Immunohistochemical Characterization of Langerhans Cells in the Skin of Three Amphibian Species.

The amphibian taxon includes three orders that present different morphological characteristics: Anura, Caudata, and Apoda. Their skin has a crucial role: it acts as an immune organ constituting a physical, chemical, immunological, and microbiological barrier to pathogen insult and conducts essential physiological processes. Amphibians have developed specialized features to protect the vulnerable skin barrier, including a glandular network beneath the skin surface that can produce antimicrobial and toxic substances, thus contributing to the defense against pathogens and predators. This study aims to characterize Langerhans cells in the skin of Lithobates catesbeianus (order: Anura; Shaw, 1802), Amphiuma means (order: Caudata; Garden, 1821), and Typhlonectes natans (order: Apoda; Fischer, 1880) with the following antibodies: Langerin/CD207 (c-type lectin), Major Histocompatibility Complex (MHC)II, and Toll-like receptor (TLR)2 (expressed by different types of DCs). Our results showed Langerhans cells positive for Langerin CD/207 in the epidermis of the three species; moreover, some antigen-presenting cells (APCs) in the connective tissue expressed TLR2 and MHCII. The distribution of the Langerhans cells is very similar in the three amphibians examined, despite their different habitats. A greater knowledge of the amphibian immune system could be useful to better understand the phylogeny of vertebrates and to safeguard amphibians from population declines. Furthermore, the similarities between amphibians' and human skin concerning immunological features may be useful in both biology and translational medicine.

Macro- and micro morphology of the olfactory organ of African bonytongue, Heterotis niloticus (Cuvier 1829), compared with other species of the family Osteoglossidae (Teleostei).

Osteoglossiformes (bonytongue fishes) possess many morphological specializations associated with functions such as airbreathing, feeding, and electroreception. The olfactory organ also varies among species, notably in the family Osteoglossidae. Herein, we describe the olfactory organ of an osteoglossid, Heterotis niloticus, to compare it with the olfactory organs of other osteoglossiforms. We demonstrate the presence of an olfactory rosette within the olfactory chamber. This structure consists of a short median raphe surrounded by olfactory lamellae, which possess dorsal lamellar processes. On the surface of the olfactory lamellae, there are secondary lamellae formed by the olfactory epithelium. Within the olfactory epithelium, two zones can be distinguished: parallel brands of sensory cells located in the cavities between the secondary lamellae and a nonsensory area covering the remaining part of the olfactory lamellae. The olfactory epithelium is formed by ciliated and microvillus olfactory sensory neurons, supporting cells, goblet cells, basal cells and ciliated nonsensory cells. Additionally, rodlet cells were observed. The results confirm large variability in terms of the olfactory organ of Osteoglossiformes, particularly of Osteoglossidae, and support the secondary lamellae evolution hypothesis within this family.

The gas bladder of Heterotis niloticus (Cuvier, 1829).

This work reports on the structural characteristics of the respiratory gas bladder of the osteoglossiform fish Heterotis niloticus. The bladder-vertebrae relationships are also analyzed. A slit-shaped orifice in the mediodorsal pharyngeal wall is surrounded by a muscle sphincter and serves as a glottis-like opening to the gas bladder. The dorsolateral internal surface of the gas bladder is lined by a parenchyma of highly vascularized trabeculae and septa displaying an alveolar-like structure. The trabeculae contain, in addition to vessels, numerous eosinophils probably involved in immune responses. The air spaces are endowed with a thin exchange barrier indicating a good potential for respiratory gas exchange. The ventral wall of the gas bladder is a well-vascularized membrane that exhibits an exchange barrier in the luminal face and an inner structure dominated by the presence of a layer of richly innervated smooth muscle. This is suggestive of an autonomous adjustability of the gas bladder ventral wall. The trunk vertebrae show large transverse processes (parapophyses) and numerous surface openings that lead into intravertebral spaces that become invaded by the bladder parenchyma. Curiously, the caudal vertebrae show a regular teleost morphology with neural and hemal arches, but have similar surface openings and intravertebral pneumatic spaces. The African Arowana hence rivals the freshwater butterfly fish Pantodon in its exceptional role of displaying postcranial skeletal pneumaticity outside of Archosauria. The possible significance of these findings is discussed.

