Fine structure of ependymal cysts in and around the area postrema of the rat

Summary Peculiar cells forming cysts were observed in the area postrema and sometimes also in the choroid plexus and the tela chorioidea near the area postrema, and were studied in detail by electron microscopy. The cytological features of the cyst cell and its junctional relationship to neighboring cells imply that cyst cells are derived from ependymal and choroid epithelial cells. The cyst cells usually contact directly the perivascular spaces of postremal, choroidal or pial capillaries, where the cytoplasm is often considerably attenuated. The cystic lumen is commonly filled with a flocculent material. The limiting membrane of the cystic lumen, which frequently bears cilia and microvilli, has the same thickness as the surface cell membrane. In many cases, the cyst is surrounded by the cytoplasm of a single cell. In some cases, however, two cells participate in the formation of the cyst, although one is only a slender process and joined by a zonula occludens with the main cyst cell. Horseradish peroxidase (HRP) injected into the cerebrospinal fluid (CSF) space failed to enter the cystic lumen. A possible significance of the cyst in relation to the CSF and blood circulation was considered.     

Fine structure of the ependymal cells in the area postrema of the domestic fowl

Summary The ultrastructure of the ependymal cells in the area postrema of the domestic fowl was studied by scanning and transmission electron microscopy. The ependymal surface of the area postrema is covered with many furrows and ridges. These ridges consist of ependymal cells aggregated in a fan-like shape. The ependymal cell lacks clustered cilia, microvilli are few, and a long basal process extends through the parenchymal layer of the area postrema. Within the cytoplasm as well as in the basal process, a spherical body with a diameter ranging from 1.5 to 2 gmm is occasionally observed.This work was supported by a Scientific Research Grant, No. 144017, from the Ministry of Education of Japan to Professor M. YasudaThe authors are grateful to Drs. T. Fujioka and T. Watanabe for their valuable advice     

The microvascular pattern and perivascular linings of the area postrema

Summary The microvasculature and perivascular linings of the area postrema (A.P.) were studied electron microscopically with the ultrathin section and freeze-etching techniques. Special attention was given to the intercellular contacts of the different cellular entities. Two types of microvascular segments were identified. The endothelium of these vascular segments reveals fenestrations and a high pinocytotic activity. There are no significant differences in the frequency and distribution of the endothelial openings between both types of capillaries. The endothelium of the blood vessels, however, is joined by different types of tight junctions. Focal tight junctions occur between pericytes and the endothelium, and between leptomeningeal cellular elements in the perivascular space. The cell membrane of the perivascular glia shows intramembrane particles which are either distributed at random or organized in the form of membrane-associated orthogonal particle complexes (MOPC, Dermietzel, 1974). The significance of these findings is discussed with respect to the modified blood-brain barrier mechanism in the A.P.Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 114 Bionach) to R. DermietzelThe authors are indebted to Mrs. D. Schünke and Mr. R. Eichner for technical assistance and Mr. A. Stapper for preparing the diagram     

Distribution of neurotensin-like immunoreactivity in rat and cat area postrema

The distribution of neurotensin-like immunoreactive (NT-LI) fibers and cell bodies in the area postrema (AP) of rat and cat, utilizing the peroxidase, antiperoxidase (PAP) technique, is described. In the rat, the greatest accumulation of NT-LI fibers were present along the borders of the AP, while there were very few NT-LI fibers in central regions. In the cat, scattered NT-LI fibers occupied the majority of the central AP, while moderate numbers of NT-LI fibers were present at the ventromedial border. In more rostral cat AP levels, the number of NT-LI fibers decreased. NT-LI somata were present in rat AP, but were lacking in cat AP. The localization of NT within the AP suggests that the cardiovascular and gastric effects attributed to NT may be mediated, in part, through the AP.     

