Expression patterns and quantitative assessment of neurochemical markers in the lung of the gray bichir, Polypterus senegalus (Cuvier, 1829).

Anatomical and functional studies of the autonomic innervation and the putative oxygen receptors-the neuroepithelial (NEC)-like cells of the bichirs are lacking. The present paper describes the distribution of both NEC-like cells and the polymorphous granular cells (PGCs) that populate the mucociliated epithelium of the lung in the air breathing fish Polypterus senegalus. By using confocal immunohistochemistry we determined the coexpression of specific neurochemical markers. Colocalization studies showed that 5HT is coexpressed with calbindin and nNOS in the NEC-like cells and PGCs, and choline acetyltransferase (ChAT) is coexpressed with nNOS in both the two types of cells. Distribution of neurotransmitters (5HT, NO) and neurochemical marker ChAT is also investigated in the lung muscle. The role of these transmitters may be the autonomic control of circulation and respiration. However, the importance of these signals for the respiratory responses in the species studied is still not known. The present study also shows for the first time the simultaneous occurrence of piscidin 1 and 5HT in the PGCs. The function of these cells being equivalent to ones found in fish gill subepithelial parenchyma, is still not known. Due to the importance of piscidin 1 in local immune defense, more research is useful to understand a possible interaction of PGCs with immune response in the bichir lung.

Confocal imaging of autonomic preganglionic neurons in the spinal cord of the caecilian Typhlonectes natans (Amphibia: Gymnophiona).

Little is known about the spinal sympathetic organization in the caecilian amphibians. We examined for the first time the location of sympathetic preganglionic neurons (SPNs) in the spinal cord using a panel of specific markers expressed in SPNs. The SPNs of anuran amphibians form two cell columns segregated mainly in the lateral and medial marginal areas of the central gray matter. In the caecilian Typhlonectes natans immunoreactivity for galanin and ChAT is found in most laterally arranged neurons lying in spinal segments 2-7. They are encircled by TH- and nNOS-immunoreactive nerve fibers. These neurons might project specifically to a population of adrenergic sympathetic postganglionic neurons in paravertebral ganglia and/or non-adrenergic sympathetic postganglionic neurons in the celiac ganglia. However the segmental restriction and target specificity of the neurons of the species studied are not known. As mucous and granular glands in the dermis may represent one of the peripheral targets of the adrenergic ganglion cells and reflect the prominent preganglionic cell columns, an immunohistochemical study was done also on these glands. Retrograde-tracing studies are, however, needed to study the segmental localization of the preganglionic neurons and their projections to the postganglionic neurons in sympathetic ganglia.

Retrograde positive contrast urethrocystography of the fish urogenital system.

The radiological differences between the urinary tract of Dicentrarchus labrax, Sparus aurata, Tinca tinca, and Cyprinus carpio are shown. In fresh water teleosts the urinary bladder is sigmoid and a short urethra leads to the urinary pore. Genital and anal pores are present. In Sparus aurata the urinary bladder has a globoid shape. In Dicentrarchus labrax the urinary bladder is smaller and elongate. In both marine teleosts a single urogenital pore is visible. Positive contrast was used to survey the urogenital system and evaluate shape and size of the bladder, urethra, ureter, and gonadal ducts. Results demonstrate the morphological variability of the urinary bladder and the craniodorsal entry of the ureters into the bladder. It is envisaged that this work will provide baseline information for further imaging studies for investigating the urogenital morphology and can be applied to identify disorders in fishes. Furthermore, the main interest of this study is that it demonstrates the morphological variability of the lower urinary system that exists between different species of fishes.

Extracts deriving from olive mill waste water and their effects on the liver of the goldfish Carassius auratus fed with hypercholesterolemic diet.

