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The subcommissural organ (SCO) is a well-developed gland present in the brain of vertebrates. The SCO secretes glycoproteins into the circulating cerebrospinal fluid and these assemble to form Reissner's fibre. It also plays an important function in neurogenesis and axonal guidance during embryogenesis. This study delves into the microscopic anatomy of the SCO in the adult greater cane rat (GCR), shedding light on its histoarchitectural characteristics. By utilizing histological techniques and microscopic analysis, we investigated the SCO's location and cellular composition within the brain of adult GCR. Our findings showed that the SCO in this species is located ventrally to the posterior commissure (PC) and dorsally to the third ventricle. The SCO consists of specialized ependymal or nuclear cell layer and apical processes lining the third ventricle. Moreover, the SCO's proximity to the PC and the third ventricle highlights its strategic position within the brain's ventricular system. With immunohistochemical analyses, the SCO cells expressed glial fibrillary protein when immunolabelled with Glial fibrillary acid protein (GFAP) antibody, a marker for astrocytes/astrocytic-like cells. Few microglia-like cells were immuno-positive for Ionized calcium-binding adapter molecule 1 (Iba1) antibody, that are existing within the SCO. However, the SCO in the GCR showed a negative immunostaining to NeuN antibody. This study contributes to our understanding of the microscopic anatomy of the SCO in a lesser-studied mammalian species. Further research into the SCO's functional significance especially during development in the GCR, may hold promise for more insights into neurological health and pathology., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

The subcommissural organ (SCO) is an ancient and conserved brain gland secreting into cerebrospinal fluid (CSF) glycoproteins that form the Reissner fiber (RF). The present investigation was designed to further investigate the dynamic of the biosynthetic process of RF glycoproteins prior and after their release into the CSF, to identify the RF proteome and N-glycome and to clarify the mechanism of assembly of RF glycoproteins. Various methodological approaches were used: biosynthetic labelling injecting 35 S-cysteine and 3 H-galactose into the CSF, injection of antibodies against galectin-1 into the cerebrospinal fluid, light and electron microscopical methods; isolated bovine RF was used for proteome analyses by mass spectrometry and glycome analysis by xCGE-LIF. The biosynthetic labelling study further supported that a small pool of SCO-spondin molecules rapidly enter the secretory pathways after its synthesis, while most of the SCO-spondin molecules are stored in the rough endoplasmic reticulum for hours or days before entering the secretory pathway and being released to assemble into RF. The proteomic analysis of RF revealed clusterin and galectin-1 as partners of SCO-spondin; the in vivo use of anti-galectin-1 showed that this lectin is essential for the assembly of RF. Galectin-1 is not secreted by the SCO but evidence was obtained that it would be secreted by multiciliated ependymal cells lying close to the SCO. Further, a surprising variety and complexity of glycan structures were identified in the RF N-glycome that further expands the potential functions of RF to a level not previously envisaged. A model of the macromolecular organization of Reissner fiber is proposed.

The present investigation was designed to clarify the role of the subcommissural organ (SCO) in the pathogenesis of hydrocephalus occurring in the HTx rat. The brains of non-affected and hydrocephalic HTx rats from embryonic day 15 (E15) to postnatal day 10 (PN10) were processed for electron microscopy, lectin binding and immunocytochemistry by using a series of antibodies. Cerebrospinal fluid (CSF) samples of non-affected and hydrocephalic HTx rats were collected at PN1, PN7 and PN30 and analysed by one- and two-dimensional electrophoresis, immunoblotting and nanoLC-ESI-MS/MS. A distinct malformation of the SCO is present as early as E15. Since stenosis of the Sylvius aqueduct (SA) occurs at E18 and dilation of the lateral ventricles starts at E19, the malformation of the SCO clearly precedes the onset of hydrocephalus. In the affected rats, the cephalic and caudal thirds of the SCO showed high secretory activity with all methods used, whereas the middle third showed no signs of secretion. At E18, the middle non-secretory third of the SCO progressively fused with the ventral wall of SA, resulting in marked aqueduct stenosis and severe hydrocephalus. The abnormal development of the SCO resulted in the permanent absence of Reissner's fibre (RF) and led to changes in the protein composition of the CSF. Since the SCO is the source of a large mass of sialilated glycoproteins that form the RF and of those that remain CSF-soluble, we hypothesize that the absence of this large mass of negatively charged molecules from the SA domain results in SA stenosis and impairs the bulk flow of CSF through the aqueduct.

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.

For the first time we showed the presence of CGRP immunoreactivity in the SCO of golden hamsters, Wistar rats and Mongolian gerbils. In hamsters, the intense CGRP-like immunoreactivity was detected in the whole SCO, including Reissner's fiber- and granule-like structures at the ventricular surface of the SCO. The CGRP-positive hypendymal cells were frequently in contact with local blood vessels and some of them were situated intimately to the leptomeningeal space. In the SCOs of rats and gerbils, only the supranuclear area and apical cytoplasm of the ependymal cells were positive for CGRP, whereas the basal pole of the cells and the hypendyma were CGRP-negative. The existence of CGRP in rodents SCO was confirmed by the expression of CGRP mRNA in rat SCO by RT-PCR. The present results indicate that the SCOs of rodent species contain CGRP that may be in part synthesized by ependymocytes themselves. A species difference in the CGRP distributions of rodents SCOs may therefore imply its different synthesis rates or release pathways.

