Histological, histochemical and ultrastructural studies on the bulbus arteriosus of the sticklebacks, Gasterosteus aculeatus and Pungitius pungitius (Pisces: Teleostei)

The bulbar wall has three layers. Its lining consists of squamous-columnar endothelial cells that store neutral mucopolysaccharides and are PAS-positive. They do not contain large amounts of acid phosphatase, acid mucopolysaccharides, glycogen or lipids. A morphometric analysis shows that 32% of the cell volume in Pungitius and 12% in Gasterosteus is occupied by specific granules, 100–600 nm in diameter. According to X-ray probe micro-analysis, these granules bind chromium ions, even though the endothelial cells do not contain catecholamines. Rootlets, packed with plasmalemmal vesicles, extend from the endothelial cells into the middle layer of the bulbus. Here, smooth muscle cells alternate with elastic fibres. The staining reactions of bulbar elastica are compared with those in the mammalian aorta and the ligamentum nuchae. The outer layer of the bulbus is visceral pericardium and beneath its covering mesothelial cells are numerous collagen fibres, non-myelinated nerves, occasional fibroblasts and melanocytes. Scanning electron microscopy shows that the bulbar lining is thrown into longitudinal folds, but that there are no trabeculae subdividing the lumen.
Many features of the bulbus arteriosus may be related to the low systolic pressures of teleosts and to the proximity of their heart and gills. In contrast to mammals, only a small part of the arterial system can act as a windkessel. The bulbus is thus more distensible than the mammalian aorta and must lie within the pericardial cavity so that its greater excursions can be accommodated. Perhaps because the bulbus is so distensible, it has elastic fibres rather than lamellae. This in turn may affect the organization of the smooth muscle cells which do not form "span muscles" as in some mammalian aortae. Like most cells in the bulbus, they are joined to others by desmosomes. Evidently, firm cohesion is important in highly distensible vessels.     

Carbohydrate-containing endothelial cells lining the bulbus arteriosus of teleosts and the conus arteriosus of elasmobranchs (Pisces)

The study is a survey of the shape and carbohydrate histochemistry of the endothelial cells lining the conus arteriosus of 10 species of elasmobranchs and the bulbus arteriosus of 80 species of teleosts. Intensely PAS-positive cells that were often tall, were found in many teleosts and were typical of phylogenetically advanced species. They were not seen in any elasmobranch. Most of the teleosts with strongly PAS-positive cells were marine fish or euryhaline animals that were caught in freshwater. Beyond this, it is difficult to generalize on their life-style or habitat, for they included small fish, large fish, fast swimmers, bottom-living forms, deep-water fish, littoral species and Antarctic "bloodless" forms. It is likely that the PAS-positivity of the endothelial cells can be attributed to the moderately-dense granules revealed by E.M., but as yet the significance of these is unclear.     

Heart structure of some deep-sea fish (Teleostei: Macrouridae)

The hearts of 29 species of macrourid teleosts were examined in this study. For the one species for which a length range was available ( Coryphaenoides (C.) rupestris ), the heart weight as a percentage of body weight was 0·059. This is similar to values for relatively inactive fish. The atrial myocardium was reduced and had only a sparse trabecular network. In some species it was surrounded by a highly developed epicardium, but in others there was interstitial connective tissue in the myocardium that may serve to strengthen this chamber. The ventricle was entirely spongy, and all species lacked an outer compact layer of myocardium and associated coronary vasculature. All the ventricles were sac-like in form. The bulbus arteriosus was highly complex, and in its proximal portion there was an endothelially-lined, inner tube surrounded by a spongy network of blood-filled spaces, outside which was an outer compact layer of smooth muscle and elastica. These features of the bulbus may prevent backflow of blood after ventricular systole. The endothelial cells lining the bulbus were usually PAS-positive and in some species contained acid mucopolysaccharides.     

