Secretion by ependymal cells of the neurohypophysis and saccus vasculosus of Polypterus ornatipinnis (Osteichthyes)

Electron microscopic studies of the neurohypophysis and saccus vasculosus of the bichir (Polypterus ornatipinnis) reveal an apocrine release of secretory material by ependymal cells (e.g., crown cells). The secretory material appears to migrate along the microtubular apparatus and the ciliary filaments to the cell buds. It is postulated that the formation of the buds and their release is mediated by cilial action. Secretion buds are noted in the cerebrospinal fluid and vascular sinusoids. Bulbous projections of neurohypophyseal secretory tracts extend into the ependymal lumen which also contains elementary secretion granules. Specialized “liquorkontaktneurone” are interspersed with ependymal cells. An interrelation between the hypothalamus, neurohypophysis, saccus vasculosus, and the meta-adenohypophysis is postulated.     

Scanning and transmission electron microscopy of the subfornical organ of the grass frog (Rana pipiens)

Summary The ventricular surface of the subfornical organ of the frog is made up of ependymal cells with numerous apical microvilli, occasional cytoplasmic protrusions and many vacuoles projecting into the lumen of the third ventricle. Between these cells dendrites of cerebrospinal fluid-contacting neurons reach the ventricle to terminate in bulbous enlargements. In addition, flask-shaped encephalo-chromaffin cells, containing granulated vesicles and aggregates of filaments in their cytoplasm, project into the cerebrospinal fluid. Surrounding the centrally located capillaries are enlarged dendrites and axons of heterogeneous morphology, some of which appear to originate within the subfornical organ, intermingled with dendrites and axons of normal structure. The glial cells in this region, especially the microglial cells, often contain large lipofuscin inclusions, suggestive of degeneration and subsequent phagocytosis of some of the enlarged dendrites and axons. The normally scarce neurosecretory peptidergic axons become more evident and form typical Herring bodies in stalk-transected animals. Neuronal perikarya of varying morphology are predominantly located peripheral to the region of enlarged dendrites and axons. Supraependymal macrophages are particularly numerous on the subfornical organ.Abbreviations used CSF cerebrospinal fluid - SEM scanning electron microscope, scanning electron microscopy - SFO subfornical organ - TEM transmission electron microscope, transmission electron microscopy Supported, in part, by NIH grant NB 07492The skillful technical assistance of J.G. Linner and the secretarial assistance of Ann Gerdom are gratefully acknowledged. The SEM studies were made possible through a grant from the Graduate College of Iowa State University and the use of the SEM facility in the Department of Botany     

The secretory ependymal cells of the subcommissural organ: Which role in hydrocephalus?

Ependyma in the central nervous system gives rise to several specialized cell types, including the secretory ependymal cells located in the subcommissural organ. These elongated cells show large cisternae in their cytoplasm, which are filled with material secreted into the cerebrospinal fluid and toward the leptomeningeal spaces. A specific secretion of the subcommissural organ was named SCO-spondin, regarding its marked homology with developmental proteins of the thrombospondin superfamily (presence of thrombospondin type 1 repeats). The ependymal cells of the subcommissural organ and SCO-spondin secretion are suspected to play a crucial role in cerebrospinal fluid flow and/or homeostasis. There is a close correlation between absence of the subcommissural organ and hydrocephalus in rat and mouse strains exhibiting congenital hydrocephalus, and in a number of mice transgenic for developmental genes. The ependymal cells of the subcommissural organ are under research as a key factor in several developmental processes of the central nervous system.     

The localisation of lead in the skin of light- and dark-adapted Xenopus laevis

Summary Toads pretreated for 2 months on either a dark or a light background were then exposed to lead nitrate at 50 ppm lead for 21 days, the illumination regimes being maintained. Metal analysis of dorsal skin showed significantly higher lead levels (p<0.01) in dark-adapted toads. No precipitated lead deposits were observed at the ultrastructural level, necessitating X-ray microanalysis of sections containing melanophores, gland cells and general (non-melanophore) cytoplasm. Analysis showed the lead to be concentrated within the melanosomes of the melanophores, and to be significantly higher (p<0.01) in individual melanosomes of dark-adapted toads than in light-adapted ones. Copper was also found to be concentrated in the melanosomes and was higher (p<0.01) in the melanosomes of the dark-adapted toads.The results are consistent with the known affinity of melanin for heavy metals and the documented increase in melanophore number under prolonged dark background regimes. Since all toads received the same lead exposure, the melanosome results give rise to speculation that higher melanin levels might occur in individual melanosomes of dark-adapted skin.     