Two phases may be recognized in the development of congenital hydrocephalus in the hyh mutant mouse. During embryonic life the detachment of the ventral ependyma is followed by a moderate hydrocephalus. During the first postnatal week the cerebral aqueduct becomes obliterated and a severe hydrocephalus develops. The aim of the present investigation was to elucidate the cellular phenomena occurring at the site of aqueduct obliteration and the probable participation of the subcommissural organ in this process. Electron microscopy, immunocytochemistry, and lectin histochemistry were used to investigate the aqueduct of normal and hydrocephalic hyh mice from embryonic day 14 (E-14) to postnatal day 7 (PN-7). In the normal hyh mouse, the aqueduct is an irregularly shaped cavity with 3 distinct regions (rostral, middle, and caudal) lined by various types of ependyma. In the hydrocephalic mouse, these 3 regions behave differently; the rostral end becomes stenosed, the middle third dilates, and the caudal end obliterates. The findings indicate that the following sequence of events lead to hydrocephalus: 1) denudation of the ventral ependyma (embryonic life); 2) denudation of dorsal ependyma and failure of the subcommissural organ to form Reissner fiber (first postnatal week); 3) obliteration of distal end of aqueduct; and 4) severe hydrocephalus. No evidence was obtained that NCAM is involved in the detachment of ependymal cells. The process of ependymal denudation would involve alterations of the surface sialoglycoproteins of the ependymal cells and the interaction of the latter with macrophages.

Immuno-electron microscopy specifically enhanced with silver staining has been used to demonstrate the localization of calcitonin gene-related peptide (CGRP) in the ependymocytes of the hamster subcommissural organ (SCO). Hamster SCO consists of the ependymal and hypendymal cell layers, the latter being arranged as rosette-like structure across the posterior commissure (PC) and often arranged with longitudinal axis parallel to the ventricle. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with CGRP. In the hypendymal layer, the CGRP positive hypendymal cells were frequently in contact with local blood vessels and arranged in-groups traversing the thick portion of the PC. Ultrastructurally, the CGRP-immunoreaction products were distributed at the dilated cisternae of the rough endoplasmic reticulum (rER) and secretory granules of the ependymal and hypendymal cells. The dilated cisterna of the rER was usually concentrated in the basal region of the ependymal cells and irregular in shape. These dilated cisternae were filled with a flocculent material or finely granular substance, but hardly studded with ribosomes. Labelled secretory granules were abundant in the apical pole of the ependymal cells and discharged their contents into the third ventricle in the form of a thin layer of secretion. This CGRP positive material appeared to constitute the pre-Reissner's fiber (RF). On the basis of the present ultrastructural evidences, we proposed that ependymocytic CGRP in SCO may be synthesized and stored in the cisternae of rER, then released and incorporated into the RF in the third ventricle through the secretory granules. The abundant amount of CGRP in ependymocytes of SCO and RF in the third ventricle suggests a significant endocrine function of CGRP in hamster SCO.

The subcommissural organ (SCO) is an ependymal brain gland that synthesizes and secretes glycoproteins. Very little is known about the signal transduction cascades operating in this organ and their impact on gene expression. An important transcription factor that regulates gene expression in glial cells and neurons is the cyclic-AMP-responsive element binding protein (CREB), which is activated by phosphorylation of the serine residue 133. Here, we analyzed the presence of CREB in bovine SCO cells and its phosphorylation by drugs that activate cyclic-AMP-dependent or calcium-dependent signal transduction pathways. We also investigated the effects of three natural signaling molecules, serotonin (5HT), substance P (SP) and ATP, on CREB phosphorylation and on the second messengers cyclic AMP and calcium. Investigations were performed with cell and explant cultures by using immunocytochemistry, immunoblot, enzyme-linked immunosorbent assay, and the Fura-2 technique. A strong immunosignal for total (phosphorylated and unphosphorylated) CREB was found in virtually all SCO cells. Total CREB levels did not change upon stimulation. Phosphorylated (p)CREB levels were low in unstimulated cells and significantly elevated by drugs that increase the levels of cyclic AMP or free calcium ions. pCREB was also induced by SP and ATP; both substances increased the intracellular calcium concentration but did not affect the formation of intracellular cyclic AMP. 5HT did not influence the phosphorylation of CREB, the intracellular calcium concentration, or the formation of cyclic AMP. Our data identify CREB as an SCO transcription factor that can be activated by the second messengers cAMP and calcium. SP and ATP stimulate the phosphorylation of CREB apparently via a calcium-dependent mechanism and are thus involved in the control of gene expression in the bovine SCO.

