5800x6051; fax: +1 613 562 5486.
E-mail address: [email protected] (M.G. Jonz). 0065-1281/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2008.11.003
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M.G. Jonz, G. Zaccone Efferent innervation of neuroepithelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Putative innervation of mitochondria-rich cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction The fish gill is a sophisticated organ that performs an impressive variety of homeostatic functions. These include gas exchange, ion regulation, acid–base balance and nitrogen excretion (Evans et al., 2005). The gill is also a highly perfused organ that receives the entire cardiac output, and is a major site of circulatory or vasomotor control (Sundin and Nilsson, 2002; Olson, 2002a, b). As a corollary to this vast array of processes performed by the gill, it is not surprising that the gill receives extensive innervation from the nervous system (Figure 1) in the form of afferent (sensory) and efferent (motor) pathways, and this has been reviewed previously by several authors (Burnstock, 1969; Campbell, 1970; Nilsson, 1983, 1984; Taylor et al., 1999; Sundin and Nilsson, 2002). Indeed, the control of the gill vasculature by neural (and humoral) mechanisms is critically important for the regulation and optimization of such processes as respiration and osmoregulation. It is, therefore, of interest to understand the source and nature of gill innervation in order to appreciate how the gill is controlled by the nervous system. Characterization of gill innervation may additionally provide clues regarding novel regulatory pathways. A component of efferent innervation to the gill is that of the autonomic nervous system (Figure 2A). In all vertebrates, the autonomic nervous system is composed of all efferent pathways having ganglionic synapses outside of the central nervous system (CNS), and autonomic nerves reach nearly every part of the body, thereby controlling important homeostatic mechanisms (Nilsson, 1983). In the fish gill, autonomic innervation primarily includes the nerve supply to smooth muscle of the vasculature, and thus provides a means of vasomotor control of the gill (Nilsson, 1984; Sundin and Nilsson, 2002). As pointed out by Nilsson (1983), the early view of the autonomic nervous system in vertebrates was that the sympathetic division was mediated by adrenergic nerves, whilst the parasympathetic system was mediated by cholinergic nerves. Thus adrenaline and acetylcholine, respectively, have been considered major neurotransmitters. However, it is now clear that the autonomic system is complex and is composed of a variety of
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nerve types and putative neurotransmitters. As will be discussed, much attention has recently been focused on a wide variety of nonadrenergic–noncholinergic (NANC) substances, such as amines (e.g. serotonin, dopamine), peptides, and a small gaseous molecule, nitric oxide (NO). Many autonomic nerves of teleost, elasmobranch, and dipnoan fishes contain neuropeptides, but little is known about their pattern of colocalization with other neurotransmitters. A goal of this paper is to provide an introduction to the fish gill, its innervation, and to briefly review the autonomic control of the branchial vasculature by nerves of both cranial and spinal origin. Studies using traditional histochemical and ultrastructural procedures, as well as those that have utilized immunofluorescence and confocal techniques, will be discussed. In the latter portion of the paper, we will also discuss innervation patterns observed within the gill that are not well characterized, and propose potential roles for this innervation. As a note, the designation of cranial vs. spinal autonomic innervation of the branchial region is used throughout this review. This classification has been previously employed (e.g. Campbell, 1970; Nilsson, 1983, 1984; Sundin and Nilsson, 2002), and is more appropriate for comparative studies of autonomic innervation than Langley’s mammalian designation of ‘‘parasympathetic’’ vs. ‘‘sympathetic’’ (see Nilsson, 1983). Other important aspects of gill innervation that will not be discussed are covered elsewhere in this special issue, such as the nervous control of respiratory reflexes (Schwerte, 2008; Taylor et al., 2008) and the gill as a sensory organ (Burleson, 2008).
General organization of the gills and associated vasculature In teleost fish, four bilateral pairs of gill arches are present in the pharyngeal region and each gives rise to two parallel rows of gill filaments, called hemibranchs. Gill filaments are further divided into a series of secondary (respiratory) lamellae (Figure 1). Both the filaments and lamellae are covered by a thin epithelial layer that contains a wide variety of cell types that mediate the many physiological and homeostatic processes that the gill performs (Wilson and Laurent, 2002; Evans et al., 2005). As illustrated
ARTICLE IN PRESS Nervous control of the gills
Figure 1. Illustration of the extensive innervation to the teleost gill using confocal immunofluorescence methods. A large nerve bundle courses through the centre of the gill filament (F) and gives rise to a nerve plexus and fibres of the respiratory lamellae (L). In this example, a neuronspecific antibody (zn-12) was used to label all nervous structures of the gill in zebrafish. Bar, 50 mm. Modified from Jonz and Nurse (2003).
in Figure 2A, the gill vasculature is complex and contains two major circulatory networks: the respiratory (arterioarterial) and nonrespiratory (arteriovenous) pathways (Olson, 2002a). Blood flow through the gills begins with deoxygenated blood travelling from the heart through the ventral aorta. From here, blood enters the gill arches via the afferent branchial artery, and the gill filaments via the afferent filament arteries. Blood flow continues distally along the filaments until it enters the afferent lamellar arterioles and travels through the vascular sinus of the lamellae, where gas exchange occurs. In the respiratory pathway, blood flows to and from the gill lamellae and continues into the systemic circulation via the efferent lamellar arterioles, efferent filament arteries, the efferent branchial artery, and finally the dorsal aorta. In the nonrespiratory pathway, blood flows from the respiratory pathway into small vessels, such as the nutritive vasculature and arteriovenous anastomoses, which provide perfusion and drainage of gill tissue.
Autonomic innervation of the gill Cranial There are 11 pairs of cranial nerves found in fish. However, only the facial (VII), glossopharyngeal (IX)
209 and vagus (X) nerves provide cranial innervation to the gill region, and are, therefore, called the branchial nerves (Nilsson, 1984; Sundin and Nilsson, 2002). In jawed fishes (gnathostomes), such as teleosts, the gill arches are innervated primarily by the glossopharyngeal and vagus nerves, which form large nerve trunks that enter the gills from the dorsal aspect (Figure 2B). The second, third and fourth gill arches are innervated entirely by branches of the vagus nerve, whilst the first gill arch receives both glossopharyngeal and vagal innervation. Once within the branchial region, the nerves are further divided into pretrematic and posttrematic rami (Figure 2B) that straddle the gill slits rostrally and caudally, respectively (Nilsson, 1984; Sundin and Nilsson, 2002). Thus, each gill arch is innervated by a posttrematic and pretrematic ramus from two different cranial nerve branches. The pretrematic ramus carries sensory nerve fibres, and the posttrematic ramus carries both sensory and motor fibres (Nilsson, 1984). It is noteworthy that a reduced gill-like structure, called the pseudobranch, is innervated primarily by the pretrematic ramus of the glossopharyngeal nerve and may receive additional innervation from the posttrematic ramus of the facial nerve (Laurent and Dunel-Erb, 1984; Nilsson, 1984). Glossopharyngeal innervation to the pseudobranch, however, is believed to be primarily sensory. In teleosts, cranial autonomic outflow to the gills is restricted to the vagus nerve (Figure 2A) and mediates vasomotor control of the gill vasculature (Nilsson, 1983; Taylor et al., 1999). The need for cranial autonomic vasomotor fibres in the head, as observed in elasmobranchs and some cyclostomes (e.g. lampreys), was apparently reduced in teleosts, which instead possess an increased supply of cephalic spinal autonomic fibres (Nilsson, 1983).