The present research aims to evaluate the beneficial effects of polyphenols derived from waste water from a olive mill, obtained by non-plastic molecular imprinting device, in a hypercholesterolemic diet on Carassius auratus, commonly known as goldfish that was selected as experimental model. The study was conducted with morphological and histochemical analyses and also the data were supported by immunohistochemical analysis. Results show the beneficial activity of polyphenols with a reduction of the damage in the steatotic group, confirming that they may be suggested in the treatment of diseases by lipid accumulation, and used as any addition in feed for farmed fish, in order to improve the organoleptic and nutritional quality. The beneficial effects of waste oil extract should be suggested in the contexts of research programmes focused on the products to the health system. Furthermore, the olive mill waste water polyphenols free can be used as natural fertilizers.

The structural organization and immunohistochemistry of G-protein alpha subunits in the olfactory system of the air-breathing mudskipper, Periophthalmus barbarus (Linnaeus, 1766) (Gobiidae, Oxudercinae).

The study provides the first comprehensive information on the immunohistochemistry and ultrastructure of the olfactory receptor neurons (ORNs) in the mudskipper, Periophthalmus barbarus. The olfactory sensory epithelium is in the form of islets which cover part of the olfactory canal running from the upper lip toward the eye, where large single accessory nasal sacs occur. Within the islets, microvillous, ciliated and crypt ORNs were observed as well as giant cells and sparse non-sensory ciliated cells. Around the islets and in the walls of accessory nasal sacs, there are epidermal cells with microridges typical of fish epidermis. Close to the entrance to the accessory nasal sac, in the non-sensory epithelium of the nasal cavity and the skin epithelium covering the olfactory organ, areas of solitary chemosensory cells (SCCs) are reported for the first time. The distribution of the various ORN cell types is assessed through the immunohistochemistry against olfactory receptor coupled G-proteins. The ciliated ORNs were labeled by G alpha olf/s antibody. The ORNs with microvilli and crypt cells were G alpha i-3 immunoreactive.

Morphology and innervation of the teleost physostome swim bladders and their functional evolution in non-teleostean lineages.

Swim bladders and lungs are homologous structures. Phylogenetically ancient actinopterygian fish such as Cladistians (Polypteriformes), Ginglymods (Lepisosteids) and lungfish have primitive lungs that have evolved in the Paleozoic freshwater earliest gnathostomes as an adaptation to hypoxic stress. Here we investigated the structure and the role of autonomic nerves in the physostome swim bladder of the cyprinid goldfish (Carassius auratus) and the respiratory bladder of lepisosteids: the longnose gar and the spotted gar (Lepisosteus osseus and L. oculatus) to demonstrate that these organs have different innervation patterns that are responsible for controlling different functional aspects. The goldfish swim bladder is a richly innervated organ mainly controlled by cholinergic and adrenergic innervation also involving the presence of non-adrenergic non-cholinergic (NANC) neurotransmitters (nNOS, VIP, 5-HT and SP), suggesting a simple model for the regulation of the swim bladder system. The pattern of the autonomic innervation of the trabecular muscle of the Lepisosteus respiratory bladder is basically similar to that of the tetrapod lung with overlapping of both muscle architecture and control nerve patterns. These autonomic control elements do not exist in the bladders of the two species studied since they have very different physiological roles. The ontogenetic origin of the pulmonoid swim bladder (PSB) of garfishes may help understand how the expression of these autonomic control substances in the trabecular muscle is regulated including their interaction with the corpuscular cells in the respiratory epithelium of this bimodal air-breathing fish.

Mast cells in the intestine and gills of the sea bream, Sparus aurata, exposed to a polychlorinated biphenyl, PCB 126.

The presence of mast cells has been reported in all classes of vertebrates, including many teleost fish families. The mast cells of teleosts, both morphologically and functionally, show a close similarity to the mast cells of mammals. Mast cells of teleosts, localized in the vicinity of blood vessels of the intestine, gills and skin, may play an important role in the mechanisms of inflammatory response, because they express a number of functional proteins, including piscidins, which are antimicrobical peptides that act against a broad-spectrum of pathogens. An increase in the number of mast cells in various tissues and organs of teleosts seems to be linked to a wide range of stressful conditions, such as exposure to heavy metals (cadmium, copper, lead and mercury), exposure to herbicides and parasitic infections. This study analyzed the morphological localization and abundance of mast cells in the intestine and gills of sea bream, Sparus aurata, after a 12, 24 or 72 h exposure to PCB 126, a polychlorinated biphenyl, which is a potent immunotoxic agent. In the organs of fish exposed to PCB 126, it was observed that in addition to congestion of blood vessels, there was extravasation of red blood cells, infiltration of lymphocytes, and a progressive increase in numbers of mast cells. These data confirm the immunotoxic action of PCB, and the involvement of mast cells in the inflammatory response.