Hydrocephalic hyh mice are born with moderate hydrocephalus and a normal cerebral aqueduct. At about the fifth postnatal day the aqueduct becomes obliterated and severe hydrocephalus develops. The aim of the present investigation was to investigate the mechanism of this hydrocephalus, probably starting during fetal life when the cerebral aqueduct is still patent. By use of immunocytochemistry and scanning electron microscopy, mutant (n = 54) and normal (n = 61) hyh mouse embryos were studied at various developmental stages to trace the earliest microscopic changes occurring in the brains of embryos becoming hydrocephalic. The primary defect begins at an early developmental stage (E-12) and involves cells lining the brain cavities, which detach following a well-defined temporo-spatial pattern. This ependymal denudation mostly involves the ependyma of the basal plate derivatives. There is a relationship between ependymal denudation and ependymal differentiation evaluated by the expression of vimentin and glial fibrillary acidic protein. The ependymal cells had a normal appearance before and after detachment, suggesting that their separation from the ventricular wall might be due to abnormalities in cell adhesion molecules. The process of detachment of the ventral ependyma, clearly visualized under scanning electron microscope, is almost completed before the onset of hydrocephalus. Furthermore, this ependymal denudation does not lead to aqueductal stenosis during prenatal life. Thus, the rather massive ependymal denudation appears to be the trigger of hydrocephalus in this mutant mouse, raising the question about the mechanism responsible for this hydrocephalus. It seems likely that an uncontrolled bulk flow of brain fluid through the extended areas devoid of ependyma may be responsible for the hydrocephalus developed by the hyh mutant embryos. The defect in these embryos also includes loss of the hindbrain floor plate and a delayed in the expression of Reissner fiber glycoproteins by the subcommissural organ.

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., (Copyright 2001 Wiley-Liss, Inc.)

The subcommissural organ (SCO) is a conserved brain gland present throughout the vertebrate phylum. During ontogeny, it is the first secretory structure of the brain to differentiate. In the human, the SCO can be morphologically distinguished in 7- to 8-week-old embryos. The SCO of 3- to 5-month-old fetuses is an active, secretory structure of the brain. However, already in 9-month-old fetuses, the regressive development of the SCO-parenchyma is evident. In 1-year-old infants, the height of the secretory ependymal cells is distinctly reduced and they are grouped in the form of islets that alternate with cuboid non-secretory ependyma. The regression of the SCO continues during childhood, so that at the ninth year of life the specific secretory parenchyma is confined to a few islets of secretory ependymal cells. The human fetal SCO shares the distinct ultrastructural features characterizing the SCO of all other species, namely, a well-developed rough endoplasmic reticulum, with many of its cisternae being dilated and filled with a filamentous material, several Golgi complexes, and secretory granules of variable size, shape, and electron density. The human fetal SCO does not immunoreact with any of the numerous polyclonal and monoclonal antibodies raised against RF-glycoproteins of animal origin. This and the absence of RF in the human led to the conclusion that the human SCO does not secrete RF-glycoproteins. Taking into account the ultrastructural, lectin-histochemical, and immunocytochemical findings, it can be concluded that the human SCO, and most likely the SCO of the anthropoid apes, secrete glyco- protein(s) with a protein backbone of unknown nature, and with a carbohydrate chain similar or identical to that of RF-glycoproteins secreted by the SCO of all other species. These, as yet unidentified, glycoprotein(s) do not aggregate but become soluble in the CSF. Evidence is presented that these CSF-soluble proteins secreted by the human SCO correspond to (1) a 45-kDa compound similar or identical to transthyretin and, (2) a protein of about 500 kDa., (Copyright 2001 Wiley-Liss, Inc.)

The neural control of the subcommissural organ (SCO) has been partially characterized. The best known input is an important serotonergic innervation in the SCO of several mammals. In the rat, this innervation comes from raphe nuclei and appears to exert an inhibitory effect on the SCO activity. A GABAergic innervation has also been shown in the SCO of the rat and frog Rana perezi. In the rat, GABA and the enzyme glutamate decarboxylase are involved in the SCO innervation. GABA is taken up by some secretory ependymocytes and nerve terminals, coexisting with serotonin in a population of synaptic terminals. Dopamine, noradrenaline, and different neuropeptides such as LH-RH, vasopressin, vasotocin, oxytocin, mesotocin, substance P, alpha-neoendorphin, and galanin are also involved in SCO innervation. In the bovine SCO, an important number of fibers containing tyrosine hydroxylase are present, indicating that in this species dopamine and/or noradrenaline-containing fibers are an important neural input. In Rana perezi, a GABAergic innervation of pineal origin could explain the influence of light on the SCO secretory activity in frogs. A general conclusion is that the SCO cells receive neural inputs from different neurotransmitter systems. In addition, the possibility that neurotransmitters and neuropeptides present in the cerebrospinal fluid may also affect the SCO activity, is discussed., (Copyright 2001 Wiley-Liss, Inc.)