Spinal Spinal autonomic innervation to the gills in teleosts occurs by way of the sympathetic chain ganglia (Figure 2A and B). The sympathetic chain is well developed in teleosts, as it is in tetrapods, and extends into the cephalic region, where spinal nerve fibres leave the sympathetic chain through grey rami communicantes and enter branches of the cranial nerves. In fact, the vagus nerve is often referred to as the ‘‘vagosympathetic trunk’’ in teleosts because of the rich contribution of spinal autonomic fibres, which continue into the gills and exert vasomotor effects on the gill vasculature (Nilsson, 1983; Taylor et al., 1999). In cyclostomes and elasmobranchs, a sympathetic chain per se is
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M.G. Jonz, G. Zaccone
Figure 2. (A) Current working model for innervation of the teleost gill vasculature by cranial and spinal autonomic nerves, as evident from histochemical studies, indicating potential sites of vasomotor control. The cranial (‘‘parasympathetic’’) pathway includes nerve fibres travelling through the vagus nerve and postganglionic neurons (indicated), while the spinal (‘‘sympathetic’’) pathway travels through the sympathetic chain ganglia and the vagosympathetic trunk. Adrenergic, cholinergic and serotonergic nerve fibres are indicated. A muscular ‘‘sphincter’’ is shown at the proximal region of the efferent filament artery. Black arrows indicate the flow of deoxygenated blood and grey arrows indicate oxygenated (postlamellar) blood carried through the respiratory and nonrespiratory vascular pathways. 5-HT, serotonergic nerve; ACh, cholinergic nerve; Adr, adrenergic nerve; AFA, afferent filament artery; ALa, afferent lamellar artery; AVA, arteriovenous anastomoses; BN, branchial nerve; CNS, central nervous system; CVS, central venous sinus; EFA, efferent filament artery; ELa, efferent lamellar artery; NV, nutritive vasculature; SC, sympathetic chain; SL, secondary (respiratory) lamellae; Sph, sphincter; X, vagus nerve. Modified from Nilsson and Sundin (1998). (B) Simplified scheme of innervation to the gill region in fish. The gills are innervated by nerve fibres (both sensory and motor) originating from the cranial nerves, and by fibres that originate from the sympathetic chain and enter the cranial nerve trunk via grey rami communicantes. Each gill arch is innervated by a pretrematic ramus (anterior to the gill slit) and a posttrematic ramus (posterior to the gill slit) originating from different cranial nerve branches. Pretrematic rami carry sensory fibres, while posttrematic rami carry sensory and motor fibres. Based partly on Nilsson (1984).
not observed. However, in the latter an irregular series of paravertebral ganglia is present but these do not extend into the head (Burnstock, 1969; Campbell, 1970; Nilsson, 1983; Taylor et al., 1999). There is no evidence for branchial vasomotor control by spinal innervation in either cyclostomes or elasmobranchs (Taylor et al., 1999).
Catecholaminergic nerves and vascular control Control of the branchial vasculature by catecholaminergic (adrenergic) nerves of spinal autonomic origin is found primarily in the teleosts (Figure 2A). With the notable exception of the brown trout (Salmo trutta) and carp (Cyprinus carpio), the afferent and efferent branchial arteries of the gill arches in most species generally do not
receive innervation from catecholaminergic nerves (Donald, 1984, 1987). Thus, the control of blood flow through the gills is controlled primarily via innervation of the respiratory and nonrespiratory pathways of the gill filaments. The afferent filament arteries and afferent lamellar arterioles, which deliver deoxygenated blood to the lamellae for gas exchange, are innervated by catecholamine-containing nerves (Donald, 1984, 1987). In addition, the efferent lamellar arterioles and the proximal region of the efferent filament arteries receive similar innervation (Donald, 1984, 1987; Dunel-Erb and Bailly, 1986). In the latter case, sympathetic innervation is concentrated at a contractile segment of the filament artery, called a sphincter, which is believed to play a role in the regulation of blood flow. The vessels regarded as a part of the nutritive vasculature of the nonrespiratory pathway are supplied by
ARTICLE IN PRESS Nervous control of the gills catecholamine-containing nerves (Donald, 1984, 1987; Dunel-Erb et al., 1989), and it has been proposed that the arteriovenous anastomoses also receive innervation (Vogel et al., 1974). These nerves are also found around the central venous sinus in the core of the gill filament (Dunel-Erb and Bailly, 1986; Dunel-Erb et al., 1989). The major effect of adrenergic vasomotor innervation of the gill vessels is due to an a-adrenoceptor-mediated vasoconstriction of the nonrespiratory pathway, thus favouring an arterioarterial flow (Sundin and Nilsson, 2002). The respiratory pathway responds mainly through b-dilatory mechanisms, primarily at the afferent lamellar arterioles, leading to perfusion of additional lamellae (Nilsson and Sundin, 1998; Evans et al., 2005).
Cholinergic and aminergic nerves and vascular control Cranial autonomic (vagal) nerves carry vasomotor fibres to the gills of teleosts and have been identified as cholinergic (Figure 2A). Ultrastructural studies have revealed the presence of cholinergic-type nerve profiles innervating the sphincter at the base of the efferent filament arteries that originate from postganglionic axons, and histochemical studies have shown high levels of acetylcholinesterase, an enzyme that hydrolyzes acetylcholine, in this region (Bailly and Dunel-Erb, 1986; Mauceri et al., 2005). Sundin and Nilsson (1997) described a general constriction of the efferent vasculature, including both the filament arteries and arterioles, by the use of acetylcholine. This resulted in a marked reduction of blood flow in the distal portion of the filaments. It was concluded in this study that this vasomotor control occurred via a cholinergic innervation of the sphincter. Pettersson and Nilsson (1979) noted a vagal NANC component of the vasoconstrictory nerve supply of the gill filament of the Atlantic cod (Gadus morhua), since neither adrenergic nor cholinergic blocking agents completely suppressed the nerveinduced vasoconstriction of the efferent arterial system. Serotonergic nerves of vagal origin also occur in the efferent vasculature of the gills of teleosts (Figure 2A) (Dunel-Erb et al., 1982, 1989; Bailly et al., 1989). In particular, indolaminergic neurons innervating the proximal part of the efferent arterial vasculature, the filament epithelium, and the central venous sinus, are found. In fish species studied by Sundin et al. (1998a, b), the serotonergic fibres reach the proximal region of the efferent filament arteries (including the sphincter)
211 and efferent lamellar arterioles, and extend in the direction of the central venous system. In Pagothenia borchgrevinki, serotonergic innervation was also observed on the afferent side of the filament. In zebrafish (Danio rerio), a series of serotonergic neurons of the filament epithelium was found in whole-mount gill preparations (Jonz and Nurse, 2003). Some of these innervated both O2-chemosensory neuroepithelial cells (NECs) and the sphincter of the efferent filament artery, while the termination of other serotonergic neurons was not determined. The cell bodies of serotonergic neurons are intrinsic to the gills (i.e. postganglionic), thus indicating that these are consistent with the general pattern of cranial autonomic innervation (Sundin and Nilsson, 2002). The main nervous control by serotonin on the gill vasculature occurs through constriction of the respiratory pathway in the distal portion of the filaments, thus leading to a redistribution of blood flow to more proximal regions of the filament (Sundin et al., 1995). This is consistent with observations that serotonin increases branchial vascular resistance in perfused preparations and in vivo (Sundin and Nilsson, 2002). Moreover, the vasomotor effects of serotonin in the fish gill appears to be due to at least two different serotonin receptor subtypes, since the receptor antagonists which abolished the serotonin-induced vasoconstriction of gill vessels (Sundin et al., 1995) had no effect on the arteriovenous vasodilatory effect of serotonin (Sundin, 1995).
