Continuous delivery of a monoclonal antibody against Reissner's fiber into CSF reveals CSF-soluble material immunorelated to the subcommissural organ in early chick embryos.

The subcommissural organ (SCO) is an ependymal differentiation located in the dorsal midline of the caudal diencephalon under the posterior commissure. SCO cells synthesize and release glycoproteins into the cerebrospinal fluid (CSF) forming a threadlike structure known as Reissner's fiber (RF), which runs caudally along the ventricular cavities and the central canal of the spinal cord. Numerous monoclonal antibodies have been raised against bovine RF and the secretory material of the SCO. For this study, we selected the 4F7 monoclonal antibody based on its cross-reactivity with chick embryo SCO glycoproteins in vivo. E4 chick embryos were injected with 4F7 hybridoma cells or with the purified monoclonal antibody into the ventricular cavity of the optic tectum. The hybridoma cells survived, synthesized and released antibody into the CSF for at least 13 days after the injection. E5 embryos injected with 4F7 antibody displayed precipitates in the CSF comprising both the monoclonal antibody and anti-RF-positive material. Such aggregates were never observed in control embryos injected with other monoclonal antibodies used as controls. Western blot analysis of CSF from E4-E6 embryos revealed several immunoreactive bands to anti-RF (AFRU) antibody. We also found AFRU-positive material bound to the apical surface of the choroid plexus primordia in E5 embryos. These and other ultrastructural evidence suggest the existence of soluble SCO-related molecules in the CSF of early chick embryos.

Hydrocephalus induced by immunological blockage of the subcommissural organ-Reissner's fiber (RF) complex by maternal transfer of anti-RF antibodies.

Stenosis of the cerebral aqueduct seems to be a key event for the development of congenital hydrocephalus. The causes of such a stenosis are not well known. Overholser et al. in 1954 (Anat Rec 120:917-933) proposed the hypothesis that a dysfunction of the subcommissural organ (SCO) leads to aqueductal stenosis and congenital hydrocephalus. The SCO is a brain gland, located at the entrance of the cerebral aqueduct, that secretes glycoproteins into the cerebrospinal fluid that, upon release, assemble into a fibrous structure known as Reissner's fiber (RF). By the permanent addition of new molecules to its rostral end, RF grows and extends along the aqueduct, fourth ventricle, and central canal of the spinal cord. The immunological blockage of the SCO-RF complex has been used to test Overholser's hypothesis. The following was the sequence of events occurring in pregnant rats that had been immunized with RF glycoproteins: the mother produced anti-RF antibodies and transferred them to the fetus through the placenta and to the pup through the milk, and the antibodies reached the brain of the fetus and pup and blocked the SCO-RF complex. This resulted in a permanent absence of RF that was followed by stenosis of the cerebral aqueduct, and then by the appearance of hydrocephalus. The latter was patent until the end of the 6-month observation period. The chronic hydrocephalic state appeared, in turn, to induce new alterations of the SCO. It is concluded that a selective immunological knock out of the SCO-RF complex leads to hydrocephalus.

Changes in the cerebrospinal-fluid monoamines in rats with an immunoneutralization of the subcommissural organ-Reissner's fiber complex by maternal delivery of antibodies.

The subcommissural organ (SCO) is a brain gland secreting glycoproteins into the cerebrospinal fluid (CSF), where they aggregate forming the Reissner's fiber (RF). By the continuous addition of newly released glycoproteins, RF grows along the cerebral aqueduct, fourth ventricle, and central canal of the spinal cord. At the filum, RF-glycoproteins escape from the central canal and reach the local blood vessels. Despite a century of research, the function of the SCO remains elusive. The aim of the present investigation was to test the hypothesis that RF-glycoproteins, by binding and transporting monoamines out of the CSF, participate in the clearance of these compounds. A protocol was designed that led to the permanent immunoneutralization of the SCO through the maternal delivery of antibodies. This was achieved by transplacental transfer to the fetuses, and through the milk to the pups, of specific antibodies against SCO secretory proteins. The antibodies reached the CSF of the fetuses and pups and blocked the RF formation during the first months of life. Some of these animals died during the first postnatal weeks; those who survived displayed a rise in the CSF concentration of several monoamines, l-DOPA being the one with the highest rise. Adult rats transiently deprived of RF by a single injection of anti-RF antibodies into the CSF showed a transient rise in the CSF concentration of l-DOPA. All these results support the hypotheses that the SCO-RF complex participates in the clearance of monoamines from the CSF.

Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner's fiber.

The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner's fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.

Rostral floor plate (flexural organ) secretes glycoproteins immunologically similar to subcommissural organ glycoproteins in dogfish (Scyliorhinus canicula) embryos.

