Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.     

Insulin-, glucagonand somatostatin-like immunoreactivity in the endocrine pancreas of the lungfish,Neoceratodus forsteri

Summary The endocrine pancreas of the Australian lungfish,Neoceratodus forsteri, was investigated immunocytochemically for the presence of polypeptide hormone-producing cells. Three cell types were identified, namely insulin-, glucagon-, and somatostatin-immunoreactive elements. The insulin cells are confined solely to the center of the islets. Glucagon and somatostatin cells are distributed peripherally around the central mass of the insulin cells. Isolated cells or clusters of glucagon and somatostatin cells are also dispersed within the exocrine parenchyma. The immunoreactive cell types are compared with those staining with standard histological procedures. The spatial relationships of the different cell populations are examined.     

Immunocytochemical localization and characterization of mammalian prolactin- and somatotropin-like material in the pituitary of the Australian lungfish,Neoceratodus forsteri

The aim of the present study was to define at the light-microscopic level expression of prolactin and somatotropin material in the pituitary gland of the Australian lungfish, Neoceratodus forsteri, by use of polyclonal antibodies against ovine prolactin (oPRL) and bovine somatotropin (bSTH). Substances immunologically related to mammalian oPRL as well as bSTH were detected in two morphologically different cell types in the distal lobe, corresponding to the acidophilic cells. The specificity of the antibodies was initially confirmed in a porcine tissue control system. First, our absorption studies confirm that in Neoceratodus the anti-oPRL identifies part of an oPRL-like molecule different from bSTH. Secondly, the anti-bSTH identifies both part of a bSTH-like molecule proper to bovine and Neoceratodus STH, and part of a bSTH-like molecule having antigenic determinants in common with both bSTH and oPRL. This part of the oPRL is, however, not shared with the Neoceratodus PRL as revealed by the anti-oPRL. Altogether these observations support the concepts: (1) that mammalian PRL and STH, or part of those, were established early in evolution, and (2) that dipnoans as living sarcopterygians have an ancestor in common with the early amphibians. The exact nature and physiological functions of the substances detected remain to be defined.     

Vascularization of the pituitary of the Australian lungfish and Neoceratodus forsteri

The vascularization of the brain and the pituitary region of the Australian lungfish, Neoceratodus forsteri is described from serial section reconstruction. The distal lobe has no direct arterial blood supply and receives blood solely from a pituitary portal system basically similar to that of other sarcopterygians. The primary capillary plexus of the median eminence receives its arterial blood from the infundibular arteries, which on their way distribute some small branches to the prechiasmatic region. The primary plexus also receives capillaries from the adjacent pial hypothalamic plexus. The primary capillary plexus of the median eminence comprises a rostral 'uncovered' and caudal 'covered' part which are not sharply delineated. Distinct portal vessels connect the 'uncovered' rostral part of the primary plexus with the secondary capillary plexus supplying the rostral subdivision of the pars distalis. The 'covered' caudal part of the primary plexus merges into the proximal subdivision of the pars distalis, apparently without formation of distinct portal vessels. The primary plexus has some connections with the plexus intermedius via a hypophysial stem capillary plexus. The plexus intermedius has a substantial arterial supply and gives off capillaries to the parenchyma of the pars intermedia. The adenohypophysis is drained into an unpaired hypophysial vein. The significance of the vascular pathways is discussed from comparative, functional, and evolutionary viewpoints.     

The steroidogenic response to angiotensin II in the Australian lungfish,Neoceratodus forsteri

Corticosterone, aldosterone and cortisol were found to be present in lungfish plasma. Plasma levels of these hormones were measured in lungfish following separate single intramuscular injections of three forms of angiotensin II; [Asp¹, Ile⁵], [Asp¹, Val⁵] and [Asn¹, Val⁵]. Aldosterone levels were significantly elevated in response to [Asp¹, Ile⁵] AII and [Asn¹, Val⁵] AII injection. [Asp¹, Val⁵] AII increased plasma corticosterone levels. The difference between these data and the negative results previously reported by Blair-West et al. (1977) are discussed.Abbreviations AII angiotensin II - bw body weight - DOC deoxycorticosterone - RAS renin-angiotensin system - RIA radioimmuno assay     

Australian lungfish (Neoceratodus forsteri: Dipnoi) have low genetic variation at allozyme and mitochondrial DNA loci: a conservation alert?