Nitrergic and peptidergic nerves and vascular control NANC candidates in the fish gill are also represented by the so-called ‘‘gasotransmitters’’, e.g. nitric oxide (NO; see Olson and Donald, 2008 this issue) and neuropeptides (Zaccone et al., 2006). Zaccone et al. (2003, 2006) reported the presence of a rich supply of nerve fibres that were immunopositive for neuronal nitric oxide synthase (nNOS), an isoform of the enzyme involved in the production of NO, and these fibres were associated with the efferent filament arteries, the pillar cell system of the lamellae, and efferent lamellar arterioles in the gills of catfish (Heteropneustes fossilis). In the air sac of the same species, which originates from the fusion of gill filaments and lamellae, the vasculature of the main efferent vessels is innervated by nNOS-immunopositive nerve fibres. In addition, Gibbins et al. (1995) showed that postganglionic neurons positive for the NADPH-diaphorase reaction (an indicator of NOS activity) were present in the posttrematic branches
ARTICLE IN PRESS 212 of the branchial nerves in G. morhua, suggesting that these may be part of the cranial autonomic pathway. Studies by Funakoshi et al. (1997, 1999) provided considerable information about the nitrergic (nNOS-immunopositive) innervation of the fish gill. They examined the presence of nNOS and NADPH-diaphorase activity in the sensory system of the glossopharyngeal and vagus nerves of a pufferfish (Takifugu niphobles). In the vagal ganglion, positive neurons were found in subpopulations of the branchial rami. Nitrergic afferent terminals were found in the glossopharyngeal lobe, vagal lobe and commissural nucleus. Distribution of NADPHdiaphorase and nNOS-immunoreactive nerve fibres were traced not only along the efferent filament arteries of the gill, but also in the afferent filament arteries, the efferent lamellar arterioles and the central venous sinus. Although NO induces vasodilation in the systemic vessels of fish, there is apparently no clear evidence for the direct effects of NO on the gill vasculature, despite its vasoactive effects and the presence of nNOS in this tissue (Evans et al., 2005). Neuropeptide-containing nerve fibres have been found in the vessel walls of a range of nonmammalian vertebrate species (Holmgren, 1995). Neuropeptides have been described in vasomotor nerves in teleost fish, while evidence is lacking in cyclostomes (Morris and Nilsson, 1994). However, there have been few studies dealing with the distribution of neuropeptides in the teleost vasculature, particularly the respiratory pathway of the gill. Nerve fibres immunoreactive for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP7 38 and 27) are distributed along the main part of the filament in the catfish, H. fossilis (Zaccone et al., 2003, 2006). These fibres run to the efferent filament arteries. VIP produced a concentration-dependent vasodilation in perfused gills of the brown trout, Salmo trutta (Bolis et al., 1984). In addition, neuropeptide Y (NPY)-like immunoreactivity was demonstrated in the gill vessels in an elasmobranch, producing constriction of the afferent branchial artery (Bjenning et al., 1993). A dense network of leu- and met-enkephalin immunoreactive nerve fibres have also been traced along the walls of the efferent filament arteries and efferent lamellar arterioles. The origin of the nerve fibres expressing these types of neuropeptides remains unclear. In spite of the presence of NANC substances in the gill perivascular nerves, NANC neurotransmission has not been demonstrated in the gill vessels. In addition, the function of perivascular nerves (either sensory or vasomotor) is not clear.
M.G. Jonz, G. Zaccone
Other putative roles for efferent innervation in the gill Innervation of the respiratory lamellae It is now well established from recent studies that in some teleost species, such as zebrafish, goldfish (Carassius auratus) and catfish, the respiratory lamellae of the gills receive a dense supply of innervation (Jonz and Nurse, 2003; Saltys et al., 2006; Zaccone et al., 2003; Coolidge et al., 2008). As shown in Figure 1, the successful labelling of lamellar nerve fibres of gill lamellae appears to be due to the recent use of a specific neuronal marker derived from zebrafish (zn-12; Trevarrow et al., 1990; Jonz and Nurse, 2003) that identifies an apparently conserved neuronal surface antigen, since previous studies using traditional techniques did not produce any clear evidence of lamellar innervation (Laurent and Dunel, 1980; Donald, 1984, 1987; Nilsson, 1984). It is also evident that the presence of nerve fibres in the lamellae is species-specific, since the above studies also showed they are absent in other teleosts, such as rainbow trout (Oncorhynchus mykiss), trairao (Hoplias lacerdae) and traira (H. malabaricus) (Saltys et al., 2006; Coolidge et al., 2008). Though at present the source of lamellar innervation has not been determined, in zebrafish these nerve fibres appear to emanate from the nerve plexus of the gill filaments, and nerve sectioning experiments have demonstrated that parent cell bodies of lamellar nerve fibres are located extrinsic to the gills (Jonz and Nurse, 2003). In addition, there is preliminary evidence that at least some lamellar nerve fibres contain catecholamines (Arlotta and Jonz, unpublished observations), suggesting a potential spinal origin. Both afferent and efferent roles for lamellar innervation may be considered. Serotonergic NECs of the lamellae, which resemble O2-chemoreceptive NECs of the filaments, are innervated and may relay sensory information regarding arterial or water O2 tension to the CNS via afferent postsynaptic fibres (Jonz and Nurse, 2003; Jonz et al., 2004), thereby playing a potential role in the sensing of hypoxia. Alternatively, this innervation may be efferent and control the activity of NECs (see below) or other cell types. A group of structurally supportive cells found within the respiratory lamellae, called ‘‘pillar cells’’, separates the epithelial layers of the lamellae and produces the vascular sinus through which blood passes during gas exchange (Olson, 2002a). Pillar cells contain actin and myosin filaments that apparently allow these cells to contract and
ARTICLE IN PRESS Nervous control of the gills increase vascular resistance within the gill (Smith and Chamley-Campbell, 1981; Stensløkken et al., 1999; Mistry et al., 2004), and although their innervation has not been confirmed it has been suggested (Gilloteaux, 1969; Jonz and Nurse, 2003). Such contact between efferent nerve fibres and these cells may permit mediation of pillar cell contraction and vascular control within the lamellae, and aid in the vasomotor control of the gill vasculature by autonomic fibres. However, the question arises of how blood flow within the vascular spaces of the lamellae is controlled by pillar cells, if at all, in fish that do not possess lamellar innervation. Endocrine or paracrine mechanisms of pillar cell contraction have also been suggested (Olson, 2002a), and may operate instead in these cases.
Efferent innervation of neuroepithelial cells O2-chemoreceptive NECs of the gill filaments are believed to establish synapses with at least three types of nerve fibres. The first type of nerve ending is catecholaminergic (Bailly et al., 1992), suggesting that NECs are modulated by spinal autonomic nerves. The second type of nerve profile close to NECs likely belongs to nearby serotonin-containing neurons, since they remain intact after symapthectomy, and may be responsible for relaying sensory information to the CNS during hypoxia (Bailly et al., 1992). These two types of nerve profiles observed at the ultrastructural level correspond to the extrinsic and intrinsic innervation patterns, respectively, described in the zebrafish gill (Jonz and Nurse, 2003). A third type of nerve fibre innervating gill NECs is that of nitrergic nerve fibres that may be of cranial origin (Zaccone et al., 2003). It is worth noting that NECs may also receive afferent innervation from an additional extrinsic source, since O2-sensory responses appear functional in zebrafish that lack intrinsic serotonergic neurons of the gill filaments during early larval stages (Jonz and Nurse, 2005). However, despite the afferent innervation of NECs that is prerequisite for a functional O2-chemosensory system in the gill, the contribution of efferent innervation to NEC function has been considered (Bailly et al., 1992; Jonz and Nurse, 2003; Zaccone et al., 2006). Many nerve fibres associated with gill NECs, especially the intrinsic fibres, contain synaptic vesicles (Jonz and Nurse, 2003) and may represent presynaptic nerve endings that potentially secrete neurotransmitters (e.g. serotonin or NO) onto NECs or surrounding tissues. These results may indicate an additional neuroendocrine role for
213 NECs in the gill, where induction of NEC neurosecretion by hypoxia, for example, would lead to compensatory changes in local vascular resistance within the gill. Alternatively, a potential role for efferent nerve fibres in directly modulating the chemosensory responses of NECs to hypoxia is possible. The well-studied type I cell chemoreceptors of the mammalian carotid body are O2sensitive and are considered to be homologues of gill NECs (Jonz and Nurse, 2008). Type I cells receive both afferent and efferent sources of innervation, and a mechanism has been described in which autonomic (probably parasympathetic) nerve fibres mediate efferent inhibition of type I cells within the carotid body during hypoxic stimulation (Campanucci and Nurse, 2007). In this model, glossopharyngeal neurons release NO onto carotid body chemoreceptors, thereby reducing their excitability and attenuating their response to hypoxia. It is, at present, unknown if a homologous mechanism of efferent inhibition is functional within the O2-chemosensory system of the fish gill.