Functional morphology of the bulbus arteriosus of rainbow trout (Salmo gairdneri Richardson)

The rate of expansion in volume of the bulbus arteriosus with increase in pressure is measured. From this it is calculated the elastic rebound of the bulbus can account for approximately 25 % of blood flow in the ventral aorta; this proportion decreases as cardiac output increases. The structure of the wall of the bulbus is shown to consist of a compact outer layer with a series of separate longitudinal elements on the inner surface. These elements are connected to the compact layer by numerous radial fibres. This structure equalizes strain in all the structural elements of the wall during large changes in volume. Evidence is discussed which shows that the bulbus arteriosus in teleosts is morphologically and biochemically distinct from the ventral aorta. The bulbus is probably of cardiac origin rather than an expansion of the posterior end of the aorta as generally supposed.     

Scanning-beam electron microscopy of the conidia of the brown and albino filamentous varieties of Histoplasma capsulatum

Aspects of the surface appearance and external morphology of the conidial forms of the albino and brown filamentous varieties ofHistoplasma capsulatum as seen by scanning-beam electron microscopy are described and illustrated by electron micrographs. Septal areas between the hyphal cells of the supporting mycelium are seen as slightly elevated annular rings or ridges. The smooth micro- and macroconidia of the albino filamentous variety show a fine wrinkling and delicate irregularities of surface texture. Macroconidia of the brown filamentous variety are illustrated showing variations in numbers and respective length of the conspicuous wall projections or tubercles. The 3-dimensional perspective, unusual depth of focus, and high resolving power of the stereoscan technique permitted observations of external conidia morphology unattainable by other methods of study.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Identification and distribution of nitric oxide synthase in the brain of adult Antarctic teleosts

We investigated the presence of nitric oxide synthase (NOS) in brain of adult Antarctic teleosts by indirect immunofluorescence technique using a synthetic rat neuronal NOS (nNOS) antibody. The following species were examined: Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni (haemoglobin-rich), Chionodraco hamatus and Pagetopsis macropterus (haemoglobin-free). Immunoreactive cell bodies were localized in dorsal telencephalon, in hypothalamus, in optic tectum of the mesencephalon as well as in Purkinje cells of the cerebellum. No differences were observed in the localization of the nNOS immunopositivity in the Antarctic teleosts brains examined and NOS distribution was similar to that described in other teleosts, suggesting that nitric oxide (NO) may also function as a neurotransmitter in the brain of Antarctic teleosts. A strong immunopositivity was observed in the cerebral blood vessels of the icefishes suggesting that NO may play a pivotal role in the regulation of the cerebral blood flow especially in these haemoglobin-free species.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

Summary The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve, bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

The structure and ultrastructure of the sinus venosus in the mature dogfish (Scyliorhinus canicula): the endocardium, the epicardium and the subepicardial space

The sinus venosus of fish is the most caudal chamber of the heart. It is often reduced in teleosts but well developed in elasmobranchs. The sinus venosus of the dogfish (Scyliorhinus canicula) is vital, since it harbours key elements such as a little known neuroendocrine system and the nodal tissue. However, the study of its structure is still incomplete. We examined the endocardium, epicardium and subepicardium of the sinus venosus in mature dogfishes. The wall is 100-250 microm thick and comprises three main layers. Large bundles of myocardial cells occupy the middle layer. The endothelial ensheathing is composed of thin endocardial cells with prominent nuclei towards the lumen, whose cytoplasm contains numerous dense bodies and moderately dense bodies, 150-800 nm in diameter and large vacuoles. The possible functions of these organelles are discussed. The outermost layer is made of a robust sheet of cuboidal epicardial cells separated from the subepicardium by a conspicuous basal lamina. Numerous microvilli towards the pericardial cavity and elliptical vesicles are located in the apex of epicardial cells. A thick layer richly endowed with dense bundles of collagen fibres forms the subepicardial space. This structure should be contrasted with the venous return mechanism of elasmobranchs.     

