The effect of dehydration on the ultrastructure and cholinesterase activity of the subcommissural organ in the rat

Summary Dehydration affected certain cytological features of the subcommissural organ in the albino rat suggesting a strong secretory stimulation of the ependymal and hypendymal cells of this organ in dehydrated animals.The cytoplasm of the secretory cells of the subcommissural organ in the dehydrated rats was filled with dilated and empty sacs and vacuoles of endoplasmic reticulum. The membrane system of the Golgi apparatus was also dilated, and more numerous vesicles and vacuoles of the Golgi complex were noticed after dehydration.In brains of the dehydrated animals, Reissner's fibre was not found in the lumen of the third ventricle, and only a few vesicles containing homogeneous secretory material were seen in the cytoplasm of the subcommissural secretory cells.In control animals, the activities of the specific and non-specific cholinesterases were localized in the cytoplasmic and nuclear membranes as well as in the rough and smooth endoplasmic reticulum. After dehydration, the activities of the specific and non-specific cholinesterases were strongly decreased.     

The secretory ependymal cells of the subcommissural organ: Which role in hydrocephalus?

Ependyma in the central nervous system gives rise to several specialized cell types, including the secretory ependymal cells located in the subcommissural organ. These elongated cells show large cisternae in their cytoplasm, which are filled with material secreted into the cerebrospinal fluid and toward the leptomeningeal spaces. A specific secretion of the subcommissural organ was named SCO-spondin, regarding its marked homology with developmental proteins of the thrombospondin superfamily (presence of thrombospondin type 1 repeats). The ependymal cells of the subcommissural organ and SCO-spondin secretion are suspected to play a crucial role in cerebrospinal fluid flow and/or homeostasis. There is a close correlation between absence of the subcommissural organ and hydrocephalus in rat and mouse strains exhibiting congenital hydrocephalus, and in a number of mice transgenic for developmental genes. The ependymal cells of the subcommissural organ are under research as a key factor in several developmental processes of the central nervous system.     

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.     

Possible relationship between the activity of the adrenal gland and the subcommissural organ in the lizard Lacerta s. sicula raf.

Summary In order to study the possible functional relationship between the adrenal gland and the subcommissural organ (SCO) in the lizard Lacerta s. sicula Raf., ACTH was administered to some specimens of this species in January when both the adrenal gland and the subcommissural organ have a very low activity. In comparison to untreated controls, the adrenals of animals treated with ACTH showed clear signs of stimulation, presenting enlarged blood vessels, very few lipid droplets, numerous polymorphic mitochondria and abundant tubular smooth endoplasmic reticulum. In addition, a distinct increase in secretory material was observed in the subcommissural cells of specimens treated with ACTH. These cells showed large cisternae of the rough endoplasmic reticulum filled with granular material in the basal region, numerous secretory granules of two types in the apical region and a reduced number of microvilli on the free cell surface. These findings, together with the results of preceding studies, lead the authors to the consideration that steroid hormones might play a role in the regulation of the secretory activity of the SCO.     

Activity of specific and non-specific cholinesterases in the subcommissural organ of guinea pig and albino rat

Summary The localizations of specific and non-specific cholinesterases were demonstrated by light and electron microscopical methods in the secretory cells of the subcommissural organ of the guinea pig and albino rat.The activity of non-specific cholinesterase at light microscopical level appeared slightly stronger compared to the activity of the specific cholinesterase. No differences in the localizations of the both enzymes could be noticed.In electron microscopic specimens no differences could be observed between the localization or intensity of the specific and non-specific cholinesterase reactions except some nerve fibres round the secretory hypendymal cells in which only a specific cholinesterase reaction product was noticed. The reaction product was found mainly in the cytoplasmic and nuclear membranes, in the rough and smooth surfaced endoplasmic reticulum and around some secretory granules in the ependymal and hypendymal secretory cells of the subcommissural organ in guinea pig and albino rat.The possible role of cholinesterases in the secretory cells of the subcommissural organ is further discussed. Their participation in the metabolism and/or secretion of the hormonal end products is suggested.     

Immunohistochemical localization of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat

Summary The indirect peroxidase-antiperoxidase immunohistochemical technique was used to investigate the possible presence of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat. Considerable numbers of VIP-immunoreactive fibers were seen in the pineal gland. A moderate amount of VIP-immunoreactive fibers was present in the median eminence, the posterior lobe of the pituitary and the area postrema, but only few fibers were found in the organum vasculosum laminae terminalis. No immunoreactivity was observed in the subfornical organ or the subcommissural organ. The circumventricular organs investigated were completely free of VIP-immunoreactive perikarya. In the circumventricular organs, VIP-immunoreactive fibers were visible between the parenchymal cells and in the perivascular spaces. The presence of coarse VIP-immunoreactive terminals in apposition to the portal vessels in the external layer of the median eminence indicates that VIP may be secreted directly into the pituitary portal circulation, thus influencing the anterior pituitary cells. The presence of large VIP-immunoreactive boutons in the posterior lobe of the pituitary suggests a secretion of VIP directly into the systemic circulation. In the pineal gland, a dense innervation by VIP-immunoreactive fibers was found in the peripheral superficial part of organ, with fibers penetrating into its central portion where they mainly terminate near in vicinity of the capillaries. In the area postrema, VIP-immunoreactive material was mainly found at the ventral border of the organ. In addition to the secretion of VIP into the bloodstream via the circumventricular organs, this study provides evidence that VIP exerts specific influence on the cellular elements of these organs.     