The functional and structural borders between the CSF-and blood-dominated milieus in the choroid plexuses and the area postrema of the rat

Summary In the borderline area between the hemal milieu of the choroid plexuses (PC) and the interstitial cerebrospinal-fluid (CSF) compartment, ground substances displaying increased amounts of basal lamina-like material and containing negatively charged sulfated glycosaminoglycans appear to be endowed with selective properties. They may function as a sieve or filtration barrier gradually controlling the passage of substances between the two milieus, depending on their charge and molecular weight. Special structural features and functional properties of ependymal cells are associated with such bordering structures. These ependymal cells are transitional elements between choroid epithelium and ciliated ependymal cells. As judged from experiments with horseradish peroxidase and conventional electron microscopy, occluding junctions at the basal pole of these cells prevent a rapid alteration in the milieu conditions, enabling gradual change from hemal to CSF composition near the bases of these transitional ependymal cells. The borderline structures between the hemal milieu of the PC and the area postrema (1) are established by leptomeningeal cells which face a hemal mileu, (2) are endowed with conspicuous tight junctions, and (3) produce a flocculent substance, the light-microscopic equivalent of which is PAS positive. These structures probably establish an effective barrier between the two milieus of different composition. The functional characteristics and the morphology of the meningeal cells facing the hemal milieu of neurohemal regions resemble closely the neurothelial cells, which are interposed between the CSF milieu and the hemal milieu in the dura mater. The present results suggest that the location between the hemal and the CSF milieu is decisive for the transformation of leptomeningeal cells into neurothelial elements.Supported by the Deutsche Forschungsgemeinschaft (Grant Kr 569/5-1)Dedicated to Professor Dr. med., Dr. med. vet. h.c., Dr. phil. h.c. Andreas Oksche on the occasion of his 60th birthday     

Protein synthesis and transport by the rat choroid plexus and ependyma

Summary Light (LM-ARG) and electron microscope (EM-ARG) autoradiographs were prepared from immature rat choroid plexus and ependyma at 5, 10, 30, and 60 min and 16 h following intraperitoneal administration of [³H]- labeled amino acid mixtures. Intracellular protein synthesis and transport were ascertained in lateral and fourth ventricle choroid plexus epithelium by quantitative EM-ARG at the several post-injection intervals. ARG were also prepared from choroid plexuses cultured for one day, pulse labeled for one hour and reincubated for various periods in nonradioactive media. Significant labeling of both attached and free apical protrusions (blebs) was observed in both choroid plexus and ependyma in vivo and in choroid plexus in vitro. This phenomenon was interpreted as a physiologically significant mechanism for protein transport (apocrine secretion) by epithelia into the cerebrospinal fluid (CSF).This study was supported in part by N.I.H. Research Grant NS 12906     

Cell junctions in the subcommissural organ of the rabbit as revealed by use of ruthenium red

Summary Tight junctions were found in the apical junctional complex of the adult rabbit subcommissural organ (SCO) in addition to zonulae adhaerentes and gap junctions of typical ependymal cells. Ventricular perfusion of ruthenium red before fixation was found to give excellent results for distinguishing between gap and tight junctions at the ependymal surface. The implication of tight junctions as a mechanical means of sealing off the SCO area from the cerebrospinal fluid and the use of ruthenium red as a tracer substance are discussed.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen.     

Patch-clamp study on membrane properties and transmitter activated currents of rabbit area postrema neurons

Using the patch-clamp technique in combination with sliced tissue preparation the membrane properties of newborn rabbit area postrema neurons were investigated. The neurons responded upon depolarization with a fast Na ⁺-current followed by an inactivating and non-inactivating K ⁺-current. GABA-activated currents were investigated resulting in a large Cl^--conductance, indicating the expression of GABA_A-receptors. The expression of glutamate receptor mRNA was studied by in situ hybridization and electrophysiological measurements of these receptors by means of the patch-clamp technique. As a main result it was found that ionotropic glutamate receptors in the area postrema are composed of flop variants of the GluA-, GluB- and GluC-subunits.Abbreviations AP area postrema - GABA -aminobutyric acid - Glu glutamate - I–V current-voltage - SFO subfornical organ     

Cholecystokinin-octapeptide like immunoreactivity in the area postrema of the rat and cat

The distribution of cholecystokinin-8 (CCK-8)-like immunoreactivity in the area postrema of the rat and cat was visualized using the peroxidase, antiperoxidase technique. In the rat the greatest amount of immunostaining occurred in peripheral regions of the area postrema at intermediate and rostral levels. Caudally, scattered immunoreactivity predominated. After colchicine treatment, numerous immunoreactive somata were observed throughout the area postrema. The cat area postrema had a different and more complex pattern of immunostaining than the rat. Moderate to dense accumulations of immunostaining occurred in the ventromedial region of the area postrema bordering the solitary tract and dorsal vagal nuclei. The central region of the area postrema possessed scattered amounts of immunoreactivity at rostral levels. Following colchicine treatment, no visible CCK-8-like immunoreactive cell bodies were observed in the cat area postrema. Results of the present investigation provide morphological evidence for the role of CCK-8 in cardiovascular regulation and satiety. The difference in the distribution of CCK-8 in the rat and cat suggest a possible role in the emetic reflex.     

