Organ culture of the bovine subcommissural organ: evidence for synthesis and release of the secretory material.

The subcommissural organ (SCO) is a brain circumventricular organ formed by ependymal and hypendymal secretory cells. It secretes glycoproteins into the cerebrospinal fluid of the third ventricle where they condense into a thread-like structure known as Reissner's fiber (RF). The present study was designed to investigate whether or not the bovine SCO continues to synthesize and release glycoproteins after a long-term culture. Cultured explants of SCO survive for several months. The content of the secretory granules present in the cultured ependymocytes displayed immunoreactive and lectin-binding properties similar to those of the core glycosylated glycoproteins found in the bovine SCO. The explants actively incorporated (35)S-cysteine. In the cultured ependymocytes, the pattern of distribution of the radioactive label and that of the immunoreactive secretory material was similar, thus indicating that this material has been synthesized during culture. At the ultrastructural level, the cultured tissue exhibited a high degree of differentiation comparable to that of the bovine SCO in situ. A striking finding was the observation of similar results when cerebrospinal fluid was used as a culture medium. The addition of antibodies against RF-glycoproteins into the culture medium allowed visualization, by means of different immunocytochemistry protocols, deposits of extracellular immunoreactive secretory material on the free surface of the cultured ependymocytes, indicating that release of secretory glycoproteins into the culture medium does occur. Primary culture of dispersed SCO ependymocytes, obtained either from fresh or organ cultured bovine SCO, showed that these cells release RF-glycoproteins that aggregate in the vicinity of each cell. The present investigation has shown that: (1) two types of secretory ependymocytes become evident in the cultured SCO; (2) under culture conditions, the SCO cells increase their secretory activity; (3) explants of bovine SCO synthesize RF-glycoproteins and release them to the culture medium; (4) after release these proteins aggregate but do not form a RF; (5) a pulse of anti-RF antibodies into the culture medium blocks the secretion of RF-glycoproteins for several days.

Spatial distribution of Reissner's fiber glycoproteins in the filum terminale of the rat and rabbit.

The subcommissural organ secretes into the third ventricle glycoproteins that condense to form the Reissner's fiber (RF). At the distal end of the central canal of the spinal cord, the RF-glycoproteins accumulate in the form of an irregular mass known as massa caudalis. Antibodies against RF-glycoproteins and a set of lectins were used at the light and electron microscopic level to investigate the spatial distribution of the massa caudalis material in the rat and rabbit filum terminale. In the sacral region of the rat, the central canal presents gaps between the ependymal cells through which RF-glycoproteins spread out. The bulk of massa caudalis material, however, escapes through openings in the dorsal wall of the terminal ventricle. In the rabbit, the massa caudalis is formed within the ependymal canal, at the level of the second coccygeal vertebra, it accumulates within preterminal and terminal dilatations of the central canal, and it escapes out through gaps in the dorsal ependymal wall of the terminal ventricle. The existence of wide intercellular spaces and a large orifice (neuroporous) in the dorsal ependymal wall of the terminal ventricle, and the passage of RF-material through them, appear to be conserved evolutionary features. After leaving the terminal ventricle of the rat and rabbit, RF-glycoproteins establish a close spatial association with the numerous blood vessels irrigating the filum terminale, suggesting that in these species the blood vessels are the site of destination of the RF-glycoproteins escaping from the central canal, thus resembling the situation found in lower vertebrates. When passing from the RF stage to the massa caudalis stage, the rabbit RF-glycoproteins lose their sialic acid residues, exposing galactose as the terminal residue. Since this sialic acid-galactose modification of RF-glycoproteins had also been described in lamprey larvae, it may be regarded as a conserved evolutionary feature associated with the formation of the massa caudalis.

Hindbrain floor plate of the rat: ultrastructural changes occurring during development.