A comparative study on the innervation and the vascularization of the bulbus arteriosus in teleost fish

Summary The structure of the bulbus arteriosus of a wide range of teleost fish is described with particular reference to the vascularization and innervation. The adventitia of the organ consists of blood vessels and large nerve bundles in a collagen matrix. The nerve bundles contain monoamines, and fluorescence studies show small terminal bundles penetrating the muscular media; this is confirmed by electron microscopy. The media consists of an extensive elastic tissue matrix with a spiral arrangement of smooth muscle cells joined end to end by desmosomes and presumed electrotonic junctions. The muscle cells are innervated only at the adventitia/media boundary and the significance of this innervation is discussed. It is proposed that there is a correlation between the degree of vascularization and innervation and the activity of a particular species offish.     

Essential features of endocardial and myocardial morphology: SEM and TEM studies

The conducting pathway of the ferret's myocardium and endocardium was studied under the electron and scanning microscope. Comparisons between the two methods showed that the scanning microscope is well suited for those dimensional demonstration of biological material. Contrary to a relative absence of interspecific differences in endocardial morphology, there is a strong variation of this morphology related to the intracardiac localization of the endocardial cells. The following findings were obtained. S.e. microscopically, it was observed that the endocardium of the sino-atrial node region is not smooth, and that, more likely, it shows rough surfaced profiles. The electron microscopic study shows that the cells of the S-A node are elongated. The S-A node is located at the junction of the superior vena cava with the right atrial wall. It consists of nodal fibres which are embedded in a richinterstitial connective tissue (Figs 1-8). The Purkinje fibres originate from large bundles in the region of the right and left atrioventricular valve in the area where heart muscle fibres were originally described by Purkinje (Purkinje, 1845); these fibres, meanwhile, have become synonymous with cells of the generalized conducting system. The Purkinje fibres consist of a poorly developed contractile apparatus and contain unorganized, fine, filamentous material (Illustration 1). The SR is poorly developed, transverse tubules are absent. S.e. microscopically, one can visualize the trabecular system and the sinusoids. The trabeculae obtain muscle fibres rich in contractile material and transverse tubules. The trabeculae appear to be tendonous (chordae tendineae), especially when they freely traverse the ventricular cavity (Fig. 16). The interventricular septum (the muscle fibres from this region) takes its origin from large bundles in the region of the right and left atrioventricular valves. The endocardium of the interventricular septum is filled with large numbers of plasma-lemma folds (Figs 17, 18). The endocardium which covers the papillary muscle has a thickness of 0.5 micron. The endocardial cells lie on the myocardium so close and so thin that the surface relief and part of the atriation of the myocardium are visible (Figs 13-15).     

Immunohistochemical and ultrastructural localisation of peptide-containing nerves and myocardial cells in the human atrial appendage

Summary The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptidecontaining nerves and myocardial cells producing ANP.     

Observations on the histology and histochemistry of the oesophagus of the perch, Perca fluviatilis L.

The anatomy and enzyme histœhemistry of the œsophagus of a freshwater teleost perch ( Perca fluviatilis L.) has been studied. The mucosa is composed of an undifferentiated layer of basal epithelial cells, mucous cells and surface epithelial cells. The submucosa is compact and contains blood vessels, nerves and bundles of striated longitudinal muscles. The outer circular muscular layer contains both striated and smooth muscle fibres. Electron microscopy shows that the surface epithelial cells are degenerative and that they surround and support pores of the mucous cells. Undifferentiated epithelial cells undergo cytoplasmic changes and eventually become surface epithelial cells. Mucous cells arise from the undifferentiated epithelial cells and their droplets contain acid and neutral mucosubstances. Alkaline phosphatase, non-specific carboxylic esterases and aminopeptidase activity is absent in the mucosa. However, acid phosphatase activity is localized in the surface epithelial cells.     