The simultaneous presence of neuroepithelial cells and neuroepithelial bodies in the respiratory gas bladder of the longnose gar, Lepisosteus osseus, and the spotted gar, L. oculatus.

Anatomical and functional studies on the autonomic innervation as well as the location of airway receptors in the air-bladder of lepisosteids are very fragmentary. These water-breathing fishes share in common with the bichirs the presence of a glottis (not a ductus pneumaticus) opening into the esophagus. In contrast to a high concentration of neuroepithelial cells (NECs) contained in the furrowed epithelium in the lung of Polypterus, these cells are scattered as solitary cells in the glottal epithelium, and grouped to form neuroepithelial bodies (NEBs) in the mucociliated epithelium investing the main trabeculae in the air-bladder of Lepisosteus osseus and L. oculatus. The present immunohistochemical studies also demonstrated the presence of nerve fibers in the trabecular striated musculature and a possible relation to NEBs in these species, and identified immunoreactive elements of this innervation. Tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), 5-HT and neuropeptide immunoreactivities were detected in the intramural nerve fibers. 5-HT and VIP immunopositive nerve fibers are apparently associated with NEBs. TH, VIP and SP immunoreactivities are also present in nerve fibers coursing in the radially arranged striated muscle surrounding the glottis and its submucosa. 5-HT positive neurons are also found in submucosal and the muscle layers of the glottis. The physiological function of the adrenergic and inhibitory innervation of the striated muscle as well as the neurochemical coding and morphology of the innervation of the NEBs are not known. Future studies are needed to provide evidence for these receptors with the capacity of chemoreceptors and/or mechanoreceptors.

Localization of neurotransmitters, peptides and nNOS in the pseudobranchial neurosecretory cell system and associated carotid labyrinth of the catfish, Clarias batrachus.

The carotid labyrinth is an enigmatic endocrine structure of unknown chemosensory function lying in the gill region of the catfishes. The carotid body is found at the carotid bifurcation of amphibians and all mammalian vertebrates on the evolutionary tree. It is a vascular expansion comprised of a cluster of glomus cells with associated (afferent and efferent) innervations. In the catfish species studied (Clarias batrachus) a neurosecretory cell system consisting of pseudobranchial neurosecretory cells connect the carotid labyrinth or large vessels (both the efferent branchial artery and dorsal aorta), and is likely akin to the glomus cells, but comparing these structures in widely divergent vertebrate species, the conclusion is that the structural components are more elaborate than those of terrestrial vertebrates. However, these cells reveal both an endocrine phenotype (such as the association with capillaries and large vessels) and the presence of regulatory substances such as neurotransmitters and neuropeptides producing good evidence for high levels of conservation of these substances that are present in the glomus cells of mammalian vertebrates. VIP-immunopositive neuronal cell bodies are detected in the periphery of the carotid labyrinth. They are presumptive local neurons that differ from pseudobranchial neurosecretory cells, the latter failing to express VIP in their soma.

Occurrence of neuropeptides and tyrosine hydroxylase in the olfactory epithelium of the lesser-spotted catshark (Scyliorhinus canicula Linnaeus, 1758).