Genetic variation at allozyme and mitochondrialDNA loci was investigated in the Australianlungfish, Neoceratodus forsteri Krefft1870. Tissue samples for genetic analysis weretaken non-lethally from 278 individualsrepresenting two spatially distinct endemicpopulations (Mary and Burnett rivers), as wellas one population thought to be derived from ananthropogenic translocation in the 1890's(Brisbane river). Two of 24 allozyme lociresolved from muscle tissue were polymorphic.Mitochondrial DNA nucleotide sequence diversityestimated across 2,235 base pairs in each of 40individuals ranged between 0.000423 and0.001470 per river. Low genetic variation atallozyme and mitochondrial loci could beattributed to population bottlenecks, possiblyinduced by Pleistocene aridity. Limited geneticdifferentiation was detected among rivers usingnuclear and mitochondrial markers suggestingthat admixture may have occurred between theendemic Mary and Burnett populations duringperiods of low sea level when the drainages mayhave converged before reaching the ocean.Genetic data was consistent with theexplanation that lungfish were introduced tothe Brisbane river from the Mary river. Furtherresearch using more variable genetic loci isneeded before the conservation status ofpopulations can be determined, particularly asanthropogenic demands on lungfish habitat areincreasing. In the interim we recommend amanagement strategy aimed at conservingexisting genetic variation within and betweenrivers.     

The epithelial sodium channel in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Epithelial sodium channel (ENaC) is a Na⁺-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na⁺ absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, β and γ proteins. The nENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.     

Ultrastructure of developing tooth plates in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

While the lungfish dentition is partially understood as far as morphology and light microscopic structure is concerned, the ultrastructure is not. Each tooth plate is associated with a dental lamina that develops from the inner layer of endodermal cells that form the oral epithelium. Dentines, bone and cartilage of the jaws differentiate from mesenchyme cells aggregating beneath the oral endothelium. Enamel, in the developing and in the mature form, has similarities to that of other early vertebrates, but unusual characters appear as development proceeds. Ameloblasts are capable of secreting enamel, and, with mononuclear osteoclasts, of remodelling the bone below the tooth plate. The forms of dentine, all based largely on an extracellular matrix of collagen and mineralised with biological apatite, differ from each other and from the underlying bone in the ultrastructure of associated cells and in the mineralised extracellular matrices produced. Cell processes emerging from the odontoblasts and from the osteoblasts vary in length, degree of branching and of anastomoses between the processes, although all of the cell types have large amounts of rough endoplasmic reticulum. Mineralisation of the extracellular matrices varies among the enamel, dentines and bone in the tooth plate. In addition, the development of the hard tissues of the tooth plates indicates that many of the similarities in fine structure of the dentition in lungfish, to tissues in other fish and amphibia, apparent early in development, disappear as the dentition matures.     

Cranial nerves in the Australian lungfish,Neoceratodus forsteri,and in fossil relatives (Osteichthyes: Dipnoi)

Three systems, two sensory and one protective, are present in the skin of the living Australian lungfish, Neoceratodus forsteri, and in fossil lungfish, and the arrangement and innervation of the sense organs is peculiar to lungfish. Peripheral branches of nerves that innervate the sense organs are slender and unprotected, and form before any skeletal structures appear. When the olfactory capsule develops, it traps some of the anterior branches of cranial nerve V, which emerged from the chondrocranium from the lateral sphenotic foramen. Cranial nerve I innervates the olfactory organ enclosed within the olfactory capsule and cranial nerve II innervates the eye. Cranial nerve V innervates the sense organs of the snout and upper lip, and, in conjunction with nerve IX and X, the sense organs of the posterior and lateral head. Cranial nerve VII is primarily a motor nerve, and a single branch innervates sense organs in the mandible. There are no connections between nerves V and VII, although both emerge from the brain close to each other. The third associated system consists of lymphatic vessels covered by an extracellular matrix of collagen, mineralised as tubules in fossils. Innervation of the sensory organs is separate from the lymphatic system and from the tubule system of fossil lungfish.     

Skin structure in the snout of the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Many fossil lungfish have a system of mineralised tubules in the dermis of the snout, branching extensively and radiating towards the epidermis. The tubules anastomose in the superficial layer of the dermis, forming a plexus consisting of two layers of vessels, with branches that expand into pore canals and flask organs, flanked by cosmine nodules where these are present. Traces of this system are found in the Australian lungfish, Neoceratodus forsteri, consisting of branching tubules in the dermis, a double plexus below the epidermis and dermal papillae entering the epidermis without reaching the surface. In N. forsteri, the tubules, the plexus and the dermal papillae consist of thick, unmineralised connective tissue, enclosing fine blood vessels packed with lymphocytes. Tissues in the epidermis and the dermis of N. forsteri are not associated with deposits of calcium, which is below detectable limits in the skin of the snout at all stages of the life cycle. Canals of the sensory line system, with mechanoreceptors, are separate from the tubules, the plexus and the dermal papillae, as are the electroreceptors in the epidermis. The system of tubules, plexus, dermal papillae and lymphatic capillaries may function to protect the tissues of the snout from infection.     