Sensory structures at the surface of fish skin I. Putative chemoreceptors

Fish skin contains solitary epidermal sensory cells which, on evidence from their cytology, are believed to be chemosensory. The external appearance of the apical sensory processes of these cells, as seen by scanning electron microscopy, is shown in four species of ostariophysan teleosts, and is compared with the morphology of the pores of external taste buds. The apical processes of the gustatory cells are simple in form in all cases so far investigated in gnathostome fishes, but in some cases the solitary sensory cells have apical processes divided distally into a number of smaller processes. In the dipnoan fish Protopterus amphibius , external taste buds have simple blunt gustatory processes protruding through a cap of mucus that covers the taste bud pore. Solitary sensory cells in this species have a bulbous undivided apical process. In the lampreys, the 'end buds' have an apical morphology different from the taste bud pores of teleost fish. Lamprey epidermis has numerous solitary sensory cells each bearing a number of microvilli.     

Histologic study of the teleost liver. II. The blood vessel system

In the liver of the teleosts investigated in the present study the sinusoidal region shows a space of DISSE, which contains numerous microvilli originating from the hepatocytes. In some species, especially in Tetraodon leiurus, there are also bundles of collagenous fibrils. In the DISSE's space of larger sinusoids (transition sinusoids) sections of filament-rich cells are found. These are sometimes interconnected by desmosomes and can be interpreted as processes of smooth muscle cells from the region of the venae hepaticae. The endothelium of smaller sinusoids is fenestrated and shows micropinocytotic activity. The endothelia of the transition sinusoids and of the venae hepaticae are endowed with structures, which can be interpreted as macrovesicles. In the sinusoidal region true KUPFFER-cells and ITO-cells could not be observed. Nevertheless, the close location of granulocytes to the sinusoidal endothelium suggests that phagocytotic processes cannot be excluded for the sinusoidal region. Exceptionally, in Hemihaplochromis multicolor there were also signs of possible phagocytosis by sinusoidal endothelial cells. The chemomorphology of the sinusoidal region, above all the evidence of alkaline phosphatase, shows great differences between species. The examination of the larger blood-vessels in the liver of Haplochromis burtoni reveals venae portae with very thin walls and venae hepaticae with thick walls, which contain smooth muscle cells. Granulocytes and melanocytes are abundant in the wall of the venae hepaticae. This phenomenon indicates that the defence-functions, which in the liver of higher vertebrates are carried out by the sinusoids (KUPFFER-cells), are undertaken by the region of the venae hepaticae in the liver of Haplochromis burtoni, which is free of KUPFFER-cells. On their extrahepatic course the venae portae are surrounded by a sleeve of exocrine pancreatic tissue, which accompanies the vessels deep into the liver. The pancreatic cells bordering the thin-walled venae portae have sparse microvilli indicating a transfer of substances between venous blood and pancreas similar to the sinusoidal region of the liver. Furthermore, the investigation resulted in a clue to the innervation of the exocrine pancreas.     

Neuropeptide Y-like immunoreactivity in the cardiovascular nerve plexus of the elasmobranchs Raja erinacea and Raja radiata

Summary The distribution of nerves showing neuropeptide Y (NPY)-like immunoreactivity in the cardiovascular system of elasmobranchs and teleosts has been investigated. Two species of teleosts, the rainbow trout (Salmo gairdneri) and the Atlantic cod (Gadus morhua), and three species of elasmobranchs, the spiny dogfish (Squalus acanthias), the little skate (Raja erinacea) and the starry ray (Raja radiata), were used in this study. An innervation of the cardiovascular system by an NPY-like substance was found only in the two species of Raja. A rich innervation was encountered in these skates, with the highest density of fibres in the wall of the ventricle, the conus arteriosus, the coeliac artery and smaller mesenterial vessels. In the vessels, the fibres formed a plexus at the adventitio-mediol border. Few fibres were found in the walls of the dorsal aorta, the sinus venosus and the atrium, and no fibres were observed in the walls of the ventral aorta. Falck-Hillarp fluorescence histochemistry showed the presence of a rich innervation of arteries and arterioles of the gut by catecholamine-containing nerve fibres.     