Most of the molecular and experimental studies on the floor plate (FP) have been performed on the FP region extending along the spinal cord. However, little is known about the hindbrain FP. The FP undergoes regional and temporal changes throughout development, but information with respect to the ultrastructural correlate of such changes is missing. The present investigation was focused on the ultrastructural developmental changes occurring in the FP of the rat hindbrain. The FP cells of the hindbrain secrete a material reacting with antibodies against the secretory glycoproteins of the subcommissural organ (AFRU). This antibody was used to perform an ultrastructural immunocytochemical analysis of the rat FP. From E-12 on, there is a progressive increase in the development of the rough endoplasmic reticulum (RER), so that by E-18, it has reached a high degree of hypertrophy. A unique feature of the hindbrain FP cells is the presence of tubular formations and 140-nm vesicles that appear to originate from RER cisternae. The labelling of these two structures with AFRU and Concanavalin A strongly suggests that they are pre-Golgi compartments containing secretory material. Since these structures are present in the basal process and in the apical cell pole of the FP cells, the possibility that they release their content at these sites, is discussed. It is proposed that a secretory mechanism bypassing the Golgi apparatus (constitutive secretion?) operates in the FP cells. The presence of apoptotic cells within the FP of E-20 embryos and newborns suggests that death, and not re-differentiation, is the fate of the FP cells.

A second look at the barriers of the medial basal hypothalamus.

The cell bodies of hypothalamic secretory neurons are localized in areas protected by the blood-brain barrier (BBB), whereas their axon terminals are localized in the median eminence, which lacks a BBB. This implies a complex barrier system, allowing neurons of the central nervous system to secrete into the blood stream without making the BBB leaky. In the present study, three experimental protocols were applied to clarify certain relevant aspects of the barriers operating in the medial basal hypothalamus of the rat. We established that the milieu of the arcuate nucleus is exposed to both the ventricular and the subarachnoidal cerebrospinal fluid (CSF). The median eminence milieu, the perivascular space of the portal vessels, and the subarachnoid space appear to be in open communication; also, beta2-tanycytes establish an efficient barrier between the median eminence milieu and the ventricular CSF. Similarly, beta1-tanycytes establish a lateral barrier, separating the intercellular space of the median eminence from that of the arcuate nucleus. We also found that the glucose transporter I (GLUT I), a BBB marker, is localized throughout the whole plasma membrane of beta1-tanycytes, but is missing from beta2-tanycytes. Expression of GLUT I by tanycytes progressively develops during the first postnatal weeks; while the degree of damage of the arcuate nucleus by administration of monosodium glutamate, at different postnatal intervals, parallels that of the GLUT I immunoreactivity of beta1-tanycytes. An explanation is offered for the selective destruction of the arcuate neurons by the parenteral administration of monosodium glutamate to infant rats.

Floor plate and the subcommissural organ are the source of secretory compounds of related nature: comparative immunocytochemical study.

The subcommissural organ of vertebrates secretes glycoproteins into the third ventricle that condense to form Reissner's fiber (RF). Antibodies raised against the bovine RF-glycoproteins reacted with the floor plate (FP) cells of two teleost (Oncorhynchus kisutch, Sparus aurata) and two amphibian (Xenopus laevis, Batrachyla taeniata) species. At the ultrastructural level, the immunoreactivity was confined to secretory granules, mainly concentrated at the apical cell pole. In the rostro-caudal axis, a clear zonation of the FP was distinguished, with the hindbrain FP being the most, or the only (Batrachyla taeniata), immunoreactive region of the FP. In all the species studied, the caudal FP lacked immunoreactivity. Both the chemical nature of the immunoreactive material and the rostro-caudal zonation of the FP appear to be conservative features. Evidence was obtained that the FP secretes into the cerebrospinal fluid a material chemically related to the RF-glycoproteins secreted by the subcommissural organ. Thus, in addition to being the source of contact-mediated and diffusible signals, the FP might also secrete compounds into the cerebrospinal fluid that may act on distant targets.

Light- and electron-microscopic immunocytochemical investigation of the subcommissural organ using a set of monoclonal antibodies against the bovine Reissner's fiber.