Pineal-retinal relationships: rhythmic biosynthesis and immunocytochemical localization of melatonin in the retina of the pike (Esox lucius)

Summary The levels of melatonin and the activities of two enzymes of the melatonin biosynthetic pathway, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were measured throughout the light-dark cycle in the retina of a teleost fish, the pike. HIOMT activity did not display significant variations, whereas NAT activity and melatonin content showed a daily rhythm, high levels occurring during the night. The profiles of the latter two rhythms did not closely match one another and differed from those previously described in the pineal organ of the same species. These results are discussed with respect to a possible paracrine role of retinal melatonin. Melatonin-like immunoreactivity was found in the photoreceptor cell layer and in the Müller cells of the inner nuclear layer. The intensity of the melatonin-like immunoreactivity varied throughout the 24 h light-dark cycle, in good correlation with the variations in the melatonin level as measured by radioimmunoassay.This article is dedicated to the memory of Dr. Klaus Hoffmann     

An immunocytochemical study of the endocrine pancreas in the Australian fat-tailed dunnart (Sminthopsis crassicaudata)

Summary The endocrine pancreas of the Australian fattailed dunnart, Sminthopsis crassicaudata, was investigated by means of electron-microscopic immunocytochemistry using the protein A-gold technique on London resin (LR) white-embedded tissue. The primary antibodies used were raised against insulin, glucagon, somatostatin and pancreatic polypeptide. The morphology of the secretory granules differed in the four cell types. The insulin cells are pleomorphic, and the secretory granules composed of an electron-dense core surrounded by an electron-lucen halo. The glucago cells possess granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells have large, less dense secretory granules. The pancreatic polypeptide cells show small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborated by immunocytochemical data at the light-or preferably electron-microscopic level. Recent developments in immuno-electron-microscopic techniques have contributed to a better knowledge of cells responsible for the secretion of a wide variety of hormones, as in this study.     

The cell types in the adenohypophysis of the South-American lungfish,Lepidosiren paradoxa,with special reference to immunocytochemical identification of the corticotropin-containing cells

The histological features and distribution of cell types in the distal lobe of Lepidosiren resemble those of Protopterus. Three "basophilic" cell types are described, whereas the identification of two acidophilic cell types is uncertain. In the intermediate lobe two cell types have been found. Anti-(1-24)ACTH IgG was used in the unlabeled antibody-enzyme method to identify corticotropin-containing cells in the adenohypophysis of Lepidosiren with light and electron microscopy. Corticotropin was demonstrated in cells of the distal lobe and the intermediate lobe. The staining reaction in the distal lobe is localized in the rostrally distributed lead-hematoxylin positive cells. At the ultrastructural level the immunoreaction in these distal lobe cells is localized on polymorphic granules ranging from 130 to 210 nm. Absorption experiments show that the immunoreactive cells in the distal lobe contain at least residues 1-3 and 14-17 of the naturally occurring corticotropin hormone, while the intermediate lobe cells contain alpha-MSH or at least residues 1-3 of ACTH. The plasma level of corticotropin was determined to be 71 ng/l by means of radioimmunoassay (RIA).     

The morphology of the lung of the African lungfish,Protopterus aethiopicus

Summary The lung of the African lungfish (Protopterus aethiopicus) is paired, long and cylindrical. It is situated on the dorsal aspect of the coelomic cavity ventral to the ribs. Much of the gas exchange tissue is found in the proximal aspect of the lung with the caudal part largely taken up by a centrally situated air-duct with a few large peripherally located alveoli. Interalveolar septa, arranged at differing hierarchical levels from the air-duct, subdivide the lung into alveoli, the gas exchange compartments. The alveolar surface is covered by some cells characterized by microvilli on their free surface, while others are devoid of such structures. The general organization of the lung of Protopterus aethiopicus is similar to that of the other genera of Dipnoi, Neoceratodus and Lepidosiren, with the interalveolar septa increasing the surface area for gas exchange through pulmonary compartmentation. The abundant septal smooth muscle fibres and elastic tissue may contribute to the physiomechanical compliance of the lung. The undifferentiated alveolar pneumocytes and the double capillary system, observed in Protopterus, in general appear to characterize the very primitive lungs of the lower air-breathing vertebrates.     