Putative innervation of mitochondria-rich cells The gill is an important site of osmotic and ion balance in fish, and the primary functional unit of these processes in the teleost gill is the mitochondria-rich cell (MRC). These cells are located in the gill filament epithelium and are characterized by a high density of mitochondria, an abundance of the enzyme, Na+/K+-ATPase, and a complement of ion channels, exchangers and pumps (Perry, 1997; Wilson and Laurent, 2002; Evans et al., 2005). Depending on aquatic habitat, MRCs of the fish gill may mediate the uptake or extrusion of Na+, Ca2+ and Cl across the epithelium between the blood and water (Perry, 1997; Marshall, 2002; Evans et al., 2005). The movement of ions across ionregulatory tissues in fish, although not completely understood, appears to be mediated by circulating hormones and neurotransmitters, such as catecholamines, adrenaline, acetylcholine, NO and peptides (Zadunaisky, 1984; McCormick, 2001; Evans, 2002; Marshall, 2003). Furthermore, some evidence has accumulated to suggest that ion transport by gill MRCs may be under control of the nervous system. Early studies demonstrated that sectioning of the branchial nerves depleted gill MRCs and altered ion transport and water permeability (Pequignot and Gas, 1971; Mayer-Gostan and Hirano, 1976), suggestive of MRC innervation. Moreover, stimulation of the branchial nerves and
ARTICLE IN PRESS 214 exogenous application of adrenaline in trout altered Ca2+ flux across the gill epithelium (Payan et al., 1981; Donald, 1989). A more recent study using confocal techniques provided morphological evidence that was suggestive of innervation of MRCs in zebrafish (Jonz and Nurse, 2006). These studies showed that nerve fibres from the filament plexus appeared to contact MRCs within the gill filament. This same nerve plexus was shown in a previous study to be of extrinsic origin following denervation and nerve fibre degeneration procedures (Jonz and Nurse, 2003). Therefore, the above studies may indicate efferent innervation of MRCs and, potentially, autonomic control of ion regulation in the fish gill. Nerve fibres in zebrafish are associated with the basolateral regions of MRCs, where Na+/K+-ATPase activity is localized (Evans et al., 2005), while apical regions are oriented toward the external environment. This arrangement may suggest that presynaptic nerve terminals of efferent fibres release neurochemicals and affect the activity of postsynaptic MRCs, thereby controlling ion regulation across the gill epithelium. The physiological significance of this putative innervation in ion regulation, however, awaits confirmation at the ultrastructural level, functional studies, and perhaps identification in other fish species. Such confirmation may not be surprising, since the neural control of ion homeostasis in the mammalian kidney is well established (DiBona and Kopp, 1997). By contrast, the presence of sensory ionoreceptors in fish has been proposed, in which rapid ion transfer may be elicited in response to changes in plasma ion concentration encountered during salinity change (Evans et al., 2005; Marshall et al., 2005). In this model, MRCs would be presynaptic and receive afferent innervation. Such receptors and sensory innervation of transport epithelia has also been described in the mammalian kidney (DiBona and Kopp, 1997).
Concluding remarks This brief review has outlined important aspects of nervous innervation of the fish gill, such as vasomotor control of the vasculature, by autonomic nerves of cranial and spinal origin. We have also attempted to assign putative roles in the gill for innervation that has more recently been described. However, due to the complexity of the gill, in both its structure and in the many physiological processes that it performs, a complete understanding of gill innervation and its functional role has not yet been achieved. This is further complicated when
M.G. Jonz, G. Zaccone one considers the diversity of fish, and the wide degree of anatomical variance that has been observed between different phylogenetic groups. Of recent interest to the field of innervation of the fish gill are those studies characterizing the distribution of nonadrenergic–noncholinergic (NANC) nerve types. In particular, the emergence of nitrergic and peptidergic nerves as potential sources of neurotransmitters that may affect vasomotor responses within the gill is of major interest. The origin and functional significance of the entire population of nerve fibres of the respiratory lamellae has not been identified. Although this type of innervation appears to be species-specific, and has only been described in teleosts, it presumably plays an important role in the gills of these species. In addition, putative efferent innervation of O2-chemoreceptive NECs and ion-regulatory MRCs may represent elements of efferent inhibition of O2-chemosensory responses or neural control of ion regulation, respectively. Determination of a functional significance of this innervation, however, will await physiological confirmation. Future studies directed toward further characterization of gill innervation in fish will clarify our understanding of the role of the nervous system in controlling the gill, thereby maintaining a static internal environment, and may aid in the identification of novel regulatory or modulatory pathways that are important for allowing the gill to perform its many physiological roles.
Acknowledgement Original research of M.G.J. was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada.
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ARTICLE IN PRESS 216 Nilsson S. Innervation and pharmacology of the gills. In: Hoar WS, Randall DJ, editors. Fish physiology, Vol. XA. San Diego: Academic Press; 1984. p. 185–227. Nilsson S, Sundin L. Gill blood flow control. Comp Biochem Physiol 1998;119A:137–47. Olson KR. Vascular anatomy of the fish gill. J Exp Zool 2002a;293:214–31. Olson KR. Gill circulation: Regulation of perfusion distribution and metabolism of regulatory. molecules 2002b;293:320–35. Olson KR, Donald JA. Nervous control of circulation – the role of gasotransmitters, NO, CO, and H2S. Acta Histochem 2008; this issue, doi:10.1016/j.acthis. 2008.11.004. Payan P, Mayer-Gostan N, Pang PK. Site of calcium uptake in the freshwater trout gill. J Exp Zool 1981;216: 345–7. Pequignot J, Gas N. Modifications histologiques de l’e ´pithe´lium branchial sous l’influence de la vagotomie chez la Tanche. C R Se´ances Soc Biol Fil 1971;165:1172–6. Perry SF. The chloride cell: Structure and function in the gills of freshwater fishes. Annu Rev Physiol 1997;59: 325–47. Pettersson K, Nilsson S. Nervous control of the branchial vascular resistance of the Atlantic cod, Gadus morhua. J Comp Physiol 1979;129:179–83. Saltys HA, Jonz MG, Nurse CA. Comparative study of gill neuroepithelial cells and their innervation in teleosts and Xenopus tadpoles. Cell Tissue Res 2006;323: 1–10. Schwerte T. Cardio-respiratory control during early development in the model animal zebrafish. Acta Histochem 2008; this issue, doi:10.1016/j.acthis. 2008.11.005. Smith DG, Chamley-Campbell J. Localization of smoothmuscle myosin in branchial pillar cells of snapper (Chrysophys auratus) by immunofluorescence histochemistry. J Exp Zool 1981;215:121–4. Stensløkken K, Sundin L, Nilsson GE. Cardiovascular and gill microcirculatory effects of endothelin-1 in Atlantic cod: Evidence for pillar cell contraction. J Exp Biol 1999;202:1151–7.