Ten monoclonal antibodies (Mabs) against glycoproteins of the bovine Reissner's fiber (RF) have been used in a structural and ultrastructural immunocytochemical investigation of the bovine subcommissural organ (SCO) and RF. The SCO of other vertebrate species has also been studied. For comparison, polyclonal antibodies against bovine RF (AFRU) were used. The SCO and RF of ox, pig and dogfish and the SCO of dog, rabbit, rat and frog were submitted to light-microscopic immunocytochemistry using AFRU and Mabs. Postembedding ultrastructural immunocytochemistry was applied to sections of bovine SCO using AFRU and Mabs. Bovine SCO consists of ependymal and hypendymal cell layers, the latter being arranged as cell strands across the posterior commissure, or as hypendymal rosette-like structures. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with AFRU. Six Mabs showed the same staining pattern as AFRU, one Mab stained RF strongly and SCO weakly, two Mabs stained RF but not SCO, and, finally, one Mab (3B1) exclusively stained the apices of the ependymal and hypendymal cells. All Mabs recognized the SCO and RF of the pig. Two Mabs bound to the SCO of the dog. One Mab stained the SCO of the rabbit and another the SCO of the rat. The SCO of frog and dogfish were totally negative. Bovine SCO stained with AFRU, showed label in the rough endoplasmic reticulum (RER) and the secretory granules (SG) of the ependymal and hypendymal cells. The former, in the form of parallel cisternae, reticulum or concentric rings, was seen throughout all cytoplasmic regions. SG were abundant in the apical pole of the ependymal and hypendymal cells. Only one Mab showed a staining pattern similar to AFRU. Five Mabs showed strong reactions in the SG but weak labeling of the RER. Mab 3B1 showed the label confined to the SG only. Our results suggest that: (i) in the bovine tissue, some epitopes are present in both precursor and processed materials, whereas others are characteristic of mature glycoproteins present in SG and the RF; (ii) the bovine SCO secretes at least two different compounds present in ependymal and hypendymal cells; (iii) both compounds coexist in the same secretory granule; (iv) there are conserved, class-specific, and species-specific epitopes in the glycoproteins secreted by the SCO 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.

Analysis of the secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula).

The subcomissural organ (SCO) is an ancient and conserved brain gland secreting glycoproteins into the cerebrospinal fluid which condense to form Reissner's fiber (RF). The SCO of an elasmobranch species, the dogfish Scyliorhinus canicula, was investigated applying morphological and biochemical methods. The SCO of 34 dogfishes were processed for the following techniques: (1) conventional transmission electron microscopy; (2) light and electron microscopy lectin histochemistry (Concanavalin A, Con A; wheat germ agglutinin, WGA; Limax flavus agglutinin, LFA); (3) light and electron microscopy immunocytochemistry using antisera raised against the glycoproteins of the bovine RF (anti-bovine RF), and the secretory material of the dogfish SCO (anti-dogfish SCO). The former reacts with the SCO of virtually all vertebrate species [19] (conserved epitopes); the latter reacts only with the SCO of elasmobranchs [Cell Tissue Res., 276 (1994) 515-522] (class-specific epitopes). At the light microscopic level both antisera immunoreacted selectively with the SCO and RF; no other structure of the central nervous system was reactive. Within the SCO the binding sites for WGA (affinity = glucosamine, sialic acid) and LFA (affinity = sialic acid) displayed the same density and intracellular distribution. At the ultrastructural level two types of granules were distinguished. Type I granules (200-400 nm) were numerous, reacted with both antisera, bound WGA but not Con A. Type II granules (0.8-1.8 microns) reacted with the anti-bovine RF serum but not with the anti-dogfish SCO serum, bound Con A and WGA. The content of dilated cisternae of the rough endoplasmic reticulum reacted with both antisera and bound Con A; it did not bind WGA. The SCOs of 4500 dogfishes were extracted in ammonium bicarbonate. This extract was used for SDS-PAGE and blotting. Blots were processed for immunolabeling using anti-bovine RF and anti-dogfish SCO sera, and for lectin binding (Con A, WGA and LFA). The anti-bovine RF revealed four compounds with apparent molecular weights of 750, 380, 145 and 35 kDa. The two former also reacted with the anti-dogfish SCO serum and bound Con A. Only the 380 kDa compound bound WGA and LFA. The findings indicate that both the conserved and the class-specific epitopes are part of the same compounds (780, 380 kDa), which would be stored in type I granules. The lectin binding properties of these compounds point to the 780 kDa compound as a precursor form and the 380 kDa polypeptide as a processed form.