Ependymal cells are specialized in the synthesis and release of different factors into the cerebrospinal fluid (CSF). The subcommissural organ (SCO) is one of the most active areas of the ventricular walls secreting into the CSF. This gland is localized in the roof of the third ventricle covering the posterior commissure. Glycoproteins synthesized in SCO cells are released into the ventricular CSF where they aggregate, in a highly ordered fashion, forming an elongated supramacromolecular structure known as the Reissner's fiber (RF). RF grows caudally and extends along the brain aqueduct, the fourth ventricle, and the whole length of the central canal of the spinal cord. The SCO cells synthesize glycoproteins of high molecular weight. A precursor form of 540 kDa is synthesized in bovine and chick SCO cells, and a transcript of 10--14 kb is expressed selectively in the bovine SCO cells. The processing of this molecule generates at least one protein of about 450 kDa (RF-Gly-I), which, after being released, is involved in the formation of RF. Additionally, biochemical data indicate that bovine SCO cells synthesize a second precursor compound of 320 kDa, which is also detected in rat, rabbit, and dog. We postulate that RF is formed by two different complexes, one of which has a very high molecular mass (700 kDa or more) and is made up of at least six polypeptides, with the polypeptide of 450 kDa being its main component. The molecules that form RF in different species have different primary structures but they express common epitopes associated to the existence of cysteine bridges, which are probably crucial for polymerization of RF. Molecular procedures involving the use of anti-RF antibodies have led to the isolation of cDNA clones encoding two proteins known as RF-GLY-I and SCO-spondin. In the last 3 years, five partial cDNA sequences encoding SCO-spondin-like proteins have been obtained (Y08560, Y08561, AJ132107, AJ132106, AJ133488). These clones along with RF-GLY-I and SCO-spondin were computer-assembled generating a cDNA consensus sequence of 14.4 kb. Analyses of the long consensus sequence revealed an extended open reading frame (ORF-1) spanning from base 1,634 to 14,400 that encodes for a putative protein of 4,256 amino acids (approximately 450 kDa). The Mr of the predicted protein is consistent with the observed Mr of the largest protein recognized with anti-RF antibodies in SCO and RF extracts. However, the absence of consensus sequences typically present near the 5J'-end of the translation initiation site suggests the existence of a second open reading frame (ORF-2) extending from base 1 to base 14,400 in frame with the ORF-1 and probably encoding for the largest protein precursor (540 kDa). An antibody raised against a peptide sequence, deduced from the open reading frame encoded by a SCO cDNA, reacted specifically with the bovine and rat SCO-RF complex, thus indicating that the protein encoded by the cloned cDNA is part of RF. Immunoblots of bovine SCO extracts using the anti-peptide serum revealed bands of 540 kDa and 450 kDa, but it did not react with the proteins of 320 and 190 kDa. These data support the existence of two precursors for the bovine RF-glycoproteins (540 and 320 kDa) with the 450-kDa protein being a processed form of the 540-kDa precursor. We postulate that the cloned cDNAs encode for a protein that corresponds to the 540-kDa precursor and that at least part of this sequence is present in the processed form of 450 kDa that is secreted to form the RF., (Copyright 2001 Wiley-Liss, Inc.)

Bovine Reissner's fiber (RF) glycoproteins were used as antigen for the production of polyclonal and monoclonal antibodies (Mabs). We also produced Mabs against intracellular secretory glycoproteins of the bovine subcommissural organ (SCO). These Mabs were used for immunodetection of secretory proteins in situ (structural and ultrastructural immunocytochemistry), in blots, and in solutions. Three different antigen-mediated ELISA were designed to evaluate the affinity of the Mabs, to study the nature of the epitopes, and for competition test among Mabs. Two double antibody sandwich ELISA were designed to detect and quantify soluble secretory materials in different samples, to study coexistence of epitopes, and to elucidate whether epitopes for Mabs are repeated or not in the RF-glycoproteins. Twenty-three Mabs recognizing the bovine RF- and SCO-glycoproteins in solutions (ELISA) as well as in tissue sections, were obtained. Nineteen of these Mabs also recognized the pig SCO, 11 the rabbit SCO, 6 the dog SCO, and 5 the rat SCO. None of the Mabs recognized the SCO of non-mammalian species. The different types of ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moiety of the secretion, (2) they coexist in the same molecular forms and, with few exceptions, they did not overlap, (3) they were not repeated in the secretory molecule(s). Three Mabs were used for immunoblotting of RF; one of them revealed the same band pattern as that shown by an anti-RF serum. It is concluded that all Mabs raised in our laboratory are directed against non-repeated sequences of RF-glycoproteins that have not been conserved in vertebrate phylogeny., (Copyright 2001 Wiley-Liss, Inc.)

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., (Copyright 2001 Wiley-Liss, Inc.)

The subcommissural organ (SCO) of the Mongolian gerbil has been studied by transmission electron microscopy. The presence of extensive rough endoplasmic reticulum replete with flocculent material, well-developed Golgi complexes, condensing vesicles, electron-opaque and dense granules in the cytoplasm of SCO cells suggests that these cells are involved in protein synthesis. The secretory granules are released at the apical surface of the ependymal cells by exocytosis into the ventricular lumen, where their product condenses to form the Reissner's fiber. From the fact that the intercellular spaces of the ependymal cells of the gerbil SCO are sealed from the ventricular lumen by tight junctions, the accumulation of secretory material within the enlarged intercellular spaces provides ultrastructural evidence of basal secretion. The secretory material observed in the prominent hypendemal intracellular canaliculi, also appears to reach the intercellular spaces. The lack of perivascular spaces and external basal lamina in the gerbil SCO apparently permits basal secretions to reach vasculature and/or leptomeningeal spaces. The presence of smooth pinocytotic and coated vesicles, multivesicular bodies and lysosomes in the cycoplasm of ependymal cells suggests that the uptake of ions and macromolecules from the cerebrospinal fluid may be an additional function of the SCO.