Ependymal specializations

Summary The ependymal cells of the toad subcommissural organ produce pale and dense secretory granules. Both types of granules are mainly concentrated in the apical cytoplasm and in the perinuclear region. Pale and dense granules are synthesized by and packed in the rough endoplasmic reticulum, bypassing the step of the Golgi apparatus. The apical cytoplasm of some subcommissural ependymal cells protrudes into the ventricle. All the cells project a few cilia and numerous slender, long microvilli into the ventricular lumen.Contacting the cilia and the microvilli there is a filamentous material identical to that observed in the fibre of Reissner at the aqueduct of Sylvius. In addition to filaments, the fibre of Reissner contains vacuolar formations. The fibre is surrounded by numerous ependymal cilia, some of which are embedded in the filamentous material of the fibre.The presence of numerous microvilli projected into the ventricle and the large number of vesicles scattered in the supranuclear cytoplasm seem to indicate that the subcommissural organ may have absorption functions. The fact that the intercellular space of the ependymal layer of the subcommissural organ is not separated from the ventricular lumen by tight junctions but by zonulae adhaerentes could indicate that the cerebrospinal fluid penetrates these intercellular spaces bathing all sides of the ependymal cells. The presence in the ependymal cells of vesicles opening into the intercellular space would be in agreement with the latter possibility.There are some ultrastructural differences between the ependymal cells of the cephalic end of the subcommissural organ and those of the caudal end. A critical analysis of Reissner's fibre formation is made.This investigation was partially supported by a Grant of the Wellcome Trust Foundation.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. The author wishes to thank the valuable help of Mr. P. Heap.     

Ultrastructural alterations in rat brain tissues under the direct action of Escherichia coli endotoxin]

The experiments on the rats with intracisternal injection of endotoxin have revealed essential differences in the mechanisms of its permeability through cerebrospinal fluid-brain and blood-cerebrospinal fluid barriers. As for ependymal cells, endotoxin shunts through the cytoplasm in the areas of tight junctions and then it reaches perineuronal spaces. The mechanism of endotoxin penetration through the epithelium of choroid plexi is associated with receptor-mediated endocytosis.     

Ultrastructure of the trophic chamber and nutritive cord of Aspidiotus hederae (Homoptera,coccoidea)

Summary Each ovariole of the coccidian Aspidiotus hederae contains a single oocyte connected by means of a nutritive cord to the trophic chamber. The trophic chamber consists of three nurse cells characterized by an enlarged, ramified nucleus with a prominent nucleolus. The perinuclear cytoplasm contains nuage material, large amounts of free ribosomes, and scattered mitochondria. Occasional cisternae of the rough endoplasmic reticulum and bacteroids are found in trophocyte cytoplasm. The nutritive cord contains many microtubules in parallel array interspersed with numerous free ribosomes and a few mitochondria. The nutritive cord is strengthened by trophocyte projections which surround it. Microtubules in the projections are oriented perpendicular to the long axis of the cord.     

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.     

Glial reactivity in dogs with visceral leishmaniasis: correlation with T lymphocyte infiltration and with cerebrospinal fluid anti-<Emphasis Type="Italic">Leishmania</Emphasis> antibody titres

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. Because glial cells are extensively associated with the immune response within the brain, we evaluated the morphology of astrocytes and microglia of dogs naturally infected with Leishmania chagasi. We used immunohistochemical and lectin-histochemical techniques for morphological analyses and we also examined the glial correlation with lymphocyte infiltration of the brain and with the presence of anti-Leishmania antibodies within the cerebrospinal fluid of the dogs. Although we did not detect a shared morphological pattern in the astrocytes or microglia in the brain tissue, these cells were more intensely labelled in infected dogs than in the control group. The density of microglia was increased in the ependymal/subependymal area, thus demonstrating a strong correlation with the presence of T lymphocytes and with cerebrospinal fluid antibody titres. Thus, our results indicate a pro-inflammatory state in the brains of dogs naturally infected with L. chagasi and strongly suggest that microglia and astrocytes are involved in the pathogenesis of the neurological disorders of visceral leishmaniasis in dogs.     