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www.elsevier.de/acthis
Nervous control of the gills Michael G. Jonza,, Giacomo Zacconeb a
Department of Biology, University of Ottawa, 30 Marie Curie, P.O. Box 450, Station A, Ottawa, ON, Canada K1N 6N5 Department of Animal Biology and Marine Ecology, Section of Comparative Neurobiology and Biomonitoring, University of Messina, Via Salita Sperone 31, I-98166 Messina, Italy
b
KEYWORDS Review; Fish; Gill; Autonomic; Innervation; Sympathetic; Parasympathetic
Summary The fish gill is a highly complex organ that performs a wide variety of physiological processes and receives extensive nervous innervation from both afferent (sensory) and efferent (motor) fibres. Innervation from the latter source includes autonomic nerve fibres of spinal (sympathetic) and cranial (parasympathetic) origin whose primary role is to induce vasomotor changes within the respiratory or nonrespiratory pathways of the gill vasculature. Autonomic control of the gill occurs by nerve fibres identified as adrenergic, cholinergic, and more recent evidence indicates that nonadrenergic–noncholinergic (NANC) nerve fibres, such as those that express amines, peptides, or nitric oxide, may also play an important role. The distribution and physiological function of NANC nerve fibres, however, is less clear. This review primarily discusses histochemical studies that have characterized the nervous innervation and autonomic control of the gill vasculature. In addition, supporting evidence from recent studies for the efferent control, or modulation, of other homeostatic processes in the gill is examined. & 2008 Elsevier GmbH. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General organization of the gills and associated vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . Autonomic innervation of the gill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cranial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spinal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catecholaminergic nerves and vascular control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cholinergic and aminergic nerves and vascular control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitrergic and peptidergic nerves and vascular control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other putative roles for efferent innervation in the gill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Innervation of the respiratory lamellae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corresponding author. Tel.: +1 613 562 5800x6051; fax: +1 613 562 5486.
E-mail address: [email protected] (M.G. Jonz). 0065-1281/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2008.11.003
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M.G. Jonz, G. Zaccone Efferent innervation of neuroepithelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Putative innervation of mitochondria-rich cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction The fish gill is a sophisticated organ that performs an impressive variety of homeostatic functions. These include gas exchange, ion regulation, acid–base balance and nitrogen excretion (Evans et al., 2005). The gill is also a highly perfused organ that receives the entire cardiac output, and is a major site of circulatory or vasomotor control (Sundin and Nilsson, 2002; Olson, 2002a, b). As a corollary to this vast array of processes performed by the gill, it is not surprising that the gill receives extensive innervation from the nervous system (Figure 1) in the form of afferent (sensory) and efferent (motor) pathways, and this has been reviewed previously by several authors (Burnstock, 1969; Campbell, 1970; Nilsson, 1983, 1984; Taylor et al., 1999; Sundin and Nilsson, 2002). Indeed, the control of the gill vasculature by neural (and humoral) mechanisms is critically important for the regulation and optimization of such processes as respiration and osmoregulation. It is, therefore, of interest to understand the source and nature of gill innervation in order to appreciate how the gill is controlled by the nervous system. Characterization of gill innervation may additionally provide clues regarding novel regulatory pathways. A component of efferent innervation to the gill is that of the autonomic nervous system (Figure 2A). In all vertebrates, the autonomic nervous system is composed of all efferent pathways having ganglionic synapses outside of the central nervous system (CNS), and autonomic nerves reach nearly every part of the body, thereby controlling important homeostatic mechanisms (Nilsson, 1983). In the fish gill, autonomic innervation primarily includes the nerve supply to smooth muscle of the vasculature, and thus provides a means of vasomotor control of the gill (Nilsson, 1984; Sundin and Nilsson, 2002). As pointed out by Nilsson (1983), the early view of the autonomic nervous system in vertebrates was that the sympathetic division was mediated by adrenergic nerves, whilst the parasympathetic system was mediated by cholinergic nerves. Thus adrenaline and acetylcholine, respectively, have been considered major neurotransmitters. However, it is now clear that the autonomic system is complex and is composed of a variety of
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nerve types and putative neurotransmitters. As will be discussed, much attention has recently been focused on a wide variety of nonadrenergic–noncholinergic (NANC) substances, such as amines (e.g. serotonin, dopamine), peptides, and a small gaseous molecule, nitric oxide (NO). Many autonomic nerves of teleost, elasmobranch, and dipnoan fishes contain neuropeptides, but little is known about their pattern of colocalization with other neurotransmitters. A goal of this paper is to provide an introduction to the fish gill, its innervation, and to briefly review the autonomic control of the branchial vasculature by nerves of both cranial and spinal origin. Studies using traditional histochemical and ultrastructural procedures, as well as those that have utilized immunofluorescence and confocal techniques, will be discussed. In the latter portion of the paper, we will also discuss innervation patterns observed within the gill that are not well characterized, and propose potential roles for this innervation. As a note, the designation of cranial vs. spinal autonomic innervation of the branchial region is used throughout this review. This classification has been previously employed (e.g. Campbell, 1970; Nilsson, 1983, 1984; Sundin and Nilsson, 2002), and is more appropriate for comparative studies of autonomic innervation than Langley’s mammalian designation of ‘‘parasympathetic’’ vs. ‘‘sympathetic’’ (see Nilsson, 1983). Other important aspects of gill innervation that will not be discussed are covered elsewhere in this special issue, such as the nervous control of respiratory reflexes (Schwerte, 2008; Taylor et al., 2008) and the gill as a sensory organ (Burleson, 2008).
General organization of the gills and associated vasculature In teleost fish, four bilateral pairs of gill arches are present in the pharyngeal region and each gives rise to two parallel rows of gill filaments, called hemibranchs. Gill filaments are further divided into a series of secondary (respiratory) lamellae (Figure 1). Both the filaments and lamellae are covered by a thin epithelial layer that contains a wide variety of cell types that mediate the many physiological and homeostatic processes that the gill performs (Wilson and Laurent, 2002; Evans et al., 2005). As illustrated
ARTICLE IN PRESS Nervous control of the gills
Figure 1. Illustration of the extensive innervation to the teleost gill using confocal immunofluorescence methods. A large nerve bundle courses through the centre of the gill filament (F) and gives rise to a nerve plexus and fibres of the respiratory lamellae (L). In this example, a neuronspecific antibody (zn-12) was used to label all nervous structures of the gill in zebrafish. Bar, 50 mm. Modified from Jonz and Nurse (2003).
in Figure 2A, the gill vasculature is complex and contains two major circulatory networks: the respiratory (arterioarterial) and nonrespiratory (arteriovenous) pathways (Olson, 2002a). Blood flow through the gills begins with deoxygenated blood travelling from the heart through the ventral aorta. From here, blood enters the gill arches via the afferent branchial artery, and the gill filaments via the afferent filament arteries. Blood flow continues distally along the filaments until it enters the afferent lamellar arterioles and travels through the vascular sinus of the lamellae, where gas exchange occurs. In the respiratory pathway, blood flows to and from the gill lamellae and continues into the systemic circulation via the efferent lamellar arterioles, efferent filament arteries, the efferent branchial artery, and finally the dorsal aorta. In the nonrespiratory pathway, blood flows from the respiratory pathway into small vessels, such as the nutritive vasculature and arteriovenous anastomoses, which provide perfusion and drainage of gill tissue.
