Topographical differences of RNA labelling in rat brain after intraventricular administration of labelled RNA precursors

Summary ¹⁴C-uridine or ¹⁴C-orotic acid was injected into the third or fourth brain ventricle of adult rats. The rate of incorporation of these precursors into the RNA of various brain regions was studied by autoradiography. 0.5 hour, 1 hour, 2 hours and 4 hours after the injection of labelled uridine and/or orotic acid into the third ventricle, a very uneven labelling of different brain regions was observed. The highest grain density was found over the ventricular walls and in the closely adjacent brain tissue; the intensity of labelling decreased sharply with distance from the ventricular lumen. 24 hours after intraventricular injection, a medio-lateral gradient of grain density was no longer observed. An intense labelling of leptomeninx (especially at the base of the brain) and of ependymal cells was observed at all time intervals investigated. At time intervals 0.5–2 hours the grain density of these structures surpassed by a considerable amount the grain density over neurons, glial cells or neuropile.Two hours after the injection of ¹⁴C-orotic acid into the fourth ventricle, the grains were mainly localised over leptomeningeal cells and vessels at the base of the brain and in the adjacent narrow strip of brain tissue. The rest of the brain was only very faintly labelled.     

Tanycyte absorption affected by the hypothalamic deafferentation in Japanese quail,Coturnix coturnix japonica

In Japanese quail, Coturnix coturnix japonica the tanycytes of the median eminence absorbed peroxidase injected into the third ventricle. The number of tanycytes showing peroxidase reaction was greater in the posterior median eminence than in the anterior median eminence. Following hypothalamic deafferentation, the tanycyte absorption was augmented both in the posterior and anterior median eminence. These findings suggest that axons of some neurons, which have inhibitory action on the tanycyte absorption, were transected by deafferentation resulting in augmentation of tanycyte absorption. A considerable number of ependymal cells lining the upper portion of the third ventricle and those of the pars nervosa also absorbed peroxidase. In birds with a deafferented hypothalamus, photostimulated ovarian growth was completely inhibited.     

Uptake of peroxidase from the third ventricle by ependymal cells of the median eminence

Summary Peroxidase injected into the subarachnoid space in mice is absorbed by ependymal cells of the median eminence. The ependymal cells of the median eminence of the rat and Japanese quail absorb peroxidase injected into the third ventricle. The processes of these ependymal cells terminate at the capillaries of the primary plexus or those surrounding the ventromedial nucleus of the hypothalamus. In all three species, peroxidase is absorbed by the ependymal cells of the paraventricular organ and by those in close proximity to it. Some ependymal cells send processes to the capillaries in the lateral nucleus of the hypothalamus. These phenomena are discussed in relation to adenohypophysial function.A part of this investigation was effected while the senior author held a Visiting Professorship at the University of Giessen [Department of Anatomy (Professor A. Oksche, Director)].     

Ependymal Absorption of Peroxidase into the Third Ventricle of the Hagfish Eptatretus burgeri (Girard)

Abstract The ependymal cells of both dorsal and ventral walls lining the lumen of the neurohypophysis of the hagfish, Eptatretus burgeri, absorb peroxidase injected into the third ventricle. Peroxidase diffuses rapidly into the connective tissue separating the neurohypophysis from the adenohypophysis, and also into the connective tissue located between the cell nests of the adenohypophysis.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

Effect of Methotrexate On Cells In A Keratinized Epithelium With an Active Thymidine Salvage Pathway Assessed By Autoradiography*

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m², injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [³H]TdR and [³H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [³H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)     

The adsorptive and transport capacity of tanycytes during the perinatal period of the rat

Summary Transport of ferritin and horseradish peroxidase from the 3rd ventricle to the median eminence was examined in rats during the perinatal life, the time when functional interrelations between hypothalamus and hypophysis are established. Protein tracers injected into the lateral ventricle are adsorbed on the apical surface of the tanycyte, mainly on its protrusions or in indentations. On the 18th day of prenatal life a few small bleblike protrusions are observed. After birth microvilli appear. In time their concentration increases to result in an increase of adsorbed substances. They are taken up by smooth and coated pinocytotic vesicles and transported to the basal portion of the cell or to the intercellular space bypassing junctional complexes. In addition to pinocytotic vesicles protein tracers fill channels of smooth ER or Golgi complex and multivesicular bodies illustrating a process probably involved in metabolic or secretory processes.     