Immunohistochemistry of the Gut-Associated Lymphoid Tissue (GALT) in African Bonytongue (Heterotis niloticus, Cuvier 1829).

Heterotis niloticus is a basal teleost, belonging to the Osteoglossidae family, which is widespread in many parts of Africa. The digestive tract of H. niloticus presents similar characteristics to those of higher vertebrates, exhibiting a gizzard-like stomach and lymphoid aggregates in the intestinal lamina propria. The adaptive immune system of teleost fish is linked with each of their mucosal body surfaces. In fish, the gut-associated lymphoid tissue (GALT) is generally a diffuse immune system that represents an important line of defense against those pathogens inhabiting the external environment that can enter through food. The GALT comprises intraepithelial lymphocytes, which reside in the epithelial layer, and lamina propria leukocytes, which consist of lymphocytes, macrophages, granulocytes, and dendritic-like cells. This study aims to characterize, for the first time, the leukocytes present in the GALT of H. niloticus, by confocal immuno- fluorescence techniques, using specific antibodies: toll-like receptor 2, major histocompatibility complex class II, S100 protein, serotonin, CD4, langerin, and inducible nitric oxide synthetase. Our results show massive aggregates of immune cells in the thickness of the submucosa, arranged in circumscribed oval-shaped structures that are morphologically similar to the isolated lymphoid follicles present in birds and mammals, thus expanding our knowledge about the intestinal immunity shown by this fish.

Gill structure and neurochemical markers in the African bonytongue (Heterotis niloticus): A preliminary study.

We have conducted a morphological and immunohistochemical study of the gills of juvenile specimens of the obligate air-breathing fish Heterotis niloticus. The study has been performed under normoxic and hypoxic conditions. The gills showed a reduced respiratory surface area by development of an interlamellar cellular mass (ILCM). The ILCM persisted without changes under both normoxia and hypoxia. Neuroepithelial cells (NECs), the major oxygen and hypoxia sensing cell type, were located in the distal end of the gill filaments and along the ILCM edges. These cells expressed 5HT, the neuronal isoform of the nitric oxide synthase (nNOS) and the vesicular acetylcholine transporter (VAChT). Furthermore, NECs appeared associated with nitrergic nerve fibres. The O2 levels did not modify the location, number or the immunohistochemical characteristics of NECs. Pavement cells covering the ILCM were also positive to nNOS and VAChT. The mechanisms of O2 sensing in the gills of Heterotis appears to involve several cell populations, the release of multiple neurotransmitters and a diversity of excitatory, inhibitory and modulatory mechanisms.

The Alimentary Tract of African Bony-Tongue, Heterotis niloticus (Cuvier, 1829): Morphology Study.

A morphological study of the alimentary tract, from the oropharyngeal cavity to the rectum, including the attached glands, of African bony-tongue, Heterotis niloticus (Cuvier, 1829) was carried out by gross anatomy, and light microscope analysis. This study aimed to give a deeper knowledge of the alimentary tract morphological features of this species of commercial interest. H. niloticus is distinguished by individual morphological characteristics showing a digestive tract similar to that of reptiles and birds. Within the oropharyngeal cavity, two tubular structures with digitiform ends are arranged on both lateral sides of the triangular tongue. The oropharyngeal cavity connects the stomach by a short esophagus. This latter is adapted to mechanical trituration, and it is divided into a pars glandularis and a thick-walled pars muscularis. The gizzard flows into the anterior intestine and two blind pyloric appendages, which exhibit specific functions, including immune defense for the presence of secondary lymphoid organs. The anterior intestine continues with the middle and posterior tracts up into the rectum. According to the histological observations, all regions of the alimentary tract have common structural features, typical of hollow organs, with differences in the mucosa structure that reflects the different functions of the apparatus, from mouth to anus. Within this study, we provided the first basis for future studies on optimizing rearing conditions, feed conversion ratio, and the digestive capacity, improving the growth performance of this species, and ensuring its conservation.

Expression of the Antimicrobial Peptide Piscidin 1 and Neuropeptides in Fish Gill and Skin: A Potential Participation in Neuro-Immune Interaction.

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.

Neuroepithelial cells (NECs) and mucous cells express a variety of neurotransmitters and neurotransmitter receptors in the gill and respiratory air-sac of the catfish Heteropneustes fossilis (Siluriformes, Heteropneustidae): a possible role in local immune defence.