Autonomic innervation of the gill Cranial There are 11 pairs of cranial nerves found in fish. However, only the facial (VII), glossopharyngeal (IX)
209 and vagus (X) nerves provide cranial innervation to the gill region, and are, therefore, called the branchial nerves (Nilsson, 1984; Sundin and Nilsson, 2002). In jawed fishes (gnathostomes), such as teleosts, the gill arches are innervated primarily by the glossopharyngeal and vagus nerves, which form large nerve trunks that enter the gills from the dorsal aspect (Figure 2B). The second, third and fourth gill arches are innervated entirely by branches of the vagus nerve, whilst the first gill arch receives both glossopharyngeal and vagal innervation. Once within the branchial region, the nerves are further divided into pretrematic and posttrematic rami (Figure 2B) that straddle the gill slits rostrally and caudally, respectively (Nilsson, 1984; Sundin and Nilsson, 2002). Thus, each gill arch is innervated by a posttrematic and pretrematic ramus from two different cranial nerve branches. The pretrematic ramus carries sensory nerve fibres, and the posttrematic ramus carries both sensory and motor fibres (Nilsson, 1984). It is noteworthy that a reduced gill-like structure, called the pseudobranch, is innervated primarily by the pretrematic ramus of the glossopharyngeal nerve and may receive additional innervation from the posttrematic ramus of the facial nerve (Laurent and Dunel-Erb, 1984; Nilsson, 1984). Glossopharyngeal innervation to the pseudobranch, however, is believed to be primarily sensory. In teleosts, cranial autonomic outflow to the gills is restricted to the vagus nerve (Figure 2A) and mediates vasomotor control of the gill vasculature (Nilsson, 1983; Taylor et al., 1999). The need for cranial autonomic vasomotor fibres in the head, as observed in elasmobranchs and some cyclostomes (e.g. lampreys), was apparently reduced in teleosts, which instead possess an increased supply of cephalic spinal autonomic fibres (Nilsson, 1983).
Spinal Spinal autonomic innervation to the gills in teleosts occurs by way of the sympathetic chain ganglia (Figure 2A and B). The sympathetic chain is well developed in teleosts, as it is in tetrapods, and extends into the cephalic region, where spinal nerve fibres leave the sympathetic chain through grey rami communicantes and enter branches of the cranial nerves. In fact, the vagus nerve is often referred to as the ‘‘vagosympathetic trunk’’ in teleosts because of the rich contribution of spinal autonomic fibres, which continue into the gills and exert vasomotor effects on the gill vasculature (Nilsson, 1983; Taylor et al., 1999). In cyclostomes and elasmobranchs, a sympathetic chain per se is
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Figure 2. (A) Current working model for innervation of the teleost gill vasculature by cranial and spinal autonomic nerves, as evident from histochemical studies, indicating potential sites of vasomotor control. The cranial (‘‘parasympathetic’’) pathway includes nerve fibres travelling through the vagus nerve and postganglionic neurons (indicated), while the spinal (‘‘sympathetic’’) pathway travels through the sympathetic chain ganglia and the vagosympathetic trunk. Adrenergic, cholinergic and serotonergic nerve fibres are indicated. A muscular ‘‘sphincter’’ is shown at the proximal region of the efferent filament artery. Black arrows indicate the flow of deoxygenated blood and grey arrows indicate oxygenated (postlamellar) blood carried through the respiratory and nonrespiratory vascular pathways. 5-HT, serotonergic nerve; ACh, cholinergic nerve; Adr, adrenergic nerve; AFA, afferent filament artery; ALa, afferent lamellar artery; AVA, arteriovenous anastomoses; BN, branchial nerve; CNS, central nervous system; CVS, central venous sinus; EFA, efferent filament artery; ELa, efferent lamellar artery; NV, nutritive vasculature; SC, sympathetic chain; SL, secondary (respiratory) lamellae; Sph, sphincter; X, vagus nerve. Modified from Nilsson and Sundin (1998). (B) Simplified scheme of innervation to the gill region in fish. The gills are innervated by nerve fibres (both sensory and motor) originating from the cranial nerves, and by fibres that originate from the sympathetic chain and enter the cranial nerve trunk via grey rami communicantes. Each gill arch is innervated by a pretrematic ramus (anterior to the gill slit) and a posttrematic ramus (posterior to the gill slit) originating from different cranial nerve branches. Pretrematic rami carry sensory fibres, while posttrematic rami carry sensory and motor fibres. Based partly on Nilsson (1984).
not observed. However, in the latter an irregular series of paravertebral ganglia is present but these do not extend into the head (Burnstock, 1969; Campbell, 1970; Nilsson, 1983; Taylor et al., 1999). There is no evidence for branchial vasomotor control by spinal innervation in either cyclostomes or elasmobranchs (Taylor et al., 1999).
Catecholaminergic nerves and vascular control Control of the branchial vasculature by catecholaminergic (adrenergic) nerves of spinal autonomic origin is found primarily in the teleosts (Figure 2A). With the notable exception of the brown trout (Salmo trutta) and carp (Cyprinus carpio), the afferent and efferent branchial arteries of the gill arches in most species generally do not
receive innervation from catecholaminergic nerves (Donald, 1984, 1987). Thus, the control of blood flow through the gills is controlled primarily via innervation of the respiratory and nonrespiratory pathways of the gill filaments. The afferent filament arteries and afferent lamellar arterioles, which deliver deoxygenated blood to the lamellae for gas exchange, are innervated by catecholamine-containing nerves (Donald, 1984, 1987). In addition, the efferent lamellar arterioles and the proximal region of the efferent filament arteries receive similar innervation (Donald, 1984, 1987; Dunel-Erb and Bailly, 1986). In the latter case, sympathetic innervation is concentrated at a contractile segment of the filament artery, called a sphincter, which is believed to play a role in the regulation of blood flow. The vessels regarded as a part of the nutritive vasculature of the nonrespiratory pathway are supplied by
ARTICLE IN PRESS Nervous control of the gills catecholamine-containing nerves (Donald, 1984, 1987; Dunel-Erb et al., 1989), and it has been proposed that the arteriovenous anastomoses also receive innervation (Vogel et al., 1974). These nerves are also found around the central venous sinus in the core of the gill filament (Dunel-Erb and Bailly, 1986; Dunel-Erb et al., 1989). The major effect of adrenergic vasomotor innervation of the gill vessels is due to an a-adrenoceptor-mediated vasoconstriction of the nonrespiratory pathway, thus favouring an arterioarterial flow (Sundin and Nilsson, 2002). The respiratory pathway responds mainly through b-dilatory mechanisms, primarily at the afferent lamellar arterioles, leading to perfusion of additional lamellae (Nilsson and Sundin, 1998; Evans et al., 2005).