The cells of pseudostratified columnar ciliated ependyma of the subcommissural organ in the goat were classified into two types on the basis of the distribution of chromatin material and nuclear clefts. Amongst the cell organelles the endoplasmic reticulum was highly developed and formed three types of Nebenkerne systems. The type-I Nebenkerne had spiral concentric lamellae and was associated with round lipid droplets. The type-II Nebenkerne, with widely spaced coils, was expanded towards its central and peripheral parts. The type-III Nebenkerne, composed of a meshwork of lamellae, was modified into a vacuolated form. The concentration of mitochondria was greatly increased towards the basal processes of the ependymal cells. The inclusion bodies included small electron-dense bodies, osmiophilic asteroid droplets, large round to spherical bodies and large round osmiophilic bodies with inner structures.

The subcommissural organ (SCO) secretes glycoproteins into the cerebrospinal fluid (CSF) that aggregate and form Reissner's fiber (RF). The factors involved in this aggregation are not known. One factor may be the hydrodynamics of the CSF when flowing through the aqueduct. This hypothesis was tested by isografting rat SCO and xenografting bovine SCO into the lateral ventricle of rats. Xenografts were either fresh bovine SCO or explants cultured for 30 days before transplantation. The grafts were investigated by electron microscopy and immunocytochemistry using antibodies against RF glycoproteins, serotonin and the glucose transporter I. Maximal time of transplantation was 43 days for isografts and 14 days for xenografts. The isografts were not reinnervated but were revascularized; they secreted into the ventricle RF glycoproteins that became progressively packed into pre-RF and RF structures identical to those formed by the SCO in situ. RF was confined to the host ventricle and at its distal end the constituent proteins disassembled. Xenografts were neither reinnervated nor revascularized and secreted into the host ventricle a material that never formed an RF. These findings indicate that the CSF factor responsible for the formation of RF is species specific, and that this process does not depend on the hydrodynamics of the CSF. The blood vessels revascularizing the isografted SCO acquired the characteristics of the vessels irrigating the SCO in situ, namely, a tight endothelium displaying glucose transporter I, and a perivascular space containing long-spacing collagen, thus indicating that basal release of glycoproteins may also occur in the grafted SCO.

The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner's fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner's fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1. 2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner's fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner's fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.

The subcommissural organ (SCO) is a phylogenetically ancient and conserved structure. During ontogeny, it is one of the first brain structures to differentiate. In many species, including the human, it reaches its full development during embryonic life. The SCO is a glandular structure formed by ependymal and hypendymal cells highly specialized in the secretion of proteins. It is located at the entrance of the aqueduct of Sylvius. The ependymal cells secrete into the ventricle core-glycosylated proteins of high molecular mass. The bulk of this secretion is formed by glycoproteins that would derive from two different precursors of 540 and 320 kDa and that, upon release into the ventricle aggregate, form a threadlike structure known as Reissner's fiber (RF). By addition of newly released glycoproteins to its proximal end, RF grows caudally and extends along the aqueduct, fourth ventricle, and the whole length of the central canal of the spinal cord. RF material continuously arrives at the dilated caudal end of the central canal, known as the terminal ventricle or ampulla. When reaching the ampulla, the RF material undergoes chemical modifications, disaggregates, and then escapes through openings in the dorsal wall of the ampulla to finally reach local blood vessels. The SCO also appears to secrete a cerebrospinal fluid (CSF)-soluble material that is different from the RF material that circulates in the ventricular and subarachnoidal CSF. Cell processes of the ependymal and hypendymal cells, containing a secretory material, terminate at the subarachnoidal space and on the very special blood capillaries supplying the SCO. The SCO is sequestered within a double-barrier system, a blood-brain barrier, and a CSF-SCO barrier. The function of the SCO is unknown. Some evidence suggests that the SCO may participate in different processes such as the clearance of certain compounds from the CSF, the circulation of CSF, and morphogenetic mechanisms.

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.

The subcommissural organ is an ependymal gland located at the entrance of the cerebral aqueduct. It secretes glycoproteins into the cerebrospinal fluid, where they aggregate to form Reissner's fiber. This fiber grows along the aqueduct, fourth ventricle, and central canal. There is evidence that the subcommissural organ is involved in the pathogenesis of congenital hydrocephalus. This organ was investigated in the mutant mouse hyh developing a congenital hydrocephalus. The central nervous system of normal and hydrocephalic hyh mice, 1 to 40 days old, was investigated using antibodies recognizing the subcommissural organ secretory glycoproteins, and by transmission and scanning electron microscopy. At birth, the affected mice displayed open communications between all ventricles, absence of a central canal in the spinal cord, ependymal denudation of the ventricles, stenosis of the rostral end of the aqueduct, and hydrocephalus of the lateral and third ventricles and of the caudal end of the aqueduct. Around the 5th postnatal day, the communication between the caudal aqueduct and fourth ventricle sealed, and hydrocephalus became severe. It is postulated that the hyh mice carry a genetic defect affecting the ependymal cell lineage. The subcommissural organ showed signs of increased secretory activity; it released to the stenosed aqueduct a material that aggregated, but it did not form a Reissner's fiber. A large area of the third ventricular wall differentiated into a secretory ependyma synthesizing a material similar to that secreted by the subcommissural organ. It is concluded that the subcommissural organ changes during hydrocephalus; whether these changes precede hydrocephalus needs to be investigated.