Intercellular translocation of molecules via plasmodesmata in the multiseriate filamentous brown alga,Halopteris congesta (Sphacelariales,Phaeophyceae)

Despite the high number of studies on the fine structure of brown algal cells, only limited information is available on the intercelluar transportation of molecules via plasmodesmata in brown algae. In this study, plasmodesmatal permeability of Halopteris congesta was examined by observing the translocation of microinjected fluorescent tracers of different molecular sizes. The tip region of H. congesta consists of a cylindrical apical cell, while the basal region is multiseriate. Fluorescein isothiocyanate‐dextran (FD; 3, 10, and 20 kDa) and recombinant green fluorescent protein (27 kDa) were injected into the apical cell and were observed to diffuse into the neighboring cells. FD of 40 kDa was detected only in the injected apical cell. The plasmodesmatal size exclusion limit was considered to be more than 20 kDa and less than 40 kDa. The extent of translocation of 3 and 10 kDa FD from the apical to neighboring cells 2 h postinjection was estimated based on the fluorescence intensity. It was suggested that the diffusing capacity of plasmodesmata varied according to molecular size. In order to examine acropetal and/or basipetal direction of molecular movement, 3 and 10 kDa FD were injected into the third cell from the apical cell. Successive observations indicated that the diffusion of fluorescence in the acropetal direction took longer than that in the basipetal direction. No ultrastructural difference in plasmodesmata was noted among the cross walls.     

High and low molecular weight tracers for the electron microscopical detection of sialoglycoconjugates

Summary Hydrazide-derivative tracers of different molecular weights have been synthesized for use in the electron microscopical detection of sodium periodate-oxidized sialyl residues of glycoconjugates in various tissues and cells. Haemundecapeptide hydrazide, horseradish peroxidase hydrazide, andLimulus polyphemus haemocyanin hydrazide were obtained by coupling adipic acid dihydrazide to the tracers with the aid of water-soluble carbodiimide. The enzymatic tracers thus prepared retained their peroxidatic activity. On conversion to the hydrazide derivative, the haemocyanin molecule dissociated into its hexameric subunits. In order to test by transmission electron microscopy the ability of the conjugates to bind to the sialoglycoconjugates of endothelial cell surfaces, each tracer was perfusedin situ into rat pancreatic vasculature previously oxidized with 1mm sodium periodate. The three tracers characteristically labelled the various microdomains of the luminal cell coat of the capillary endothelial cell. The electron opacity of the haemocyanin subunits allowed their easy detection when bound to the cell surface or to components of the extracellular matrix. The bound markers were not displaced by a high ionic strength buffer, and did not label desialylated cell surfaces. These results indicate that the three hydrazide-derivative tracers may be useful tools for the electron microscopical detection of cellular and extracellular sialoglycoconjugates.     

Choroid plexus and paraphysis in lower vertebrates

Cytoarchitecture of the choroid plexus of the third ventricle and the paraphysis was investigated in some lower vertebrates to compare the histologic characteristics of these organs. Both epithelia are similar in appearance in the same class. Minor microscopic variations exist in the different classes of vertebrates, but do not provide a fundamental distinction between the two organs. The epithelia, moreover, have similar staining properties, contain mucicarmine- and PAS-reactive materials, and are derived from a common neuroepithelium. Tubules are identified in the choroid plexus and in the paraphysis; all are similarly formed by simple folding of epithelium on the surface into the stroma. The paraphyses in all vertebrates studied contain villi similar to those seen in the choroid plexus. Cilia are identified in both choroidal and paraphyseal epithelia, and are not an indication of degree of epithelial differentiation. Many types of epithelium are noted in both organs during histologic differentiation as well as in the mature stage. Functionally, the choroid plexus is active in both secretion and absorption. Accumulation of particulate material within the epithelial cytoplasm may indicate phagocytic as well as absorptive activity of cells. Based on a common neuroepithelial origin and similar histochemical properties, we conclude that the paraphysis is a modified choroid plexus. The velum transversum is an arbitrary boundary between diencephalon and telencephalon, and is itself formed of choroid plexus. The medial telencephalic ventricle is the rostral portion of the third ventricle. All neuroepithelial infoldings at the rostral end of the diencephalic roof including the velum transversum are intraventricular choroid plexuses; the neuroepithelial outpouchings in this region are the extraventricular choroid plexuses (paraphysis) of the diencephalon.     