Heteropneustes fossilis is an air-breathing teleost inhabiting environments with very poor O2 conditions, and so it has evolved to cope with hypoxia. In the gills and respiratory air-sac, the sites for O2 sensing and the response to hypoxia rely on the expression of acetylcholine (Ach) acting via its nicotinic receptor (nAChR). This study examined the expression patterns of neuronal markers and some compounds in the NECs of the gills and respiratory air sac having an immunomodulatory function in mammalian lungs. Mucous cells, epithelial cells and neuroepithelial cells (NECs) were immunopositive to a variety of both neuronal markers (VAChT, nAChR, GABA-B-R1 receptor, GAD679) and the antimicrobial peptide piscidin, an evolutionary conserved humoral component of the mucosal immune system in fish. We speculate that Ach release via nAChR from mucous cells may be modulated by GABA production in the NECs and it is required for the induction of mucus production in both normoxic and hypoxic conditions. The presence of piscidin in mucous cells may act in synergy with the autocrine/paracrine signals of Ach and GABA binding to GABA B R1B receptor that may play a local immunomodulatory function in the mucous epithelia of the gills and the respiratory air sac. The potential role of the NECs in the immunobiological behaviour of the gill/air-sac is at moment a matter of speculation. The extent to which the NECs as such may participate is elusive at this stage and waits investigation.

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod, Gadus morhua (Linnaeus, 1758).

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.

Expression of acetylcholine, its contribution to regulation of immune function and O2 sensing and phylogenetic interpretations of the African butterfly fish Pantodon buchholzi (Osteoglossiformes, Pantodontidae).

Acetylcholine (Ach) is the main neurotransmitter in the neuronal cholinergic system and also works as a signaling molecule in non-neuronal cells and tissues. The diversity of signaling pathways mediated by Ach provides a basis for understanding the biology of the cholinergic epithelial cells and immune cells in the gill of the species studied. NECs in the gill were not found surprisingly, but specialized cells showing the morphological, histochemical and ultrastructural characteristics of eosinophils were located in the gill filaments and respiratory lamellae. Much remains unknown about the interaction between the nerves and eosinophils that modulate both the release of acetylcholine and its nicotinic and muscarinic receptors including the role of acetylcholine in the mechanisms of O2 chemosensing. In this study we report for the first time the expression of Ach in the pavement cells of the gill lamellae in fish, the mast cells associated with eosinophils and nerve interaction for both immune cell types, in the gill of the extant butterfly fish Pantodon buchholzi. Multiple roles have been hypothesized for Ach and alpha nAChR in the gills. Among these there are the possible involvement of the pavement cells of the gill lamellae as O2 chemosensitive cells, the interaction of Ach positive mast cells with eosinophils and interaction of eosinophils with nerve terminals. This could be related to the use of the vesicular acetylcholine transporter (VAChT) and the alpha 2 subunit of the acetylcholine nicotinic receptor (alpha 2 nAChR). These data demonstrate the presence of Ach multiple sites of neuronal and non-neuronal release and reception within the gill and its ancestral signaling that arose during the evolutionary history of this conservative fish species.

The gas bladder of Pantodon buchholzi: Structure and relationships with the vertebrae.

We report here on the histological and structural characteristics of the gas bladder, the vertebral morphology, and the bladder-vertebra relationships of the butterfly fish, Pantodon buchholzi. The bladder opens at the boundary between the pharynx and the esophagus by a middle slit. A pneumatic duct is absent. The bladder shows a dorsolateral wall that adapts to the anfractuosities of the coelomic cavity and a ventral wall in contact with the abdominal organs. The vertebral bodies are formed by an hourglass shaped autocentrum, and by an arcocentrum reduced to several longitudinal ridges. The transverse processes adopt the structure of a cage whose walls are formed by bone trabeculae of variable size and distribution pattern. The dorsolateral wall of the bladder is a membrane that covers the kidney, adapts to the irregular shape of the vertebrae, and invades the transverse processes at several points before extending laterally. However, invasion of the vertebral bodies, the presence of a labyrinth, or the formation of respiratory parenchyma were not observed. The luminal surface of this wall is a thin respiratory barrier containing a single epithelial cell type. In addition, the wall contains numerous eosinophils that may be implicated in immune defense. The bladder ventral wall is a membrane rich in collagen, vessels, smooth muscle, and nerves that lacks a respiratory barrier. Its luminal surface contains ciliated and nonciliated cells. The two cell types appear implicated in surfactant production.