The ultrastructure and distribution of 5-HT-containing neurons in the intestine of a teleost fish,Aldrichetta forsteri

Summary The intestine of Aldrichetta forsteri was examined ultrastructurally for evidence of 5-HT-containing intrinsic neurons. A population of nerve cell bodies and processes characterized by the presence of amine-containing vesicles was found after the destruction of adrenergic processes with 6-hydroxydopamine. The distribution of the neurons matched that of the 5-HT-containing neurons previously seen with formaldehyde-induced fluorescence and 5-HT immunohistochemistry in A. forsteri and other teleosts. The axons made close contacts with the circular smooth muscle layer and the muscularis mucosae. The axons also lay exposed to the epithelial cells of the mucosa but did not make contact with neurons or with the longitudinal smooth muscle layer.     

Immunocytochemical and electron-microscopic study of the elasmobranch nucleus sacci vasculosi

Summary The elasmobranch nucleus sacci vasculosi was studied by means of electron microscopy (in the dogfish) and immunocytochemistry (in the dogfish and the skate) by using antibodies against tyrosine hydroxylase, alpha-melanocyte-stimulating hormone, somatostatin, serotonin, and substance P. Ultrastructural study of the dogfish nucleus sacci vasculosi shows the presence of medium-sized cells that possess numerous mitochondria but that have no dense-core vesicles in the cytoplasm or in cell processes. Fibres of the conspicuous tractus sacci vasculosi have a beaded appearance and form conventional synapses with dendrites and cell perikarya of the nucleus sacci vasculosi. The perikarya of this hypothalamic nucleus were not immunoreactive to any of the antibodies tested, and fibres immunopositive to tyrosine hydroxylase, alpha-melanocyte-stimulating hormone, somatostatin, serotonin, and substance P were scarce within this nucleus, in both the dogfish and the skate. Dorsal to the nucleus sacci vasculosi, there are numerous positive neuronal processes in addition to many small neurons that show immunoreactivity to alpha-melanocyte-stimulating hormone, somatostatin and tyrosine hydroxylase. Two types of neuron occur in this dorsal region, displaying dense-core vesicles of either 100–160 nm or 60–100 nm diameter in their cytoplasm; they were identified as peptide-containing and monoamine-containing neurons, respectively. The neuropil of this region has a significantly different ultrastructure from that of the nucleus sacci vasculosi, with many processes containing dense-core vesicles. This group of neurons, located dorsal to the nucleus sacci vasculosi and showing (a) immunoreactivity to neuropeptides or to monoamine-synthesizing enzyme, and (b) cytoplasm with dense-core vesicles, was considered not to be a part of the nucleus sacci vasculosi but rather part of the nucleus tuberculi posterioris. These results support the non-peptidergic and non-aminergic character of the nucleus sacci vasculosi.     

C-PON immunoreactive neurons in the neostriatum of the hedgehog (Erinaceus europaeus): a correlated light- and electron-microscopic study

The present study provides light- and electronmicroscopic immunocytochemical data on the presence of neurons that are immunoreactive to the C-terminal flanking peptide of neuropeptide Y, C-PON, in the neostriatum of the hedgehog (Erinaceus europaeus). Positive neurons have mostly fusiform or round perikarya from which two to four poorly branched processes arise. Immunostained fibers and puncta are also evenly distributed throughout the neostriatum. Ultrastructurally, each neuron exhibits a deeply invaginated nucleus surrounded by abundant cytoplasm with a well-developed rought endoplasmic reticulum and Golgi apparatus. Positive neurons receive symmetric and asymmetric synapses from unlabeled terminals. The results of this study can be correlated with previous findings, as the C-PON-positive neurons of the hedgehog resemble medium-sized neostriatal neurons that are known to be local circuit neurons exhibiting C-PON in the rat. Thus, a high degree of C-PON neuronal system phylogenetic conservation and function can be postulated for the neostriatum of mammals.     

Immunocytochemical localization of glycine and glycine receptors in the retina of the frog <Emphasis Type="Italic">Rana ridibunda</Emphasis>

Glycine and glycine receptors (GlyRs) were analyzed immunocytochemically in the retina of the frog Rana ridibunda. Glycine was localized to somata of glycinergic amacrine and interplexiform cells. Approximately 50% of the cells in the amacrine cell layer were found to be glycinergic. GlyRs of the inner plexiform layer (IPL) were localized to brightly fluorescent puncta, probably representing postsynaptic clusters of GlyRs. GlyR clusters were not evenly distributed across the IPL but showed patterns of stratification specific for the various GlyR subunits. Clusters containing the 1 subunit formed four narrow strata within the IPL. Clusters containing the 3 subunit were more abundant and covered the whole IPL, with a band of higher density in stratum 3. Clusters of GlyRs were also observed in the outer plexiform layer. Thus, several isoforms of synaptic GlyRs involved with different synapses and inhibitory circuits are present in the frog retina.This work was supported by the Deutsche Forschungsgemeinschaft SFB269/B4