Cerebrospinal fluid-contacting area in the pineal recess of the vole (Microtus agrestis), Guinea Pig (Cavia cobaya), and rhesus monkey (Macaca mulatta)

Summary The ventricular lining in the pineal recess of the vole (Microtus agrestis), guinea pig (Cavia cobaya) and Rhesus monkey (Macaca mulatta) was investigated light and electron microscopically. Deep in the pineal recess of all three species the ependymal lining exhibits interruptions. A varying proportion of pinealocytes penetrates through this ependymal area, so that the surface of the protruding cells is directly exposed to the cerebrospinal fluid (CSF). At their base, these cells are anchored in the hypependymal tissue by means of processes. It is conjectured that these pinealocytes are engaged in secreting pineal substances into the CSF, as various physiological findings appear to indicate.     

Electron-microscopic study of the secretion of the ependymal cells in the domestic cat (ependymin-beta cells).

We have studied, by electron microscopy, the ultrastructural aspects of secretion (neurosecretion) of the ependyma of the third ventricle of the domestic cat. We have found cytoplasmic protrusions and isolated masses of cytoplasm, some with homogeneous cytoplasm and others with very dense granulation (protein-beta?). Axons, synaptic terminals and free secretory granules in the ventricular lumen were also seen. The existence of ependymin-beta cells (ependymocyte-beta) and axohormonal buttons is suggested. The ependymal cells are classified into seven types: (1) covering ependymocytes, (2) tanycyt ependymocytes, (3) secretory ependymocytes, (4) ependymocytes-beta, (5) neurosecretory ependymocytes, (6) neurosensorial ependymocytes (crown-like) and (7) supraependymal microgial ependymocytes. A neurohormonal hypothesis and the possible existence of one or more cerebral hormones (neurohormones) are suggested. These hormones would flow into the CSF through some of the ependymal cells (by microapocrine secretion, liberation of neurosecretion granules, or by axohormonal buttons): this could be the most important link in the endocrine system, assuring the functional unity throughout the ventricular system of the cerebrospinal axis which it winds through, although its basic influence is exercised) on the hypophysis level as a vertex of the classical endocrine system.     

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat

Summary Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2–3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.     

Histology and ultrastructure of the neural lobe of the lizard,Klauberina riversiana

Summary The pars nervosa of Klauberina riversiana belongs to a primitive tetrapod type which is characterized by the deep penetration of the infundibular recess, a thin-walled structure, and the virtual absence of pituicytes. The differential response of this gland to aldehyde fuchsin and periodic acid Schiff suggests the presence of two types of neurosecretory nerve endings. Ultrastructurally four kinds of nerve endings are distinguishable. Type I, probably a cholinergic nerve ending, contains only small clear vesicles ca. 400 Å in diameter. The relative abundance of cholinergic nerve endings in this pars nervosa may be related to the necessity of transporting hormone through the ependymal cell. Type II, containing granulated vesicles about 1,000 Å in diameter and probably aminergic, is very rare. The two remaining types apparently secrete neurohypophysial hormones. They are Type III, containing dense granules ca. 1,500 Å in diameter and Type IV containing pale granules ca. 1,500 Å in diameter. Evidence is reviewed which suggests that Type III nerve endings may secrete arginine vasotocin while Type IV endings may secrete (an)other hormone(s).All these axons end only on the ependymal cells, the vascular processes of which form a continuous cuff over the basement membranes of the blood vessels. Hence the ependymal cells link the cerebrospinal fluid, the nerve endings and the blood vessels. Particles resolvable with the electron microscope are traced through a possible transport pathway from the granules, through the ependymal cells to the basement membrane. It is suggested that pituicytes replace ependymal cells and assume their transport functions in animals with massive neural lobes containing large numbers of nerve endings and blood vessels.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina.This investigation was supported in part by a Public Health Service fellowship 1 FZ HD 32,949-01 REP from the national Institute of Child Health and Human Development.The authors wish to thank Professor H. Heller for his constant interest and constructive criticism.     

Distribution of intraventricularly injected horseradish peroxidase in cerebrospinal fluid compartments of the rat spinal cord