Expression of Acetylcholine- and G protein coupled Muscarinic receptor in the Neuroepithelial cells (NECs) of the obligated air-breathing fish, Arapaima gigas (Arapaimatidae: Teleostei).

The air-breathing specialization has evolved idependently in vertebrates, as many different organs can perfom gas exchange. The largest obligate air-breathing fish from South America Arapaima gigas breathe air using its gas bladder, and its dependence on air breathing increases during its growth. During its development, gill morphology shows a dramatic change, remodeling with a gradual reduction of gill lamellae during the transition from water breathing to air breathing . It has been suggested that in this species the gills remain the main site of O2 and CO2 sensing. Consistent with this, we demonstrate for the first time the occurrence of the neuroepithelial cells (NECs) in the glottis, and in the gill filament epithelia and their distal halves. These cells contain a broader spectrum of neurotransmitters (5-HT, acetylcholine, nNOS), G-protein subunits and the muscarininic receptors that are coupled to G proteins (G-protein coupled receptors). We report also for the first time the presence of G alpha proteins coupled with muscarinic receptors on the NECs, that are thought as receptors that initiate the cardiorespiratory reflexes in aquatic vertebrates. Based on the specific orientation in the epithelia and their closest vicinity to efferent vasculatures, the gill and glottal NECs of A. gigas could be regarded as potential O2 and CO2 sensing receptors. However, future studies are needed to ascertain the neurophysiological characterization of these cells.

Air- breathing in fish: Air- breathing organs and control of respiration: Nerves and neurotransmitters in the air-breathing organs and the skin.

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

Identification and distribution of neuronal nitric oxide synthase and neurochemical markers in the neuroepithelial cells of the gill and the skin in the giant mudskipper, Periophthalmodon schlosseri.

Mudskippers are amphibious fishes living in mudflats and mangroves. These fishes hold air in their large buccopharyngeal-opercular cavities where respiratory gas exchange takes place via the gills and higher vascularized epithelium lining the cavities and also the skin epidermis. Although aerial ventilation response to changes in ambient gas concentration has been studied in mudskippers, the localization and distribution of respiratory chemoreceptors, their neurochemical coding and function as well as physiological evidence for the gill or skin as site for O2 and CO2 sensing are currently not known. In the present study we assessed the distribution of serotonin, acetylcholine, catecholamines and nitric oxide in the neuroepithelial cells (NECs) of the mudskipper gill and skin epithelium using immunohistochemistry and confocal microscopy. Colocalization studies showed that 5-HT is coexpressed with nNOS, Na+/K+-ATPase, TH and VAChT; nNOS is coexpressed with Na+/K+-ATPase and TH in the skin. In the gill 5-HT is coexpressed with nNOS and VAhHT and nNOS is coexpressed with Na+/K+-ATPase and TH. Acetylcholine is also expressed in chain and proximal neurons projecting to the efferent filament artery and branchial smooth muscle. The serotonergic cells c labeled with VAChT, nNOS and TH, thus indicating the presence of NEC populations and the possibility that these neurotransmitters (other than serotonin) may act as primary transmitters in the hypoxic reflex in fish gills. Immunolabeling with TH antibodies revealed that NECs in the gill and the skin are innervated by catecholaminergic nerves, thus suggesting that these cells are involved in a central control of branchial functions through their relationships with the sympathetic branchial nervous system. The Na+/K+-ATPase in mitochondria-rich cells (MRCs), which are most concentrated in the gill lamellar epithelium, is colabeled with nNOS and associated with TH nerve terminals. TH-immunopositive fine varicosities were also associated with the numerous capillaries in the skin surface and the layers of the swollen cells. Based on the often hypercapnic and hypoxic habitat of the mudskippers, these fishes may represent an attractive model for pursuing studies on O2 and CO2 sensing due to the air-breathing that increases the importance of acid/base regulation and the O2-related drive including the function of gasotransmitters such as nitric oxide that has an inhibitory (regulatory) function in ionoregulation.