Cholinergic and aminergic nerves and vascular control Cranial autonomic (vagal) nerves carry vasomotor fibres to the gills of teleosts and have been identified as cholinergic (Figure 2A). Ultrastructural studies have revealed the presence of cholinergic-type nerve profiles innervating the sphincter at the base of the efferent filament arteries that originate from postganglionic axons, and histochemical studies have shown high levels of acetylcholinesterase, an enzyme that hydrolyzes acetylcholine, in this region (Bailly and Dunel-Erb, 1986; Mauceri et al., 2005). Sundin and Nilsson (1997) described a general constriction of the efferent vasculature, including both the filament arteries and arterioles, by the use of acetylcholine. This resulted in a marked reduction of blood flow in the distal portion of the filaments. It was concluded in this study that this vasomotor control occurred via a cholinergic innervation of the sphincter. Pettersson and Nilsson (1979) noted a vagal NANC component of the vasoconstrictory nerve supply of the gill filament of the Atlantic cod (Gadus morhua), since neither adrenergic nor cholinergic blocking agents completely suppressed the nerveinduced vasoconstriction of the efferent arterial system. Serotonergic nerves of vagal origin also occur in the efferent vasculature of the gills of teleosts (Figure 2A) (Dunel-Erb et al., 1982, 1989; Bailly et al., 1989). In particular, indolaminergic neurons innervating the proximal part of the efferent arterial vasculature, the filament epithelium, and the central venous sinus, are found. In fish species studied by Sundin et al. (1998a, b), the serotonergic fibres reach the proximal region of the efferent filament arteries (including the sphincter)
211 and efferent lamellar arterioles, and extend in the direction of the central venous system. In Pagothenia borchgrevinki, serotonergic innervation was also observed on the afferent side of the filament. In zebrafish (Danio rerio), a series of serotonergic neurons of the filament epithelium was found in whole-mount gill preparations (Jonz and Nurse, 2003). Some of these innervated both O2-chemosensory neuroepithelial cells (NECs) and the sphincter of the efferent filament artery, while the termination of other serotonergic neurons was not determined. The cell bodies of serotonergic neurons are intrinsic to the gills (i.e. postganglionic), thus indicating that these are consistent with the general pattern of cranial autonomic innervation (Sundin and Nilsson, 2002). The main nervous control by serotonin on the gill vasculature occurs through constriction of the respiratory pathway in the distal portion of the filaments, thus leading to a redistribution of blood flow to more proximal regions of the filament (Sundin et al., 1995). This is consistent with observations that serotonin increases branchial vascular resistance in perfused preparations and in vivo (Sundin and Nilsson, 2002). Moreover, the vasomotor effects of serotonin in the fish gill appears to be due to at least two different serotonin receptor subtypes, since the receptor antagonists which abolished the serotonin-induced vasoconstriction of gill vessels (Sundin et al., 1995) had no effect on the arteriovenous vasodilatory effect of serotonin (Sundin, 1995).
Nitrergic and peptidergic nerves and vascular control NANC candidates in the fish gill are also represented by the so-called ‘‘gasotransmitters’’, e.g. nitric oxide (NO; see Olson and Donald, 2008 this issue) and neuropeptides (Zaccone et al., 2006). Zaccone et al. (2003, 2006) reported the presence of a rich supply of nerve fibres that were immunopositive for neuronal nitric oxide synthase (nNOS), an isoform of the enzyme involved in the production of NO, and these fibres were associated with the efferent filament arteries, the pillar cell system of the lamellae, and efferent lamellar arterioles in the gills of catfish (Heteropneustes fossilis). In the air sac of the same species, which originates from the fusion of gill filaments and lamellae, the vasculature of the main efferent vessels is innervated by nNOS-immunopositive nerve fibres. In addition, Gibbins et al. (1995) showed that postganglionic neurons positive for the NADPH-diaphorase reaction (an indicator of NOS activity) were present in the posttrematic branches
ARTICLE IN PRESS 212 of the branchial nerves in G. morhua, suggesting that these may be part of the cranial autonomic pathway. Studies by Funakoshi et al. (1997, 1999) provided considerable information about the nitrergic (nNOS-immunopositive) innervation of the fish gill. They examined the presence of nNOS and NADPH-diaphorase activity in the sensory system of the glossopharyngeal and vagus nerves of a pufferfish (Takifugu niphobles). In the vagal ganglion, positive neurons were found in subpopulations of the branchial rami. Nitrergic afferent terminals were found in the glossopharyngeal lobe, vagal lobe and commissural nucleus. Distribution of NADPHdiaphorase and nNOS-immunoreactive nerve fibres were traced not only along the efferent filament arteries of the gill, but also in the afferent filament arteries, the efferent lamellar arterioles and the central venous sinus. Although NO induces vasodilation in the systemic vessels of fish, there is apparently no clear evidence for the direct effects of NO on the gill vasculature, despite its vasoactive effects and the presence of nNOS in this tissue (Evans et al., 2005). Neuropeptide-containing nerve fibres have been found in the vessel walls of a range of nonmammalian vertebrate species (Holmgren, 1995). Neuropeptides have been described in vasomotor nerves in teleost fish, while evidence is lacking in cyclostomes (Morris and Nilsson, 1994). However, there have been few studies dealing with the distribution of neuropeptides in the teleost vasculature, particularly the respiratory pathway of the gill. Nerve fibres immunoreactive for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP7 38 and 27) are distributed along the main part of the filament in the catfish, H. fossilis (Zaccone et al., 2003, 2006). These fibres run to the efferent filament arteries. VIP produced a concentration-dependent vasodilation in perfused gills of the brown trout, Salmo trutta (Bolis et al., 1984). In addition, neuropeptide Y (NPY)-like immunoreactivity was demonstrated in the gill vessels in an elasmobranch, producing constriction of the afferent branchial artery (Bjenning et al., 1993). A dense network of leu- and met-enkephalin immunoreactive nerve fibres have also been traced along the walls of the efferent filament arteries and efferent lamellar arterioles. The origin of the nerve fibres expressing these types of neuropeptides remains unclear. In spite of the presence of NANC substances in the gill perivascular nerves, NANC neurotransmission has not been demonstrated in the gill vessels. In addition, the function of perivascular nerves (either sensory or vasomotor) is not clear.
M.G. Jonz, G. Zaccone
Other putative roles for efferent innervation in the gill Innervation of the respiratory lamellae It is now well established from recent studies that in some teleost species, such as zebrafish, goldfish (Carassius auratus) and catfish, the respiratory lamellae of the gills receive a dense supply of innervation (Jonz and Nurse, 2003; Saltys et al., 2006; Zaccone et al., 2003; Coolidge et al., 2008). As shown in Figure 1, the successful labelling of lamellar nerve fibres of gill lamellae appears to be due to the recent use of a specific neuronal marker derived from zebrafish (zn-12; Trevarrow et al., 1990; Jonz and Nurse, 2003) that identifies an apparently conserved neuronal surface antigen, since previous studies using traditional techniques did not produce any clear evidence of lamellar innervation (Laurent and Dunel, 1980; Donald, 1984, 1987; Nilsson, 1984). It is also evident that the presence of nerve fibres in the lamellae is species-specific, since the above studies also showed they are absent in other teleosts, such as rainbow trout (Oncorhynchus mykiss), trairao (Hoplias lacerdae) and traira (H. malabaricus) (Saltys et al., 2006; Coolidge et al., 2008). Though at present the source of lamellar innervation has not been determined, in zebrafish these nerve fibres appear to emanate from the nerve plexus of the gill filaments, and nerve sectioning experiments have demonstrated that parent cell bodies of lamellar nerve fibres are located extrinsic to the gills (Jonz and Nurse, 2003). In addition, there is preliminary evidence that at least some lamellar nerve fibres contain catecholamines (Arlotta and Jonz, unpublished observations), suggesting a potential spinal origin. Both afferent and efferent roles for lamellar innervation may be considered. Serotonergic NECs of the lamellae, which resemble O2-chemoreceptive NECs of the filaments, are innervated and may relay sensory information regarding arterial or water O2 tension to the CNS via afferent postsynaptic fibres (Jonz and Nurse, 2003; Jonz et al., 2004), thereby playing a potential role in the sensing of hypoxia. Alternatively, this innervation may be efferent and control the activity of NECs (see below) or other cell types. A group of structurally supportive cells found within the respiratory lamellae, called ‘‘pillar cells’’, separates the epithelial layers of the lamellae and produces the vascular sinus through which blood passes during gas exchange (Olson, 2002a). Pillar cells contain actin and myosin filaments that apparently allow these cells to contract and
ARTICLE IN PRESS Nervous control of the gills increase vascular resistance within the gill (Smith and Chamley-Campbell, 1981; Stensløkken et al., 1999; Mistry et al., 2004), and although their innervation has not been confirmed it has been suggested (Gilloteaux, 1969; Jonz and Nurse, 2003). Such contact between efferent nerve fibres and these cells may permit mediation of pillar cell contraction and vascular control within the lamellae, and aid in the vasomotor control of the gill vasculature by autonomic fibres. However, the question arises of how blood flow within the vascular spaces of the lamellae is controlled by pillar cells, if at all, in fish that do not possess lamellar innervation. Endocrine or paracrine mechanisms of pillar cell contraction have also been suggested (Olson, 2002a), and may operate instead in these cases.