Summary The circulation of the cerebrospinal fluid along the central canal and its access to the parenchyma of the spinal cord of the rat have been analyzed by injection of horseradish peroxidase (HRP) into the lateral ventricle. Peroxidase was found throughout the central canal 13 min after injection, suggesting a rapid circulation of cerebrospinal fluid along the central canal of the rat spinal cord. It was cleared from the central canal within 2 h, in contrast with the situation in the brain tissue, where it remained in the periventricular areas for 4 h. In the central canal, HRP bound to Reissner's fiber and the luminal surface of the ependymal cells; it penetrated through the intercellular space of the ependymal lining, reached the subependymal neuropil, the basement membrane of local capillaries, and appeared in the lumen of endothelial pinocytotic vesicles. Furthermore, it accumulated in the labyrinths of the basement membrane contacting the basolateral aspect of the ependymal cells. In ependymocytes, HRP was found in single pinocytotic vesicles. The blood vessels supplying the spinal cord were classified into two types. Type-A vessels penetrated the spinal cord laterally and dorsally and displayed the tracer along their external wall as far as the gray matter. Type-B vessels intruded into the spinal cord from the medial ventral sulcus and occupied the anterior commissure of the gray matter, approaching the central canal. They represented the only vessels marked by HRP along their course through the gray matter. HRP spread from the wall of type-B vessels, labeling the labyrinths, the intercellular space of the ependymal lining, and the lumen of the central canal. This suggests a communication between the central canal and the outer cerebrospinal fluid space, at the level of the medial ventral sulcus, via the intercellular spaces, the perivascular basement membrane and its labyrinthine extensions.     

The localisation of lead in the skin of light- and dark-adapted Xenopus laevis

Toads pretreated for 2 months on either a dark or a light background were then exposed to lead nitrate at 50 ppm lead for 21 days, the illumination regimes being maintained. Metal analysis of dorsal skin showed significantly higher lead levels (p less than 0.01) in dark-adapted toads. No precipitated lead deposits were observed at the ultrastructural level, necessitating X-ray microanalysis of sections containing melanophores, gland cells and general (non-melanophore) cytoplasm. Analysis showed the lead to be concentrated within the melanosomes of the melanophores, and to be significantly higher (p less than 0.01) in individual melanosomes of dark-adapted toads than in light adapted ones. Copper was also found to be concentrated in the melanosomes and was higher (p less than 0.01) in the melanosomes of the dark-adapted toads. The results are consistent with the known affinity of melanin for heavy metals and the documented increase in melanophore number under prolonged dark background regimes. Since all toads received the same lead exposure, the melanosome results give rise to speculation that higher melanin levels might occur in individual melanosomes of dark-adapted skin.     

Cytochemical localization of alkaline phosphatase in the ependyma of the rat medulla oblongata

Summary The ependyma of the IVth ventricle and the central canal of the rat medulla oblongata was investigated using the cytochemical technique for alkaline phosphatase (AlPase) which revealed two types of ependymal cells in the medulla. The central canal type of the ependymal cell occupying the dorsal part of the central canal in the lower medulla exhibited intense AlPase activity with light microscopy. These cells had reaction products in all plasma membranes, including the microvilli and the cilia at the luminal cell surface. Some cells appeared to be tanycytes, since the process reached the basement membrane of the parenchymal blood vessel. The ventricular type of ependymal cells, which form the floor of the IVth ventricle and the central canal, contained no reaction products in any structure of the luminal cell surface.The possible relationship between the cerebrospinal fluid and the nervous tissues through the ependymal linings is discussed.     

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina

By gently scraping off the surface of the lateral ventricles of adult bovine brains, we obtained sheets containing the ependymal layer and some attached sub-ependymal cells. Explants were cultured in serum-free medium or in two media enriched with 20% fetal calf serum or 20% adult bovine cerebrospinal fluid, and processed for different time intervals from 4 h to 60 days. For characterization of the ependymal cells we used antisera against S-100 protein, vimentin and glial fibrillary acidic protein (GFAP). For comparison, the ependyma of adult bovines and of fetuses from days 60 to 120 post coitum was studied in situ. The adult ependyma consisted of a ciliated, cuboid cell monolayer with short basal processes; it displayed S-100 immunoreactivity but only scarce deposits of vimentin and no GFAP. The fetal ependyma had the appearance of a pseudostratified epithelium with elongated nuclei and basal processes containing S-100 and vimentin from day 80 post coitum and GFAP from day 100 post coitum. In explants, no differences were seen between the three culture media; the ependyma became pseudostratified, developed basal processes and showed increasing amounts of S-100 and vimentin first, and subsequently also GFAP. These changes were concomitant with the onset of mitotic activity in the subependymal layer leading to the production of numerous cells. The morphological and immunocytochemical features of ependymal cells in cultured explants resembled those of fetal ependyma. Our results indicate that the culture of ependymal explants from adult bovine lateral ventricles is an useful model system for morphological and functional studies of the ependyma and for the analysis of cell proliferation in the subependymal layer.     