The subcommissural organ of vertebrates secretes glycoproteins into the cerebrospinal fluid of the third cerebral ventricle. This material polymerizes in Reissner's fiber. During ontogenetic development, besides the subcommissural organ, the ependyma lining the pontine flexure constitutes an additional Reissner's fiber-secreting gland named flexural organ. We have studied the secretion of the flexural organ and the subcommissural organ in dogfish (Scyliorhinus canicula) embryos using three different antisera and the lectins concanavalin A and wheat germ agglutinin. AFRU is an antiserum against the bovine Reissner's fiber, Ab-600 is an antiserum against 600 kDa dogfish subcommissural organ glycoproteins; and APSO is an antiserum against immunoaffinity purified bovine subcommissural organ secretory glycoproteins. These three antisera immunostained the flexural organ indicating that it contains epitopes similar to those present in bovine and dogfish subcommissural organ glycoproteins. It seems highly probable that the flexural organ and the subcommissural organ of dogfish embryos secrete similar compound(s). Other ependymal regions were also immunostained with Ab-600 and APSO antisera. Then, Reissner's fiber-like glycoproteins were transiently expressed by most embryonary ependymal cells. These glycoproteins might play a role in the development of the central nervous system of vertebrates.

Antibody-mediated lysis of the bovine subcommissural organ maintained in culture.

The subcommissural organ (SCO) is a brain gland that secretes glycoproteins into the cerebrospinal fluid (CSF). It is an ancient and conserved secretory structure of the brain, developing very early in ontogeny. However, the function of the SCO is unknown. The secretory cells of the SCO are arranged into a single or double, irregularly shaped layer located at the interface of the CSF and nervous tissue. This has prevented its selective surgical destruction. The present investigation was designed to destroy the secretory cells of 30-day-old explants of bovine SCO by use of an immunological approach. A membrane preparation enriched with plasma membrane of the secretory cells of the bovine SCO was obtained. This preparation was further processed to separate the structural proteins. A similar procedure was applied to obtain a fraction of integral proteins of the plasma membrane of a nonsecretory ciliated ependyma. Antisera were prepared against both preparations of integral proteins. The antiserum against the fraction obtained from the SCO cells immunostained the plasma membrane of the bovine SCO cells and in immunoblot it reacted with several proteins of the membrane preparation from SCO cells. When added to the culture medium this antibody bound to the apical plasma membrane of the secretory ependyma of the bovine SCO kept in culture; it caused the lysis of these cells when used together with complement. None of these properties were displayed by the antiserum raised against the integral proteins of the plasma membrane of the ciliated ependyma. This antiserum, however, immunostained the bovine ciliated ependyma neighboring the SCO. These results indicate that immunological surgery of the SCO in living animals may be possible to achieve.

Monoclonal antibodies as probes for the analysis of the secretory ependymal differentiation in the subcommissural organ of the chick embryo.

Monoclonal antibodies directed against components of the subcommissural organ (SCO) of the chick embryo were produced by immunizing mice with SCO homogenate. In three series of production, 788 hybridomas were screened by immunofluorescence microscopy. Four hybridoma cell lines producing antibodies that specifically recognize both SCO cells and Reissner's fiber (RF) were selected and cloned. Using these immunological probes, the ontogenetic development of the SCO and RF was investigated in the chick embryo. Immunoreactive material could be detected in the SCO anlage from stage 17 on and RF was first observed in the central canal of the thoracal part of the spinal cord in 10-day-old embryos. Monoclonal antibodies can be useful as markers for analyzing molecular mechanisms involved in the specific function of these ependymal cells.

Pinealocytes immunoreactive with antisera against secretory glycoproteins of the subcommissural organ: a comparative study.

By means of light-microscopic immunocytochemistry two polyclonal antibodies (AFRU, ASO; see p. 470) directed against secretory glycoproteins of the subcommissural organ were shown to cross-react with cells in the pineal organ of lamprey larvae, coho salmon, a toad, two species of lizards, domestic fowl, albino rat and bovine (taxonomic details, see below). The AFRU-immunoreactive cells were identified as pinealocytes of the receptor line (pineal photoreceptors, modified photoreceptors or classical pinealocytes, respectively) either due to their characteristic structural features or by combining AFRU-immunoreaction with S-antigen and opsin immunocytochemistry in the same or adjacent sections. Depending on the species, AFRU- or ASO-immunoreactions were found in the entire perikaryon, inner segments, perinuclear area, and in basal processes facing capillaries or the basal lamina. In most cases, only certain populations of pinealocytes were immunolabeled; these cells were arranged in a peculiar topographical pattern. In lamprey larvae, immunoreactive pinealocytes were observed only in the pineal organ, but not in the parapineal organ. In coho salmon, the immunoreaction occurred in S-antigen-positive pinealocytes of the pineal end-vesicle, but was absent from S-antigen-immunoreactive pinealocytes of the stalk region. In the rat, AFRU-immunoreaction was restricted to S-antigen-immunoreactive pinealocytes found in the deep portion of the pineal organ and the habenular region. These findings support the concept that several types of pinealocytes exist, which differ in their molecular, biochemical and functional features. They also indicate the possibility that the AFRU- and ASO-immunoreactive material found in certain pinealocytes might represent a proteinaceous or peptide compound, which is synthesized and released from a specialized type of pinealocyte in a hormone-like fashion. This cell type may share functional characteristics with peptidergic neurons or paraneurons.