Ultrastructural immunocytochemistry and lectin histochemistry of the subcommissural organ in the snake Natrix maura with particular emphasis on its vascular and leptomeningeal projections.

The ependymal cells of the subcommissural organ (SCO) of the snake Natrix maura display long basal processes which terminate either on blood vessels or on the leptomeninges. The cell body and the basal processes contain a secretory material detectable immunocytochemically at the light-microscopic level using an antibody raised against bovine Reissner's fiber. The present investigation deals with the ultrastructural location in these cells of the (i) immunoreactive material; (ii) concanavalin A (Con A)- and wheat-germ agglutinin (WGA)-binding sites. In the subnuclear region the immunoreactive material was located within dilated cisternae of the rough endoplasmic reticulum and had affinity for Con A but not for WGA. In the supranuclear region the secretory material was exclusively located within numerous granules. Since all these granules showed affinity for WGA, they can be regarded as "post-Golgi" elements. Thus, at variance with the situation in the mammalian SCO, in the ophidian SCO most of the secretion is stored in secretory granules rather than in dilated cisternae of the rough endoplasmic reticulum. In the perivascular and leptomeningeal endings the immunoreactive material was located within granules which, because of their affinity for WGA, should also be regarded as true secretory granules derived from the Golgi apparatus. It is concluded that these granules are transported along the basal processes and accumulated in the perivascular and leptomeningeal endfeet. This observation favours the view of a local release of the content of these granules, since there is no evidence for a reverse transport of these granules all the way back from the distal termination to the apical pole, to be finally released into the ventricle.

Single injection into the cerebrospinal fluid of antibodies against the secretory material of the subcommissural organ reversibly blocks formation of Reissner's fiber: immunocytochemical investigations in the rat.

An antibody (cf. Rodríguez et al. 1984b) raised in rabbits against the glycoproteins of the bovine Reissner's fiber (RF) was injected into the lateral brain ventricle of 38 rats with the aim to interfere with RF formation. The rats were killed 20 min; 1, 4, 8, 12 h; and 1, 2, 3, 5, and 8 days after the injection. Based on the fact that the material secreted by the subcommissural organ (SCO) into the cerebrospinal fluid (CSF) first condenses on the organ surface as a distinct layer (pre-RF material) and then becomes assembled to form RF and that both structures are distinguishable in tissue sections, three immunostaining procedures were applied. They served to visualize: (i) secretory material that had not bound the injected antibody; (ii) secretory material-antibody complexes formed in vivo; and (iii) antibody not bound to its antigen and present in the ventricles and the subarachnoid space. After a single injection of the above-mentioned antibody the following events were observed: (1) The antibody was present in the brain cavities for at least 8 h. (2) The injected antibody bound selectively to the pre-RF and RF. (3) Pre-RF displayed antibody binding during the 24 h following the injection. During the 2nd and 3rd post-injection days, the pre-RF was free of antibody, indicating that it was formed by newly released secretory material. (4) Approximately 4 h after the injection, the RF detached from the SCO and underwent fragmentation. Clusters of these fragments were found in the Sylvian aqueduct and fourth ventricle. (5) In the fragmented original RF the injected antibody against Reissner's fiber remained bound throughout the entire period of observation, i.e. for 8 days. (6) In rats of the 1-, 3-, 5- and 8-day-groups, RF was missing from the central canal of the spinal cord. (7) One day after the injection, a new RF structure started to grow from the rostral end of the SCO. This newly formed fiber could be distinguished from the original RF because of (i) its normal appearance; (ii) it did not display binding of the injected antibody. (8) At day 3, the growing RF had not yet extended to the Sylvian aqueduct. (9) At day 8, the new RF reached the fourth ventricle. Control experiments involved the intraventricular administration of (i) an antibody against the secretory material extracted from the entire bovine SCO; (ii) antivasopressin; and (iii) rabbit IgG. From these only antibody (i) bound to pre-RF and RF.