The penetration of sodium into the brain following a cisternal injection of sodium chloride with particular emphasis on the area postrema

Summary Following an intracisternal injection of sodium chloride, sodium has been localized in paraventricular and subpial tissues of the posterior fossa by means of the pyroantimonate histochemical technique, with the use of a buffered pyroantimonate medium. The electron dense deposit is present in these tissues within 4 minutes after injection and is found only extracellularly except in the area postrema. This finding supports the contention that sodium is chiefly an extracellular ion and that the cerebrospinal fluid and the extracellular fluid are in equilibrium at these sites. In the area postrema, an intracellular precipitate is noted in the vesicular structures of the atypical astrocytes of this structure and in pinocytic vesicles of the large blood vessels. The intraglial localization of sodium in the area postrema is discussed in relation to a possible function of this structure as a regulator of cerebrospinal fluid ionic content.This work was supported by grant number NB-08549-02 from the National Institute of Neurological Disease and Stroke, Bethesda, Maryland.     

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.     

Cellular junctions in perifollicular contractile tissue of the rat ovary during the preovulatory period

Summary Rat ovarian perifollicular contractile tissue was examined at specified intervals prior to ovulation to determine the type, relative number, and length of cellular junctions. Rat ovaries were taken for electron-microscopic observation at 1500 h on the afternoon of proestrus (proestrus 0-h group), at 2000 h (proestrus 5 h group), at 0100 h (proestrus 10-h group) and at 1600 h on the afternoon of diestrus I. Close junctions, intermediate junctions, and gap junctions were counted and measured. The number of gap junctions 1,000 m of membrane and the number of intermediate junctions 1,000 m of membrane was significantly higher in the proestrus 10 h group as compared to the other groups. There was no difference in the number of close junctions during the periods studied. Also the length of all junctions was similar in all groups. These morphological findings are discussed in the context of a contractile role for perifollicular tissue in the ovulatory process.     

The functional and structural border between the CSF-and blood-milieu in the circumventricular organs (organum vasculosum laminae terminalis,subfornical organ,area postrema) of the rat

Summary The present study continues a previous investigation on the median eminence (EM) (Krisch et al., 1978). In rats with high levels of neurohormones (LHRH, vasopressin) a limited immunohistochemical labeling of perivascular tanycyte processes can be observed surrounding capillaries in the marginal region of the organum vasculosum laminae terminalis (OVLT) and in the inner part of the subfornical organ (SFO). This labeling extends from the perivascular space a short distance along the tanycyte processes. By conventional electron microscopy and by freeze-etching, tight junctions are demonstrated at a distance from the capillary lumen which corresponds to the borderline of the immunohistochemical labeling of perivascular tanycyte processes in light microscopic preparations. The tight junctions are arranged in several parallel and helical rows and correspond to those found in the median eminence. Consequently, the immunohistochemical labeling in the OVLT and in the SFO marks the intercellular cleft. In the circumventricular organs the immunostaining labels the extension of the perivascular space characterized by the hemal milieu. The perivascular space is separated off by tight junctions from the CSF-milieu of the adjacent neuropil. Furthermore, the present study demonstrates tight junctions in the marginal region of the area postrema (AP) between the perivascular processes of the tanycytes.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung VolkswagenwerkThe skillful technical assistance of Miss K. Bielenberg, Mrs. A. Hinz and Mrs. Helga Prien is thankfully acknowledged     

In utero exposure to tributyltin alters the expression of E-cadherin and localization of claudin-1 in intercellular junctions of the rat ventral prostate