THE INFLUENCE OF INTRAVASCULAR FLUID VOLUME ON THE PERMEABILITY OF NEWBORN AND ADULT MOUSE LUNGS TO ULTRASTRUCTURAL PROTEIN TRACERS

The permeability of the alveolar-capillary membrane of newborn and adult mice to horseradish peroxidase (HRP) and catalase was studied by means of ultrastructural cytochemistry, and the permeability to ferritin was studied by electron microscopy. The influence of varying volumes of intravenously injected fluid on the rate of leakage of the tracers from pulmonary capillaries was examined. The tracers were injected intravenously and the mice were sacrificed at timed intervals. Experiments on newborn mice with intranasally instilled HRP were also done. The tissues were fixed in formaldehyde-glutaraldehyde fixative. Chopped sections were incubated in Graham and Karnovsky's medium for peroxidase and in a modification of this medium for catalase. Tissues were postfixed in OsO₄ and processed for electron microscopy. In both newborn and adult mice, the ready passage of peroxidase through endothelial clefts was dependent on the injection of the tracer in large volumes of saline. When the tracer was injected in small volumes of saline, its passage through endothelial clefts was greatly reduced. Endothelial junctions of newborn mice were somewhat more permeable to HRP than those of adult mice. In all animals, alveolar epithelial junctions were impermeable to HRP. Catalase and ferritin did not pass through endothelial junctions. Intranasally instilled HRP in newborn mice was taken up by pinocytotic vesicles and tubules of flat alveolar cells.     

Das Verhalten des Konjunktivalepithels zu verschiedenen elektronendichten Markierungssubstanzen

Zusammenfassung Die Resorptionsfähigkeit des Konjunktivalepithels vom Meerschweinchen wurde nach verschieden langer Einwirkung (5–120 min) elektronendichter Markierungssubstanzen (Thoriumdioxyd, Goldsol, Ferritin, Peroxydase) geprüft. Alle Substanzen werden in geringen Mengen aus dem Konjunktivalsack resorbiert, Peroxydase nach 10 min, die übrigen Tracer nach 20 min. Eine Vorbehandlung mit EDTA-haltigen Spüllösungen ändert diese Ergebnisse nicht. Die Prüfung der Verteilung subepithelial injizierter Thoriumdioxydund Peroxydaselösungen 10 min nach Injektion ergibt, daß Thoriumdioxyd als umschriebenes Depot liegen bleibt, während Peroxydase gut diffundiert und die Interzellularräume bis zu den Zonulae occludentes gleichmäßig erfüllt. Eine Phagocytose der Tracer durch Epithelzellen ist auch hier nur in geringen Mengen nachweisbar.Das Konjunktivalepithel ist eine Barriere gegen die Aufnahme der verwendeten Markierungssubstanzen in die Bindehaut.

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The uptake of various electron dense tracers by the conjunctival epithelium

Summary The resorption capability of the guinea pig conjunctival epithelium was studied applying electron dense markers (thoriumdioxide, colloidal gold, ferritin, peroxidase) for 5–120 min. All these substances are resorbed in small amounts from the epithelial surface; peroxidase after 10 min, the other markers after 20 min. Pretreatment with EDTA-containing solutions does not alter these results. Studying the localization of subepithelial injected thoriumdioxide- or peroxidase solutions 10 min after the injection shows that the thoriumdioxide remains confined to the site of injection, while peroxidase penetrates the epithelial intercellular space up to the zonulae occlndentes. A tracer uptake by phagocytosis into the epithelial cells is seen very rarely under these conditions.Thus, the epithelial layer is a barrier, preventing the diffusion of the used tracers into the conjunctiva.

Herrn Prof. Dr. Gerhard Wolf-Heidegger zum 60. Geburtstag gewidmet. — Auszugsweise vorgetragen beim XIV. Symposion der Gesellschaft für Histoohemic gemeinsam mit der Niederländischen Gesellschaft für Histochemie. Köln, 25.–29. September 1970.     

The presence of substance P-immunoreactive nerve fibres in the organum vasculosum laminae terminalis of the Mongolian gerbil (Meriones unguiculatus)

Summary Serial frontal and sagittal sections through the organum vasculosum laminae terminalis (OVLT) of the male Mongolian gerbil (Meriones unguiculatus) were incubated with an antibody against substance P and stained by the indirect peroxidase anti-peroxidase technique. A high density of nerve fibres with large-sized terminals exhibiting substance P-immunoreactivity was observed in the OVLT in close relation to the blood vessels both in the internal and the external zone of the organ. In addition, a few fibres penetrated into the ependyma covering the organ. These results indicate that substance P is released into the bloodstream from the OVLT and in addition might influence the ependymal cells bordering the organ towards the third ventricle.This study was supported in part by the Carlsberg Foundation     

Scanning electron microscopy of the wall of the third ventricle in the brain of Rana temporaria. Part IV.