The subcommissural organ is an ependymal brain gland that secretes, into the ventricular cerebrospinal fluid, high molecular weight glycoproteins that form Reissner's fiber. Precursor and processed forms of secretion have been demonstrated by immunoblotting in the subcommissural organ of mammals and fish. In the chicken only a processed form has as yet been identified. In the present report, we have studied the subcommissural organ of 13-day-old chick embryos using (1) an antiserum against bovine Reissner's fiber, and (2) the lectins, concanavalin A and Limax flavus agglutinin. Paraffin sections of the subcommissural organ and blots of subcommissural organ extracts have been analyzed. The ependymal cells of sectioned subcommissural organ are strongly stained with the antiserum. Concanavalin A binds to materials in all cytoplasmatic regions, whereas Limax flavus agglutinin identifies materials confined to the apex of the ependymal cells. In the blots, a band of 540 kDa is immunostained. This band is positive for concanavalin A positive but negative for Limax flavus agglutinin and is thereby regarded as representing a precursor form of the secretion.

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.

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.

The subcommissural organ (SCO) secretes specific glycoproteins into the cerebrospinal fluid that aggregate to constitute Reissner's fiber (RF), a thread-like structure running along the central canal of the spinal cord. For further identification of the gene(s) encoding these secretions, we have prepared a cDNA library in the vector IGT11 from bovine embryonic SCO. The screening of this library was performed using a polyclonal antibody raised against bovine RF. Three positive clones were isolated and purified and one of these lambda RF101 comprising an insert of #400 nucleotides was undercloned into pBluescript plasmid and mapped. After labeling with 35S (ATP) this cDNA fragment served as a probe to analyse the presence of specific transcripts in the subcommissural organ of the embryonic bovine by in situ hybridization. A labeling signal was observed in the embryonic SCO both in the secretory ependymal and hypendymal cells. This labeling is specific since the ependymal layer bordering the ventricular cavity as well as the surrounding nervous tissue remained negative. Thus, the embryonic SCO contains specific transcripts that are colocalized with the specific glycoproteins as shown after the use of a specific monoclonal antibody C1B8A8. In addition, the pattern of labeling with the specific SCO cDNA is different from those of beta actin cDNA and tear lipocalin cDNA, which, respectively, served as positive and negative controls. In a subsequent set of experiments the expression pattern was compared in embryos at two different stages of development (4-month-old and 8-month-old embryos).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The fine structural features and regional differences of the ependyma in adult pigeons have been investigated by scanning electron microscopy. Pigeons of either sex were fixed with buffered glutaraldehyde (3%) and formaldehyde (0.5%) by intravascular perfusion. The brain was dissected using section planes adequate to expose each part of the ventricular system. The specimens were then dehydrated, critical point dried and sputtered with gold. Depending upon the distribution of cilia, microvilli and single cilia, different areas were recognised in the 4 ventricles. The topographic locations of these areas were determined using the atlas of Karten & Hodos (1967). The medial surfaces of the 1st and 2nd lateral ventricles are more densely ciliated than the lateral surfaces. In the floor of the 4th ventricle the medial part is less ciliated than the lateral parts. The circumventricular organs (subseptal organ, organum vasculosum of the lamina terminalis, infundibulum, choroid plexus, subcommissural organ, area postrema) show very characteristic surfaces and are surrounded by a transitional zone with the nonspecialized ependyma. In contrast, in the paraventricular organ the transition to the nonspecialized ependyma is rather abrupt. The ependyma covering the trochlear nucleus appears densely ciliated, differing from that of the classic circumventricular organs. Finally, the existence of openings in the caudal medullary velum, which represent direct communications between the ventricles and the subarachnoid space, was demonstrated.

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.

Ultrastructural and cytochemical studies were undertaken with special reference to the apical secretion in the ependymal cells of the subcommissural organ in the domestic chicken. Glucose-6-phosphatase (G6Pase) and acid phosphatase (AcPae) were used as cytochemical markers for the endoplasmic reticulum (ER) and lysosomes, respectively. Periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) cytochemistry was employed to detect glycoconjugates. Most dense bodies were negative with AcPase staining. Dilated cisternal of ER were reacted with G6Pase cytochemistry. Golgi saccules and several kinds of spherical bodies were stained with PA-TCH-SP cytochemistry. Our results suggest that: 1) most dense bodies in the apical cytoplasm belong to the secretory granules discharged into the ventricular cavity, 2) dilated cisternae of the ER serve as the storage site of the substance which eventually becomes the secretory product, however, contents of the ER are not directly released into the ventricular cavity. Findings of this studies lead us to speculate that there are two intracellular transportation via different compartments: one is a transport via small vesicles from the perinuclear ER to the Golgi apparatus and the other is a transport bypassing the Golgi apparatus via cisternae of ER from the perinuclear ER to the supra-Golgi region. The substances in these two structures seem to be intermingled with each other and matured into secretory granules. This granules may be discharged into the ventricular cavity by exocytosis.