Tributyltin (TBT) is an environmental contaminant, exhibiting well-established toxicity to reproductive systems in aquatic organisms. Little information exists regarding the effects of TBT on mammalian reproduction. Cellular junctions are crucial for sperm development and maturation. Intercellular tight junctions are formed by transmembrane proteins such as claudins (Cldns), while the formation of tight junctions involves signaling components of adhering junctions, comprised of cadherins. The objectives of this study were to determine the effects of in utero exposure to TBT on the rat ventral prostate. Pregnant Sprague-Dawley rats were given doses of TBT (2.5, 10, or 20 mg/kg) throughout gestation and sacrificed at Day 91. Ventral prostate weights of TBT-treated rats were decreased in all treatment groups. Results of gene expression macro-array analysis indicated that numerous genes related to cellular adhesion and cell polarity were affected. Cldn-1 mRNA levels decreased after exposure to TBT. Cldn-1 was immunolocalized to the apical lateral margins of adjacent prostatic epithelial cells in controls, but was increasingly dispersed along the lateral plasma membrane with increasing TBT dose, suggesting that the targeting of Cldn-1 or its localization to tight junctions was altered as a result of fetal TBT exposure. E-cadherin mRNA levels and immunolocalization were decreased in a dose-dependent manner. These data indicate that in utero TBT exposure results in permanent alterations in ventral prostate and that these are associated with alterations in the expression and distribution of cell adhesion and tight junctional proteins.     

Intermitochondrial junctions in the extraocular muscle of the rat

Summary Intermitochondrial junctions with a spacing of 17–21 nm were observed in the superior rectus muscle of a rat. Periodic rounded densities are aligned midway between the apposed outer mitochondrial membranes at some of these junctions. Such densities have a diameter of about 8–10 nm and a center-to-center spacing of about 26–30 nm. These junctions occur in cases where one mitochondrial profile is enclosed within another or where two profiles are interlocked so that their combined overall form has a smoothly contoured profile. Intermitochondrial junctions seem not to have been previously described in muscle, but have been reported in other kinds of tissues. In agreement with those previous reports, the presently observed intermitochondrial junctions usually involve mitochondria that display atypical features indicative of tissue abnormality or stress. Such junctions were never observed in normal extraocular muscle.This study was supported by Grants EY-00309, EY-01297 and EY-01842 from the National Eye Institute (USPHS) and in part by a grant from the Eye Surgery Fund, Inc. and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc.     

Alterations in intercellular junctions of the uterine epithelium during the preimplantation phase in the rabbit

Summary In the rabbit, the pseudopregnant uterus has been used as a model for studying alterations characteristic of the preimplantation phase. Alterations in intercellular junctions of the uterine epithelium were investigated during early pseudopregnancy (day 0 to day 6) by means of the freeze-fracture technique.In the uterine epithelium of oestrous females the zonula occludens belongs to the tight type of tight junctions. During pseudopregnancy an impressive proliferation of tight junctional belts can be observed. The basal strands proliferate, forming loops perpendicular to the luminal surface, whereas the more or less parallel arrangement of the luminal strands is maintained. At day 4 of pseudopregnancy macular tight junctions begin to develop on the lower portions of the lateral plasmalemma and are extensive by day 6 post hCG.Small gap junctions are infrequent between cells of the uterine epithelium and show no significant changes during the preimplantation phase.The physiological significance of the present morphological observations is discussed in the light of changes occurring during the preimplantation period.Supported by grant Kü 210/9 from the Deutsche Forschungsgemeinschaft     

A morphological,enzyme-cytochemical,and physiological study of the blood-gonad barrier in the hermaphroditic snail Lymnaea stagnalis