Immunohistochemical studies using antisera against various neuropeptides (Substance P, vasoactive intestinal polypeptide, and cholecystokinin octapeptide) and tyrosine hydroxylase revealed both olfactory sensory neuron (OSN) polymorphisms and transepithelial-subepithelial nerves in the olfactory epithelium of the cartilaginous fish, Scyliorhinus canicula. This study provides the first evidence of three morphological types of OSNs within the olfactory epithelium of cartilaginous fish that are similar to those found in the teleosts. In fishes there is evidence that OSNs differ functionally, including their differential olfactory bulb projections and molecular properties. The Substance P positive olfactory neurons in S. canicula may have a separate bulbar projection site that is not known, but may indicate a characteristic found in olfactory neuron subtypes in both lampreys and teleost fish. Numerous Substance P immunopositive nerves are found at the base of and in the olfactory epithelium. Some of them were observed to extend outwards almost reaching the epithelial surface. Their presumptive origin from the trigeminal nerve and their interrelationship with chemosensory cells in the nasal passages of vertebrates are discussed.

Immunolocalization of neurotransmitter-synthesizing enzymes and neuropeptides with associated receptors in the photophores of the hatchetfish, Argyropelecus hemigymnus Cocco, 1829.

Anatomical and functional studies of the autonomic innervation of the photophores of luminescent fishes are scarce. The present immunohistochemical study demonstrated the presence of nerve fibers in the luminous epithelium and lens epithelium of the photophores of the hatchet fish, Argyropelecus hemigymnus and identified the immunoreactive elements of this innervation. Phenylethanolanine N-methyltransferase (PNMT) and catecholamine (CA)-synthesizing enzymes were detected in nerve varicosities inside the two epithelia. Neuropeptides were localized in neuropeptide Y (NPY) and substance P (SP)- and its NK11 receptor-immunopositive nerves in the lens epithelium. Neuropeptides were also localized in non-neural cell types such as the lens cells, which displayed immunoreactivities for pituitary adenylate cyclase activating peptide (PACAP) and their receptors R-12 and 93093-3. This reflects the ability of the neuropeptide-containing nerves and lens cells to turn on and off the expression of selected messengers. It appears that the neuropeptide-containing nerves demonstrated in this study may be sensory. Furthermore, neuronal nitric oxide synthase-immunopositive axons associated with photocytes in the luminous epithelium have previously been described in this species. Whereas it is clear that the photophores receive efferent (motor) fibers of spinal sympathetic origin, the origin of the neuropeptide sensory innervation remains to be determined. The functional roles of the above neuropeptides or their effects on the bioluminescence or the chemical nature of the terminals, either sensory or postganglionic neurons innervating the photophores, are still not known.

Nervous control of photophores in luminescent fishes.

Functional studies of the autonomic innervation in the photophores of luminescent fishes are scarce. The majority of studies have involved either the stimulation of isolated photophores or the modulatory effects of adrenaline-induced light emission. The fish skin is a highly complex organ that performs a wide variety of physiological processes and receives extensive nervous innervations. The latter includes autonomic nerve fibers of spinal sympathetic origin having a secretomotor function. More recent evidence indicates that neuropeptide-containing nerve fibers, such as those that express tachykinin and its NK1 receptor, neuropeptide Y, or nitric oxide, may also play an important role in the nervous control of photophores. There is no anatomical evidence that shows that nNOS positive (nitrergic) neurons form a population distinct from the secretomotor neurons with perikarya in the sympathetic ganglia. The distribution and function of the nitrergic nerves in the luminous cells, however, is less clear. It is likely that the chemical properties of the sympathetic postganglionic neurons in the ganglia of luminescent fishes are target-specific, such as observed in mammals.

Complex innervation patterns of the conus arteriosus in the heart of the longnose gar, Lepisosteus osseus.

Anatomical and functional studies of the autonomic innervation in the conus arteriosus of the garfishes are lacking. This study reveals that the conus arteriosus of the longnose gar is primarily myocardial in nature, but additionally, large numbers of smooth muscle cells are present in the subendocardium. A well-developed system of adrenergic, cholinergic, substance P (SP) and neuronal nitric oxide synthase (nNOS) positive nerve terminals are found in the wall of the conus arteriosus. Coronary blood vessels running in the adventitia receive a rich supply of nNOS positive nerve fibers, thus suggesting their importance in the nitrergic control of blood flow in the conus arteriosus. The present data show that the patterns of autonomic innervation of the garfish conus arteriosus are more complex than previously appreciated.