Light and electron microscopical demonstration of concanavalin A and wheat-germ agglutinin binding sites by use of antibodies against the lectin or its label (peroxidase).

Three straining protocols for the ultrastructural visualization of concanavalin A (ConA) and wheat germ agglutinin (WGA) binding sites were applied to samples of nervous tissue embedded in Lowicryl K4M. The hypothalamo-neurohypophysial neurosecretory system was chosen for this investigation because it has two major neuronal populations, one secreting vasopressin, whose precursor is glycosylated, and the other secreting oxytocin whose precursor form is not glycosylated. The series of incubations of the tissue sections for the three protocols were: Protocol 1: i) non labeled ConA or WGA; ii) ConA or WGA antibody; iii) protein A-gold; Protocol 2: i) pre-prepared WGA-anti-WGA complex; ii) protein A-gold; Protocol 3: i) peroxidase-labeled ConA or WGA; ii) anti-peroxidase; iii) protein A-gold. The three methods allowed to detect fine differences in the distribution of sugar residues. This, in turn, made it possible to distinguish vasopressin granules containing precursor forms from those containing processed precursor. At the light microscopic level the three methods were successfully applied to paraffin and 1-micron methacrylate sections by using a second antibody, PAP complex and the diaminobenzidine reaction.

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.

Immunocytochemistry and ultrastructure of the neuropil located ventral to the rat supraoptic nucleus.

The neuropil located ventral to the SON was investigated by the use of immunoperoxidase staining for neurophysins, oxytocin and vasopressin, and electron microscopy. The study was performed in six groups of rats: 1) control; 2) infusion of isotonic saline into the CSF; 3) infusion of hypertonic saline into the CSF; 4) drinking hypertonic saline for 4 days; 5) same as group 4 but injection of colchicine into the CSF on second day of dehydration; 6) salt loading for 3 months. In the control rats the ventral neuropil contained a few immunoreactive processes, the general morphology of which was completely different from that of the neurosecretory axons emerging from the SON at its dorsal aspect. In rats of groups 3 to 6 the ventral processes (VP) became loaded with neurosecretory granules, whereas the perikarya and axons were depleted. Based on their general morphology and reactivity pattern it is suggested that the VP are dendrites. Most of these "dendrites" were embedded in a glial cushion formed by the processes of a particular type of marginal glia. Some of these "dendrites" enveloped an arteriole penetrating the optic tract. All VP were rich in synaptic contacts. The possibility that the VP of neurosecretory cells may be functionally related to the subarachnoid CSF and the arteriolar blood flow is discussed.

Standardization of various applications of methacrylate embedding and silver methenamine for light and electron microscopy immunocytochemistry.

The use of butyl-methyl-methacrylate embedding and the application of the silver methenamine (SM) method as a poststaining of the immunoperoxidase-DAB (IP) procedure led to the standardization of several useful methods for the visualization of tissue antigens at the light and electron microscope level. These procedures included: 1) Standardization of the actual methacrylate embedding; 2) The IP-SM method with and without periodic acid oxidation, which provided 100% intensification of the IP staining; 3) The IP-SM method made it possible to stain semithin sections (0.5 micron), and this in turn, permitted a) clear visualization under the light microscope of the intracellular distribution of antigens and, b) staining, in several adjacent sections, of roughly the same cytoplasmic region of the same cell with different primary antisera; 4) a double immunostaining whereby the first antigen in the sequence was revealed by the IP-SM method and the second by the IP procedure; 5) standardization of the IP and the IP-SM methods for post-embedding staining of ultrathin methacrylate sections. The combined application of methacrylate embedding and the IP-SM, and the use of an appropriate fixative, resulted in an ultrastructural immunocytochemical procedure characterized by a good immunoreactivity of the tissue sections, a strong and selective immunoreaction and a well preserved ultrastructure.