The lungs of Polypterus senegalus and Erpetoichthys calabaricus: Insights into the structure and functional distribution of the pulmonary epithelial cells.

The present article is a comparative, structural study of the lung of Polypterus senegalus and Erpetoichthys calabaricus, two species representative of the two genera that constitute the Polypteriformes. The lung of the two species is an asymmetric, bi-lobed organ that arises from a slit-like opening in the ventral side of the pharynx. The wall is organized into layers, being thicker in P. senegalus. The inner epithelium contains ciliated and non-ciliated bands. The latter constitute the respiratory surface and are wider in E. calabaricus. The air-blood barrier is thin and uniform in P. senegalus and thicker and irregular in E. calabaricus. In the two species, the ciliated areas contain ciliated cells, mucous cells and cells with lamellar bodies. Additionally, P. senegalus contains polymorphous granular cells (PGCs) and neuroendocrine cells (NECs) while E. calabaricus lacks PGCs but shows granular leukocytes and a different type of NEC. Interestingly, ciliated cells and secretory cells show a dual morphology in E. calabaricus indicating the presence of cellular subtypes and suggesting more complex secretory activity. Also in E. calabaricus, cilia show a novel doublet-membrane interaction that may control the displacement of the microtubule doublets. The subepithelium is a connective layer that appears thicker in P. senegalus and contains, in the two species, fibroblasts and granulocytes. The outer layer contains bundles of richly innervated striated muscle. This layer is likely involved in the control of lung motion. In the two species, smooth muscle cells constitute a limiting layer between the subepithelium and the striated muscle compartment. The role of this layer is unclear.

The Effect of Hypoxia and Hyperoxia on Growth and Expression of Hypoxia-Related Genes and Proteins in Spotted Gar Lepisosteus oculatus Larvae and Juveniles.

We studied the molecular responses to different water oxygen levels in gills and swim bladder of spotted gar (Lepisosteus oculatus), a bimodal breather. Fish at swim-up stage were exposed for 71 days to normoxic, hypoxic, and hyperoxic water conditions. Then, all aquaria were switched to normoxic conditions for recovery until the end of the experiment (120 days). Fish were sampled at the beginning of the experiment, and then at 71 days of exposure and at 8 days of recovery. We first cloned three hypoxia-related genes, hypoxia-inducible factor 2α (HIF-2α), Na(+) /H(+) exchanger 1 (NHE-1), and NHE-3, and uploaded their cDNA sequences in the GeneBank database. We then used One Step Taqman® real-time PCR to quantify the mRNA copies of target genes in gills and swim bladder of fish exposed to different water O2 levels. We also determined the protein expression of HIF-2α and neuronal nitric oxide synthase (nNOS) in the swim bladder by using confocal immunofluorescence. Hypoxic stress for 71 days significantly increased the mRNA copies of HIF-2α and NHE-1 in gills and swim bladder, whereas normoxic recovery for 8 days decreased the HIF-2α mRNA copies to control values in both tissues. We did not found significant changes in mRNA copies of the NHE-3 gene in either gills or swim bladder in response to hypoxia and hyperoxia. Unlike in normoxic swim bladder, double immunohistochemical staining in hypoxic and hyperoxic swim bladder using nNOS/HIF-2α showed extensive bundles of HIF-2α-positive nerve fibers in the trabecular musculature associated with a few varicose nNOS immunoreactive nerve terminals.

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.

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.

The mechanism of olfactory organ ventilation in Periophthalmus barbarus (Gobiidae, Oxudercinae).

Periophthalmus barbarus Linnaeus, 1766 has many adaptations for amphibious life as a consequence of tidal zone occupation. One of them is the ability to keep a little amount of water and air in mouth while on land or in hypoxic water, correlated with closing a gill lid for gas exchange improvement. It causes that mechanisms of olfactory organ ventilation described in other species of actinopterygians (compression of accessory nasal sac(s) by the skull and jaw elements while mouth and gill lid moving) are not in operation. There is a specific mechanism of olfactory organ ventilation independent on jaw and skull elements movements. Compression of accessory nasal sacs is possible by a0 contraction and it is a movement effect on bones combined by ligaments. This process can be observed on P. barbarus as lifting the rostral part of the head. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00435-012-0167-y) contains supplementary material, which is available to authorized users.