Light- and electron-microscopic immunocytochemistry and lectin histochemistry of the subcommissural organ: evidence for processing of the secretory material.

The subcommissural organ (SCO) of the rat was investigated by use of histochemical and immunocytochemical methods at the light- and electron-microscopic levels. Consecutive thin methacrylate sections were stained with the pseudoisocyanin (Psi), immunoperoxidase (IMC; employing an antiserum against Reissner's fiber, AFRU), periodic acid-Schiff (PAS) and periodic acid-silver methenamine (SM) techniques, and reacted with six types of lectins. Psi, SM, concanavalin A (Con A) and IMC were also used for double and triple sequential staining of the same section. Increasing dilutions of AFRU (from 1:1000 to 1:200 000) were used for immunostaining of serial paraffin sections. In addition, ultrastructural localization of (i) Con A-binding sites and (ii) immunoreactive secretory material was performed. Some of these procedures were also applied to the ophidian and canine SCO. Con A-positive, Psi-positive and immunoreactive materials coexisted within the same cisternae of the rough endoplasmic reticulum. The Golgi apparatus lacked Con A-positive and immunoreactive substances. Apical secretory granules and secreted material lying on the surface of the SCO showed (i) the highest affinity for AFRU, but were (ii) Con A-negative, and (iii) wheat-germ agglutinin-, PAS- and SM-positive. Reissner's fiber displayed a low affinity for AFRU. It is suggested that the SCO secretes N-linked glycoproteins, the carbohydrate and protein moieties of which undergo (i) a maturation process before being released, and (ii) some kind of modification(s) after their release into the ventricle. The perivascular secretory cells of the dog SCO might secrete a material different from that secreted by the ependymal cells.

Concanavalin A-binding glycoproteins in the subcommissural and the pineal organ of the sheep (Ovis aries). A fluorescence-microscopic and electrophoretic study.

Glycoproteins rich in mannosyl or glucosyl residues were analyzed in the subcommissural organ (SCO) and the pineal organ of the sheep (Ovis aries). By use of concanavalin A labelled with fluorescein isothiocyanate, fluorescent material was found both in ependymal and hypendymal cells of the SCO. In the pineal organ, either isolated or grouped parenchymal cells showed a marked fluorescence. These cells may correspond to ependymal elements also called "interstitial cells" or "supporting cells". In addition, scarce slender, fluorescent processes were observed in the pineal parenchyma. The techniques of electrophoresis and electrotransfer on nitrocellulose paper have been applied to analyze the glycopeptide content of the SCO and the pineal organ in comparison to cerebellar and cerebral fractions solubilized by use of Triton X 100. Approximately 30 different concanavalin A-reactive glycopeptides were revealed in each fraction. In the SCO extract four glycopeptides (30, 54, 72, 100 kd) might correspond to subunits of the glycoprotein(s) characteristically stored in the ependymal cells of the SCO. In addition, two glycopeptides (32/33, 115 kd) are specific to the pineal organ extract. The possible similarity of the concanavalin A-reactive material in both organs is discussed and a putative secretory activity of the pineal ependymal cells is postulated.

Immuno-electron-microscopic analysis of the basal route of secretion in the subcommissural organ of the rabbit.

Low-temperature-embedded tissue of the subcommissural organ (SCO) of the rabbit was analyzed for the basal route of secretory product by means of indirect immuno-metal cytochemistry (protein A-gold technique) at the electron-microscopic level. By use of (1) an antiserum against bovine Reissner's fibre (see Sterba et al. 1981) and, thereafter, (2) particulate gold-marker solution, immunoreactive sites could be clearly visualized within the extracellular matrix of both (a) the basal part of the ependymal cell layer, and (b) the hypendyma proper. Abundant secretory material was identified within (i) dilated intercellular spaces (a + b) as well as (ii) branching basal lamina labyrinths and distinct perivascular spaces (b). All these compartments are thought to belong to a system of extracellular channels, which may function in secretion directed toward hypendymal blood vessels.

[Detection of the neurotropic properties of the serum of schizophrenia patients]. / Vyiavlenie neirotropnykh svoistv syvorotki bol'nykh shizofreniei.

A method of testing neurotropic properties of the serum of schizophrenic patients is proposed. This method was tested on 27 schizophrenic patients and based on experimental data. In all cases there was a narrowing of the width of the glomerular zone in the adrenal glands of rats administered the serum of schizophrenic patients intravenously. There was a significant difference in the width of the zone depending upon the stage of the disease and exacerbation, while in experiments, depending upon the presence of antibodies to brain subcommissural organs.