Summary The surface specializations of the wall of the third cerebral ventricle of Rana temporaria were investigated with the scanning electron microscope. These specializations can be divided into three types: cilia, large bulbous protrusions, and microvillus-like protrusions.Most parts of the ventricular surface are densely ciliated. In contrast, other regions are either scantily ciliated or devoid of cilia. Four areas of the ventricular surface are studded with numerous large bulbous protrusions. These large protrusions can be divided into two types: One type consists of intraventricular end bulbs of dendrites of secretory neurons. The other type is represented by large cytoplasmic extensions of ependymal cells.In the third ventricle of Rana, microvillus-like surface specializations of ependymal cells are ubiquitous structures. Generally, filiform protrusions of varying length are the predominant type. The microvillus-like specializations are transient structures, the number of which varies according to different physiological states of the ependymal cells.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

EARLY STAGES OF INTESTINAL ABSORPTION OF SPECIFIC ANTIBODIES IN THE NEWBORN : An Ultrastructural, Cytochemical, and Immunological Study in the Pig, Rat, and Rabbit

In mammals, passive immunity is transferred from mother to offspring by transplacental passage or by intestinal absorption. The rabbit receives antibodies exclusively across the placenta, whereas intestinal absorption is the principal source of antibodies for the new-born pig. In the rat, passive immunity is transferred by both pathways. The role of the jejunal absorptive cells was investigated in these three species, by the use of specific immune globulins as tracers of protein absorption. Rabbit anti-peroxidase and anti-ferritin antibodies were injected into the jejunum of newborn pigs, rats, and rabbits, and absorption was studied over the first 2 hr. The specific antibodies were detected in glutaraldehyde-fixed tissues after in vitro treatment with the antigens, and in sera by immunological methods. Intact antibodies are transferred into the circulation of the pig and the rat, but not into that of the rabbit. In the three species, the jejunal absorptive cells take up antibodies by endocytosis. In the pig, the antibodies are transported across the epithelium in vacuoles. In the rabbit, the endocytosis of antibodies triggers a lysosomal response and all absorbed antibodies are trapped in lysosomes. In the rat, both situations are found; there is no evidence of transfer of antibody fragments into the circulation.     

Structure,Ultrastructure and Function of the Neural Gland Complex of Ascidia interrupta (Chordata,Ascidiacea): Clarification of Hypotheses Regarding the Evolution of the Vertebrate Anterior Pituitary

Abstract The neural gland complex of Ascidia interrupta consists of three parts: dorsal tubercle, ciliated duct, and neural gland. The dorsal tubercle protrudes above the pharyngeal lining and bears a ciliated funnel. The funnel opens into a ciliated duct which opens into the neural gland, a blind sac in a blood sinus below the brain. Funnel and duct cells are joined by adhaerens junctions and, apically, by putative tight junctions. The neural gland wall is a loose, irregular, non-ciliated epithelium of phagocytes. Adhaerens, but not tight, junctions join the cells. Secretory cells were not observed. Tracers delivered onto the dorsal tubercle and dissolved in seawater around undissected animals are transported unidirectionally inward into the neural gland. The continuous ciliary incurrent moves the tracers across the wall of the neural gland and into the pharyngeal blood vessels. Small particulates and large macromolecules, however, are removed from the water stream by endocytosis on neural gland cells. Large particulates delivered onto the dorsal tubercle do not enter the system but rather are rejected by cilia on the surface of the tubercle. The neural gland complex is interpreted as an organ of blood volume regulation that consists of a pump (cilia) and coarse (tubercle) and fine (gland) filters. Analogous and homologous systems are discussed.     

Peroxidases in the neural retina and pigment epithelium.

Peroxidase activity, assayed with 2 mM-H₂O₂ and suitable hydrogen donors (either p-phenyl-enediamine or diaminobenzidine), was demonstrated in homogenates of neural retina and pigment epithelium of both the dog and the cow. The enzyme is particle-associated in the native state, but is readily extractable by brief sonication or freeze-thawing. At optimum pH, which is between 4.0 and 4.5 for both sources, the specific activity is up to 40 times greater in pigment epithelial cells than in neural retina. Some catalase activity was detected in extracts from both bovine and canine neural retina, but catalase was essentially absent in pigment epithelium. Fractionation of bovine pigment epithelial cells showed that peroxidase activity is associated mainly with heavy organelles sedimenting at low centrifugal forces. Melanosomes, nuclei, melanolysosomes and plasma membranes were the principal organelles identified in these low speed sediments. It was not possible to separate them either by differential centrifugation or on discontinuous sucrose gradients. However, melanosomes were excluded as the only source of peroxidase activity by isolating separately the melanotic and amelanotic cell populations; equal peroxidase was found in both cell types. Since nuclei are not a likely source of this enzyme, it is suggested that most of the peroxidase activity in bovine pigment epithelial cells is localized in either the melanolysosomes, plasma membranes, or both.     