Differentiation of the cardiac outflow tract components in alevins of the sturgeon Acipenser naccarii (Osteichthyes, Acipenseriformes): implications for heart evolution

Previous work showed that in the adult sturgeon an intrapericardial, nonmyocardial segment is interposed between the conus arteriosus of the heart and the ventral aorta. The present report illustrates the ontogeny of this intermediate segment in Acipenser naccarii. The sample studied consisted of 178 alevins between 1 and 24 days posthatching. They were examined using light and electron microscopy. Our observations indicate that the entire cardiac outflow tract displays a myocardial character during early development. Between the fourth and sixth days posthatching, the distal portion of the cardiac outflow tract undergoes a phenotypical transition, from a myocardial to a smooth muscle-like phenotype. The length of this region with regard to the whole outflow tract increases only moderately during subsequent developmental stages, becoming more and more cellularized. The cells soon organize into a pattern that resembles that of the arterial wall. Elastin appears at this site by the seventh day posthatching. Therefore, two distinct components, proximal and distal, can be recognized from the fourth day posthatching in the cardiac outflow tract of A. naccarii. The proximal component is the conus arteriosus, characterized by its myocardial nature and the presence of endocardial cushions. The distal component transforms into the intrapericardial, nonmyocardial segment mentioned above, which is unequivocally of cardiac origin. We propose to designate this segment the "bulbus arteriosus" because it is morphogenetically equivalent to the bulbus arteriosus of teleosts. The present findings, together with data from the literature, point to the possibility that cells from the cardiac neural crest are involved in the phenotypical transition that takes place at the distal portion of the cardiac outflow tract, resulting in the appearance of the bulbus arteriosus. Moreover, they suggest that the cardiac outflow tract came to be formed by a bulbus arteriosus and a conus arteriosus from an early period of the vertebrate evolutionary story. Finally, we hypothesize that the embryonic truncus of birds and mammals is homologous to the bulbus arteriosus of fish.     

Gut regulatory peptides in some Antarctic notothenioids

The presence and the comparative distribution of regulatory peptides and serotonin in the gut of four species of Antarctic notothenioid fishes [Cryodraco antarcticus and Chionodraco hamatus (Channichthyidae), and Trematomus bernacchii and Trematomus newnesi (Nototheniidae)], were immunohistochemically studied. In these species, numerous immunoreactive (IR) endocrine cells and nerve elements were detected. In the nototheniids most of the IR nerve fibres were of the intrinsic type, while most of the IR nerve fibres of the channichthyid intestine, besides insulin-like IR fibres, seemed to be of the extrinsic type. The intensity and frequency of immunopositivity are not the same in channnichthyids and nototheniids; the species belonging to the first family show many differences from the teleosts living in temperate water. The finding of insulin-like endocrine cells and nerve fibres in the gut wall of Cryodraco antarcticus is exceptional for vertebrates and deserves special attention.A preliminary account of this work was presented to the VII International Ichthyology Congress, Den Haag, 26–30 August 1991     

Anatomy and ultrastructure of ventral pharyngeal organs and their phylogenetic importance in Polychaeta (Annelida)

Summary Transmission electron microscopic studies were carried out on the ventral pharyngeal organs in Ctenodrilus serratus and Scoloplos armiger. The pharyngeal organs are composed of a muscle bulbus and a tongue-like organ. In both species the muscle bulbus consists of transverse muscle fibres and interstitial cells with voluminous cell bodies and dorsoventral tonofilaments; the investing muscle runs into the tongue-like organ; the nuclei of the investing muscle fibres are located in caudal bulges; salivary glands are not present, but numerous gland cells occur in the bulbus epithelium. The tongue-like organ, however, is formed by lateral folds (C. serratus) or a bridge-like structure (S. armiger). The specific structure of the bulbus muscle is probably a homologous characteristic also occurring in several other polychaete families. The phylogenetic importance of this ventral pharynx is discussed and a hypothesis is suggested to explain the differentiation of certain other ventral pharyngeal organs from this probably primitive type.