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.

Fine structure and stereo-images of the Golgi apparatus and endoplasmic reticulum (ER) in the subcommissural organ (SCO) cells were visualized by the application of zinc-iodide osmium tetroxide (ZIO) impregnation, conventional electron microscopy and high voltage electron microscopy (HVEM). The Golgi apparatus in the SCO cells of rats, gerbils and hamsters consisted of flattened saccules stacked in parallel array. It showed a selective staining toward ZIO mixture and might form a complex network of tubular structures because of the presence of numerous fenestrations in the flattened Golgi saccules. The cytoplasm of the SCO cells in the rat and gerbil was crowded by dilated cisternae of the ER with a few flattened profiles. In the hamster SCO cells, however, the dilated cisternae of the ER were not observed. Flattened cisternae of ER in all species studied showed a positivity for ZIO impregnation and formed a complex tubular network, whereas dilated cisternae of the ER in the rats and gerbils did not show any reactivity. It was thus determined that the observation of thin and thick sections selectively stained with appropriate reagent for defined cellular organelles under conventional electron microscopy and HVEM offered valuable information about three-dimensional organization of the cell. A definite species-specific variation of SCO ultrastructure and cytochemistry was also demonstrated.

Glandular ependymocytes of the rat subcommissural organ (SCO) receive a multiple innervation: a serotonergic afference identified by radioautography and at least one other input of a hitherto unknown transmitter content. Both types of terminals make well differentiated axo-glandular synapses with SCO-ependymocytes, but display different morphological characteristics. Serotonergic terminals being considered as inhibitory, it is suggested that the other type of terminal is excitatory.

Light-and electron-microscopic radioautographic studies were carried out in rats after intraventricular injection of [3H]GABAergic elements in the subcommissural organ (SCO). This amino acid is specifically accumulated in SCO ependymocytes and numerous nerve terminals and fibres. Some labelled terminals contain clear round vesicles and large granular vesicles and sometimes display synaptic contacts on the SCO ependymocytes. They disappear after 5,7-dihydroxytryptamine (5,7-DHT) treatment or lesions of mesencephalic raphé nuclei. They are morphologically and pharmacologically similar to the serotonin-containing terminals of the basal SCO. The other labelled terminals contain exclusively clear round vesicles, are dispersed throughout the SCO and survive after the 5,7-DHT treatment or raphé nuclei lesions; some of them are in synaptic contact with neuronal elements of the basal SCO. In both SCO ependymocytes and fibres or terminals, [3H]GABA is accumulated according to the pharmacological criteria of a GABA neuronal uptake. Under the same experimental conditions [3H[glutamine and beta[3H]alanine fail to label the SCO: [3H] glutamate produces a very light labelling and [3H] serotonin is accumulated only in nerve profiles of the basal SCO. These results, and the presence of a glutamate decarboxylase activity in the SCO, suggest the existence of GABA-synthesizing elements in the SCO and could indicate the possible involvement of GABA in the secretory activity of the SCO.

Freeze-fracture studies on the tight junction of ependymal cells in the gerbil and mouse subcommissural organ (SCO) show an obvious species-specific variation. The tight junctional structure of the mouse SCO is composed of several strands (7.03 +/- 2.09 strands/cell) and occupies a total depth of 0.88 +/- 0.16 micron with a linear density of 7.12 +/- 2.11 strands/micron. The tight junction of the gerbil SCO is composed of three regions: (1) an apical region: made of 4 to 6 strands, oriented parallel to the free surface, with a high linear density (21.78 +/- 3.98 strands/micron) and small depth (0.049 +/- 0.009 micron); (2) a rather smooth and/or empty intermediate region, and (3) a basal region similar in morphology and morphometry to the junctional area of mouse SCO. These data indicate that the main difference in the SCO tight junction between the gerbil and the mouse is the presence of an apical region of high strand density in the former. We speculate that this apical region may play a role in maintaining the homeostasis of this CNS region in gerbils and possibly other desert animals, and may be part of a mechanism for survival in an extremely dry environment.

The apical surface and apical cytoplasm of the ependymal cells of the subcommissural organ (SCO) of the chicken, Gallus gallus were investigated by scanning and transmission electron microscopy. The presence of "secretory elements" formed by dilated and irregularly shaped RER cisternae in the proximity of the apical plasma membrane and located into ventricular protusions of apical cytoplasm, evidence an ependymal secretion towards the cerebrospinal fluid (CSF), which is responsible of the Reissner's fibre (RF) by means of an apocrine mechanism. The observed ependymal lysosomal population has been related with an endocytotic mechanism from the ventricular CSF which could also suggest an absorptive role for the SCO.