Summary In the hermaphroditic pulmonate snail Lymnaea stagnalis a blood-gonad (blood-testis) barrier appears to exist. Septate junctions between Sertoli cells and epithelial cells of the neck areas of the gonadal acini constitute this barrier; they separate the male from the female compartment. Experiments with tracer substances (colloidal gold particles, lanthanum nitrate, tannic acid) showed that the basal lamina around the acini hardly forms a barrier; only the larger colloidal gold particles do not pass this lamina.Physiologically, the blood-gonad barrier is apparent in studies on the composition of gonadal fluid, which differs considerably from that of haemolymph. The osmolarity and the concentration of protein and amino acids in gonadal fluid exceed those of haemolymph. As to the major ions, in the gonadal fluid Na⁺ is partly replaced by K⁺, and HCO ₃ ^- is almost totally replaced by Cl^-. Such a distribution of HCO ₃ ^- and Cl^- is indicative of metabolic acidosis. The cytochemical localization of carbonic anhydrase activity in cells lining the acinar lumen (Sertoli cells, epithelial cells) suggests that these cells are involved in the process of ion exchange. The metabolic acidosis in the gonad might result from the anaerobic production of lactate and succinate by Sertoli cells; these cells lack the enzymes cytochrome oxidase, lactate dehydrogenase, and succinate dehydrogenase. Spermatogenic cells, on the other hand, do possess these enzymes. This probably indicates that these cells metabolize lactate and succinate secreted by Sertoli cells.The authors are greatly indebted to Prof. Dr. H.H. Boer for stimulating comments during this study, to Dr. C.J.F. van Noorden and Miss Ilse M.C. Vogels for localization of dehydrogenase activity; to Mr. R. van Elk for determining amino acids, and to Dr. W.P.W. van der Knaap for performing the agglutinin assay     

Compartments in the organum vasculosum laminae terminalis of the rat and their delineation against the outer cerebrospinal fluid-containing space

Summary Using intravenously injected horseradish peroxidase (HRP) as tracer, we demonstrate, that — in contrast to other neurohemal regions — the organum vasculosum laminae terminalis (OVLT) is composed of two functionally different divisions. Both parts of the OVLT are endowed with fenestrated capillaries which, however, obviously differ in their permeability for HRP. In one of these portions the neurohemal region remains unlabeled under the experimental conditions used, while the other portion, in analogy to the majority of neurohemal regions, is labeled by the tracer. The functionally different divisions of the OVLT are separated from one another by tanycytic processes and meningeal cells establishing a barrier between the two hemal compartments. The meningeal elements penetrate the organ in the form of an uninterrupted layer; they are continuous with the pia mater and produce large amounts of basal lamina-like material. Furthermore, they provide the delineation of the OVLT against the outer cerebrospinal fluid-containing compartment, a structural feature that is characteristic of both divisions of the OVLT and corresponds to the arrangement of meninges in all other portions of the brain where a blood vessel penetrates its surface.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/5-2) and the Stiftung Volkswagenwerk     

The role of cytoskeletal components in the maintenance of intercellular junctions in an insect

Summary Accessory glands of the cockroach are composed of secretory and supportive cells, the latter providing a skeleton-like framework of attentuated cytoplasmic processes into which the former are positioned. These two cell types are associated with one another laterally by adhaering, pleated septate, and gap junctions. Hemi-adhaerens junctions are also found on both luminal and basal surfaces of the gland; the former are associated with the cuticular lining of the lumen and the latter with extracellular matrix. The adhering and septate junctions are flanked by both filaments and microtubules; the former insert into the junctional membranes and are actin-like, binding both rhodamine-conjugated phalloidin and the S₁ subfragment of rabbit heavy meromyosin. The role of this cytoskeletal protein with the cellular junctions has been explored by treatment with a disruptive agent, cytochalasin D. Dissociation of actin leads to changes in septate junctions and in microtubular distribution. This suggests that the latter act as anchors for the actin filaments which, in turn, appear bound to certain of the intramembranous junctional components.Supported by a Conicet/Royal Society Visiting Fellowship     

Fine structure of the rectum in cockroaches (Dictyoptera): General organization and intercellular junctions

The organization of the rectal pads is described in cockroaches belonging to the Groups Blattoidea (Periplaneta americana, Blatta orientalis) and Blaberoidea (Supella supellectilium, Blaberus craniifer). In the Blattoidea, each pad is composed of two layers (principal and basal cells) and is surrounded by very narrow junctional cells supporting the sclerotized cuticle of the pad frame; basally, the junctional cells abut on to the basal cells. In the Blaberoidea, the basal cell layer is discontinuous, the basal cells being interspersed between extensions of the junctional cells beneath the pad. The ultrastructural features of each cell type is described, with special reference to the intercellular junctions, which exhibit unusual complexity. Four types of junction are recognized: desmosomes (belt and spot desmosomes), gap junctions, septate junctions and scalariform (ladder-like) junctions. The last are usually closely associated with mitochondria, forming mitochondrial-scalariform junction complexes (MS). The distribution of these junctions is examined in relation to the partitioning of extracellular spaces, and to the problem of fluid transport.