Efferent innervation of neuroepithelial cells O2-chemoreceptive NECs of the gill filaments are believed to establish synapses with at least three types of nerve fibres. The first type of nerve ending is catecholaminergic (Bailly et al., 1992), suggesting that NECs are modulated by spinal autonomic nerves. The second type of nerve profile close to NECs likely belongs to nearby serotonin-containing neurons, since they remain intact after symapthectomy, and may be responsible for relaying sensory information to the CNS during hypoxia (Bailly et al., 1992). These two types of nerve profiles observed at the ultrastructural level correspond to the extrinsic and intrinsic innervation patterns, respectively, described in the zebrafish gill (Jonz and Nurse, 2003). A third type of nerve fibre innervating gill NECs is that of nitrergic nerve fibres that may be of cranial origin (Zaccone et al., 2003). It is worth noting that NECs may also receive afferent innervation from an additional extrinsic source, since O2-sensory responses appear functional in zebrafish that lack intrinsic serotonergic neurons of the gill filaments during early larval stages (Jonz and Nurse, 2005). However, despite the afferent innervation of NECs that is prerequisite for a functional O2-chemosensory system in the gill, the contribution of efferent innervation to NEC function has been considered (Bailly et al., 1992; Jonz and Nurse, 2003; Zaccone et al., 2006). Many nerve fibres associated with gill NECs, especially the intrinsic fibres, contain synaptic vesicles (Jonz and Nurse, 2003) and may represent presynaptic nerve endings that potentially secrete neurotransmitters (e.g. serotonin or NO) onto NECs or surrounding tissues. These results may indicate an additional neuroendocrine role for
213 NECs in the gill, where induction of NEC neurosecretion by hypoxia, for example, would lead to compensatory changes in local vascular resistance within the gill. Alternatively, a potential role for efferent nerve fibres in directly modulating the chemosensory responses of NECs to hypoxia is possible. The well-studied type I cell chemoreceptors of the mammalian carotid body are O2sensitive and are considered to be homologues of gill NECs (Jonz and Nurse, 2008). Type I cells receive both afferent and efferent sources of innervation, and a mechanism has been described in which autonomic (probably parasympathetic) nerve fibres mediate efferent inhibition of type I cells within the carotid body during hypoxic stimulation (Campanucci and Nurse, 2007). In this model, glossopharyngeal neurons release NO onto carotid body chemoreceptors, thereby reducing their excitability and attenuating their response to hypoxia. It is, at present, unknown if a homologous mechanism of efferent inhibition is functional within the O2-chemosensory system of the fish gill.
Putative innervation of mitochondria-rich cells The gill is an important site of osmotic and ion balance in fish, and the primary functional unit of these processes in the teleost gill is the mitochondria-rich cell (MRC). These cells are located in the gill filament epithelium and are characterized by a high density of mitochondria, an abundance of the enzyme, Na+/K+-ATPase, and a complement of ion channels, exchangers and pumps (Perry, 1997; Wilson and Laurent, 2002; Evans et al., 2005). Depending on aquatic habitat, MRCs of the fish gill may mediate the uptake or extrusion of Na+, Ca2+ and Cl across the epithelium between the blood and water (Perry, 1997; Marshall, 2002; Evans et al., 2005). The movement of ions across ionregulatory tissues in fish, although not completely understood, appears to be mediated by circulating hormones and neurotransmitters, such as catecholamines, adrenaline, acetylcholine, NO and peptides (Zadunaisky, 1984; McCormick, 2001; Evans, 2002; Marshall, 2003). Furthermore, some evidence has accumulated to suggest that ion transport by gill MRCs may be under control of the nervous system. Early studies demonstrated that sectioning of the branchial nerves depleted gill MRCs and altered ion transport and water permeability (Pequignot and Gas, 1971; Mayer-Gostan and Hirano, 1976), suggestive of MRC innervation. Moreover, stimulation of the branchial nerves and
ARTICLE IN PRESS 214 exogenous application of adrenaline in trout altered Ca2+ flux across the gill epithelium (Payan et al., 1981; Donald, 1989). A more recent study using confocal techniques provided morphological evidence that was suggestive of innervation of MRCs in zebrafish (Jonz and Nurse, 2006). These studies showed that nerve fibres from the filament plexus appeared to contact MRCs within the gill filament. This same nerve plexus was shown in a previous study to be of extrinsic origin following denervation and nerve fibre degeneration procedures (Jonz and Nurse, 2003). Therefore, the above studies may indicate efferent innervation of MRCs and, potentially, autonomic control of ion regulation in the fish gill. Nerve fibres in zebrafish are associated with the basolateral regions of MRCs, where Na+/K+-ATPase activity is localized (Evans et al., 2005), while apical regions are oriented toward the external environment. This arrangement may suggest that presynaptic nerve terminals of efferent fibres release neurochemicals and affect the activity of postsynaptic MRCs, thereby controlling ion regulation across the gill epithelium. The physiological significance of this putative innervation in ion regulation, however, awaits confirmation at the ultrastructural level, functional studies, and perhaps identification in other fish species. Such confirmation may not be surprising, since the neural control of ion homeostasis in the mammalian kidney is well established (DiBona and Kopp, 1997). By contrast, the presence of sensory ionoreceptors in fish has been proposed, in which rapid ion transfer may be elicited in response to changes in plasma ion concentration encountered during salinity change (Evans et al., 2005; Marshall et al., 2005). In this model, MRCs would be presynaptic and receive afferent innervation. Such receptors and sensory innervation of transport epithelia has also been described in the mammalian kidney (DiBona and Kopp, 1997).
Concluding remarks This brief review has outlined important aspects of nervous innervation of the fish gill, such as vasomotor control of the vasculature, by autonomic nerves of cranial and spinal origin. We have also attempted to assign putative roles in the gill for innervation that has more recently been described. However, due to the complexity of the gill, in both its structure and in the many physiological processes that it performs, a complete understanding of gill innervation and its functional role has not yet been achieved. This is further complicated when
M.G. Jonz, G. Zaccone one considers the diversity of fish, and the wide degree of anatomical variance that has been observed between different phylogenetic groups. Of recent interest to the field of innervation of the fish gill are those studies characterizing the distribution of nonadrenergic–noncholinergic (NANC) nerve types. In particular, the emergence of nitrergic and peptidergic nerves as potential sources of neurotransmitters that may affect vasomotor responses within the gill is of major interest. The origin and functional significance of the entire population of nerve fibres of the respiratory lamellae has not been identified. Although this type of innervation appears to be species-specific, and has only been described in teleosts, it presumably plays an important role in the gills of these species. In addition, putative efferent innervation of O2-chemoreceptive NECs and ion-regulatory MRCs may represent elements of efferent inhibition of O2-chemosensory responses or neural control of ion regulation, respectively. Determination of a functional significance of this innervation, however, will await physiological confirmation. Future studies directed toward further characterization of gill innervation in fish will clarify our understanding of the role of the nervous system in controlling the gill, thereby maintaining a static internal environment, and may aid in the identification of novel regulatory or modulatory pathways that are important for allowing the gill to perform its many physiological roles.
Acknowledgement Original research of M.G.J. was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada.
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