The subcommissural organ (SCO) of the Bradypus tridactylus was studied from the histological and ultrastructural point of view. The methods of GOMORI (alum-chromic-hematoxylin-phloxine), AZAN (M. Heidenhain), KLUVER and BARRERA (for nerve cells and myelinated fibers) and silver impregnation according to PALMGREN and CAJAL-DE CASTRO, were used. The material prepared for ultrastructural analize was submitted to the sequence of gluteraldehyde, osmium tetroxide, uranyl acetate and lead citrate. In function of the secretory phenomenon, a systematic study is made of different hystological and ultrastructural characteristics of the SCO and hypendymal rosettes, which were considered in the B. tridactylus as a morphofunctional unit. Fine vascular and nerve networks were observed, by the basal parts of the compact tubule alveolar system (hypendymal rosettes) which composes part of the SCO. Nerve fibers were seen between its cells reaching even up to the lumen, and they may end there in bulbar dilatations which seem to show secretory characteristics at the ultrastructural examination.

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.

The subcommissural organ and aqueduct of the red necked wallaby were examined using scanning electron microscopy. The surface of the subcommissural organ was similar to that of the possum brain and was superficially similar to that of the rat. However, on close examination, the apical protrusions composing the eutherian subcommissural organ were not present and tight clusters of microvilli were noted instead. Caudal to the subcommissural organ and on each side of the mesocoelic recess there were ciliated paramedian folds. These have been described in the possum but have not been noted in any eutherian mammalian brain. It is believed that these structures are peculiar to marsupial brains. Zones B and C (caudal to the subcommissural organ) represent a superior convex bulge into the aqueduct and then a concavity in the roof of the aqueduct, respectively. Both zones had sparsely ciliated ependyma, the cell surfaces being covered by microvilli. The most caudal zone, C, was much wider and longer than zone B. A line of cells in the roof of the aqueduct similar to zone C has been observed in eutherian mammalian brains in which a dorsal dilation is present. Furthermore, a ridge of long thin ependymal cells has been noted in the roof of the mesencephalon in prenatal rats as early as 16 days of gestation. It is postulated that this region is a circumventricular organ present in ontogeny in all mammals, persisting into the adult only in those with a dorsal dilation in the aqueduct.

Two different types of ependymal cells were found in the subcommissural organ (SCO) of Natrix maura. Most secretory cells showed morphological features resembling the general structure and ultrastructure of cells in the SCO of other vertebrates. This report describes a second population of cells lining a portion of the dorsal groove of the SCO. These cells were not selectively stained by chromalum-hematoxylin and, under the electron microscope, they were characterized by scarce surface differentiations, sparse apical cytoplasm and short basal processes. Flat, parallel cisternae of the rough endoplasmic reticulum produced vesicles that appeared to be transported to the well-developed Golgi apparatus. Dense secretory granules about 200 nm in diameter were found in the Golgi region. Similar granules were seen in the vicinity of the apical plasma membrane; some of them opened toward the ventricle. All these characteristics clearly differentiate this cell group from the other secretory cells lining the SCO laterally and ventrally.

The secretory activity of the subcommissural organ (SCO) was studied during embryogenesis of the chick (Gallus gallus) using two lectins labelled with fluorescein isothiocyanate, concanavalin A (Con A) and wheat-germ agglutinin (WGA). While WGA labels the apical or ventricular border of the organ, Con A labels both, the apical and vascular poles of the cells. Glycoproteinaceous secretory products, visualized by Con A appear early, at 5 days, in the ependymal epithelium and expand progressively in a rostrocaudal direction. A correlation is established between histofluorescence and the ultrastructural aspects of the ependymocytes. This throws light on the role of the endoplasmic reticulum in the synthesis, storage and transport of the material produced by the SCO, and points to the existence of two poles of exchange between the secretory cells and the extracellular medium, i.e., the ventricular and the vascular one. WGA reactivity at the apical border is linked up with the formation of Reissner's fibre and shows that the secretory product of the SCO cells undergoes at least partly modifications during its intracytoplasmic transport preceding apical discharge.

By means of various techniques and on the basis of a large sampling of the brush-tailed possum, Trichosurus vulpecula, a detailed description is given of the morphology of the neural elements associated with the subcommissural organ (SCO) and the leptomeninges on the dorsal surface of the midbrain. It is shown that the SCO of the possum is richly innervated, the nerves reaching the cerebral aqueduct by passing through the organ; there is, however, no evidence of a neural link between the SCO and the pineal gland. No typical axo-glandular synapses were found; however, many axo-glandular synaptoid junctions between axons and secretory cells of the SCO were demonstrable within the parenchyma of the SCO and on the aqueductal surface of the organ. The possible functional implications of the findings are: (1) The secretory activity of the SCO may be modified by nerves associated with it; this is in accord with some recently published experimental studies. (2) The nerves that innervate the SCO may also be responsible for ciliary activity on the aqueductal surface of the SCO, thus directing the apical secretory product of the SCO and debris flowing caudally in the cerebrospinal fluid into the poorly ciliated median groove where Reissner's fibre is found.

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.

The endoplasmic reticulum, the distribution of ribosomes attached to its membranes and the topographic relation of mitochondria have been studied in ependymal cells of the subcommissural organ of Cebus, Saimiri, and Callicebus. Occasionally, these organelles appear to be distinctly organized in restricted areas. They may be either irregularly arranged, formed into stacks, or organized into individualized corpuscules. The constituents of these organized areas are the same. Topographic relations with the GOLGI apparatus which would indicate a close interaction between these organelles, were not observed. The assemblage of substructures known to be involved in the synthesis of secretions of the subcommissural organ indicates a specific function of those areas.