The fine structure of ependymal cells part II: An electron microscopic study of the soft-shelled turtle paraventricular organ,with special reference to the fine structure of ependymal cells and so-called albuminous substance

Summary The present study deals with the electron microscopic observations on the softshelled turtle paraventricular organ, with special reference to the relationship of the ependymal cells and the so-called albuminous substances. It is shown that the so-called albuminous substances consist of the tips of neuronal processes extending into the ventricular lumen. They probably arise from the nerve cells lying within the hypendymal or the underlying tissue. The ependymal cells of the PVO themselves are basically similar in structure to those of any other animal.The processes observed contain two types of vesicles, namely: the clear vesicle, 500 to 1600 Å in diameter, and the cored vesicle, measuring 600 to 1500 Å in diameter, which has a distinct membrane enclosing an extremely dense core of variable sizes. The functional significance of these vesicles is discussed in relation to that of inclusions in the neurosecretory and the autonomic nerve fibers in the hypothalamus.The findings indicate that in the terminal endings of the processes a production or formation of vesicles might occur and that these vesicles might be discharged into the cerebrospinal fluid by microapocrine secretion.The author's grateful thanks are due to Prof. E. Yamada for his continuously kind guidances and due to Prof. T. Sakurai for his constant encouragement.     

DEVELOPMENT OF PERIPHERAL VACUOLES IN PLANT CELLS

The vacuolar apparatus of various plant cells consists of two distinct features: the large central vacuole and peripheral vacuoles which are derived from invaginations of the plasma membrane. Peripheral vacuoles are conspicuous structures in both living and fixed hair or filament cells of Tradescantia virginiana. They occur as spherical structures along the inner boundary of the peripheral cytoplasm and can be recognized as projections into the central vacuole. These structures are variable in size and number within a cell and can represent a significant proportion of the volume of the vacuole. Peripheral vacuoles most frequently are observed in motion with the streaming cytoplasm although their velocity is usually somewhat slower that that of the cytoplasmic organelles. Ultrastructural studies show two closely approximated membranes, one for each vacuole, in areas where a peripheral vacuole projects into the central vacuole. These are separated by an intermembrane zone continuous with the peripheral cytoplasm. The movement of organelles over the perimeter of the peripheral vacuole is presumed to occur along this intermembrane zone. The internal area of the peripheral vacuoles may appear empty although some contain a vesicular content of unknown origin and function.     

Histology and ultrastructure of the neurohypophysis of the south american lungfish,Lepidosiren paradoxa

Summary The neurohypophysis of the South American lungfish Lepidosiren paradoxa has been studied with light and electron microscopy, including the Falck-Hillarp technique for catecholamines. The pars nervosa hypophyseos is a well-marked, dorsally located subdivision of the pituitary gland composed of lobes or follicles, each one constituted of a central core of ependymal cells, a subependymal hilar region made up of nerve fibers and a peripheric palisade zone of nerve endings which contact capillary vessels. Four types of neurosecretory axons can be distinguished under the electron microscope. Type I, the most common, contains spherical elementary granules of high electron density, 1500–1800 Å in diameter. The scarce type II axons contain irregularly-shaped elementary granules. Type III contains only small clear vesicles, 400–600 Å in diameter. Type IV, mostly present in regions of the gland contacting the pars intermedia, contain large granulated vesicles, 900–1000 Å in diameter. The Falck-Hillarp technique revealed axons with a positive reaction for catecholamines at sites corresponding approximately to the location of type IV of the electron microscope.Ependymal cells are of large size, linking the cerebrospinal fluid, the nerve endings and the blood vessels. A conspicuous membrane-bound, spherical dense material, 1400–2000 Å in diameter, is observed in both the apical and vascular processes of these cells. The ependymal processes which traverse the hilar and palisade regions contain structures resembling degenerated neurosecretory axons. These results are discussed in relation with the currently available information on the comparative anatomy of the pars nervosa. The possible functional significance of ependymal cells and of each type of axon are also discussed.This study was aided by the following grants: NIH NS 06953 to Prof. De Robertis, Consejo Nacional de Investigaciones Científicas y Técnicas to Prof. Zambrano, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires and Comisión de Investigaciones Cientificas de la Universidad Nacional de la Plata: to Prof. Iturriza.The authors are indebted to Prof. De Robertis for his generosity in granting us his laboratory facilities, and to Dr. F. J. J. Risso and Mr. A. Fernández (Resistencia, Chaco) who provided the specimens used in this study. The able microtechnical assistance of Miss L. Riboldazzi and Mrs. R. Raña and the photographic work of Mr. A. Saenz are much appreciated.Members of the Scientific Career, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.