An autoradiographic investigation of the proliferative activity of pyloric caeca and gondas of Asterias rubens

The proliferative activity of the pyloric caeca of Asterias rubens was investigated. Autoradiographic experiments using intracoelomically injected (methyl-³H)-thymidine were performed throughout the year and incorporation into pyloric caeca and into gonads was studied. Tritiated thymidine was found to be incorporated mainly in the coelomic lining of both organs. Cell divisions in the coelomic lining may be necessary for the growth of these organs, for the production of coelomocytes or, in the case of the pyloric caeca, for growth of the digestive epithelium. Proliferative activity of the digestive epithelium of the pyloric caeca was only observed in the median duct. It is hypothesized that new cells, arising from mitosis, grow from the median duct to the side lobes and differentiate into storage cells, for example. The existence of a mitosis-inducing or mitosis-stimulating substance is discussed. In the ovaries follicle cells were found to incorporate (methyl-³H)-thymidine; in the testis, proliferation of the germinal epithelium occurred simultaneously in all spermatogenic columns. First, the spermatogonia and then later the spermatocytes became labeled. Absorption of substances from the coelomic fluid is discussed.     

Vascular permeability (problem of the blood-brain barrier) in the pineal organ of the rainbow trout,Salmo gairdneri

Summary The problem of the blood-brain barrier in the pineal organ of the rainbow trout, Salmo gairdneri, was investigated following intraperitoneal or intracardial injections of several tracers and dyes with different molecular weights. As demonstrated at the light-microscopic level, repeated injections of trypan blue or horseradish peroxidase (HRP) resulted in an accumulation of these substances in the pineal epithelium (parenchyma). By use of the electron microscope, HRP was found in electron-dense bodies, probably lysosomes, in (i) the endothelial cells and perivascular macrophages 4 h after intraperitoneal injection, (ii) the supporting cells and intrapineal or luminal macrophages 8 h after injection, and (iii) the receptor cells 24 h after injection of the tracer. Ferritin particles penetrated the fenestrated endothelium of pineal capillaries. They were confined to vesicles, vacuoles and the smooth endoplasmic reticulum of the supporting cells as well as to the synaptic vesicles and the smooth endoplasmic reticulum of the pineal photoreceptors. The intercellular passage of tannic acid mixed with the fixative was blocked at the luminal junctional complex separating the pineal lumen from the basal portion of the pineal epithelium. The passive intercellular transport of substances with high molecular weight from the bloodstream to the cerebrospinal-fluid compartment is thus prevented. However, no blood-brain barrier exists for exogenously administered proteins, which are rapidly taken up by pineal cells and actively transported in a transcellular manner.The findings on the blood-brain barrier of the pineal organ of the rainbow trout are discussed with particular reference to the endocrine capacity of pineal sensory organs.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany.     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

Summary The endodermal trophotaenial epithelium in goodeid embryos acts as a placental exchange site. Fine structural and cytochemical data indicate that the trophotaenial absorptive cells are endocytotically highly active. To test their micropinocytotic capacity and characterize the cellular mechanisms involved in membrane, solute and ligand movements, living embryos of Xenoophorus captivus were incubated in saline media containing horseradish peroxidase (HRP) and/or cationized ferritin (CF) in vitro, and the uptake of these tracer proteins examined by both time sequence analysis and pulse-chase procedures. In some embryos, the effects of prolonged exposure to CF injected into the ovarian cavity, was also investigated.Labelling of the free cell surface was detectable with CF only, but interiorization of both probes was quick from all incubation media. Adsorptive pinocytosis of CF and fluid-phase uptake of HRP sequentially labelled pinocytic vesicles, endosomes, and lysosome-like bodies. In addition, CF-molecules were sequestered within apical tubules and small vesicles. HRP was largely excluded from both organelles and ended up in the lysosomal compartment. For CF, two alternative pathways were indicated by the pulse-chase experiments; transcellular passage and regurgitation of tracer molecules to the apical cell surface. The latter procedure involves membrane and receptor recycling, in which apical tubules are thought to mediate.In double-tracer experiments, using an 81 excess of HRP, external labelling with CF was light or lacking after 1–3 min, and the initial uptake-phase produced pinocytic vesicles and endosomes that mainly contained HRP-reaction product. Prolonged incubation, however, resulted in densely CF-labelled plasmalemmal invaginations and pinocytic vesicles that predominantly carried ferritin granules. After 60 min, the vacuoles of the endosomal compartment contained either high concentrations of HRP-reaction product, both tracers side by side, or virtually exclusively CF.