Ependymal specializations

Summary The ependymal cells of the toad subcommissural organ produce pale and dense secretory granules. Both types of granules are mainly concentrated in the apical cytoplasm and in the perinuclear region. Pale and dense granules are synthesized by and packed in the rough endoplasmic reticulum, bypassing the step of the Golgi apparatus. The apical cytoplasm of some subcommissural ependymal cells protrudes into the ventricle. All the cells project a few cilia and numerous slender, long microvilli into the ventricular lumen.Contacting the cilia and the microvilli there is a filamentous material identical to that observed in the fibre of Reissner at the aqueduct of Sylvius. In addition to filaments, the fibre of Reissner contains vacuolar formations. The fibre is surrounded by numerous ependymal cilia, some of which are embedded in the filamentous material of the fibre.The presence of numerous microvilli projected into the ventricle and the large number of vesicles scattered in the supranuclear cytoplasm seem to indicate that the subcommissural organ may have absorption functions. The fact that the intercellular space of the ependymal layer of the subcommissural organ is not separated from the ventricular lumen by tight junctions but by zonulae adhaerentes could indicate that the cerebrospinal fluid penetrates these intercellular spaces bathing all sides of the ependymal cells. The presence in the ependymal cells of vesicles opening into the intercellular space would be in agreement with the latter possibility.There are some ultrastructural differences between the ependymal cells of the cephalic end of the subcommissural organ and those of the caudal end. A critical analysis of Reissner's fibre formation is made.This investigation was partially supported by a Grant of the Wellcome Trust Foundation.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. The author wishes to thank the valuable help of Mr. P. Heap.     

The infundibular organ of the lancelet (Branchiostoma lanceolatum,Acrania): an immunocytochemical study

Reissner's fibers are secretions produced by different ependymal areas of the chordate brain, viz., in adult vertebrates, by the dorsal subcommissural organ, and in all stages of cephalochordates (Branchiostoma lancelets), by the ventral infundibular organ. Fibers produced by these different organs are seemingly identical and the two fiber sources also share some immunocytochemical and lectin-binding properties. The secretions in these two glands are, however, not identical; the infundibular organ cells are strongly reactive with antibodies against vertebrate Reissner's fibers, but they do not react with antibodies raised against the source of the vertebrate fibers, viz., the subcommissural organ. The results support the possibility that, in adult vertebrates, the Reissner's fibers are composed of material not only from the subcommissural organ, but also from another, not yet identified, source that is identical or equivalent to the infundibular organ of the lancelet. There are indications that the infundibular organ is immunocytochemically closely akin to some secretory cells in the vertebrate embryonic brain and also to those that produce the juvenile vertebrate Reissner's fibers, viz., secretory cells in the flexural organ.     

Evidence for basal secretion in the subcommissural organ of the adult rabbit

Summary Morphological evidence is presented supporting the possibility of basal secretion into hypendymal capillaries of the adult rabbit subcommissural organ (SCO). The synthetic apparatus of the SCO cell is described as well as the heterogeneous granules and vesicles which are concentrated in the basal processes bordering a widened perivascular space. The origin of the electron dense granules, of which two fairly distinct subgroups are found, is discussed.A binding of secretory sacs to the lateral plasma membrane is seen. The possibility of a lateral secretion is supported by the presence of a system of extracellular channels between SCO cells which are filled with a flocculent material resembling that of the secretory sacs.Nerve perikarya which are separated from the SCO by only a few glial fibers are demonstrated. Synapses are described in nerve fascicles bordering on the hypendymal capillaries. The possibility of an innervation of the hypendymal region is discussed as well as possible nervous connections with the pineal gland.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen.     

Reissner’s fibre supports the survival of chick cortical neurons in primary mixed cultures

Reissner's fibre, a thread-like structure present in the central canal of the spinal cord, is a product of the condensation of specific glycoproteins that are released by specialized ependymal cells into the cerebrospinal fluid. These secretory ependymocytes constitute the subcommissural organ, a circumventricular organ that lines the roof of the third ventricle of the brain. The subcommissural organ/Reissner's fibre complex is a permanent structure in the vertebrate central nervous system. The addition of bovine Reissner's fibre itself or of soluble material released by Reissner's fibre to primary mixed cultures of chick cerebral cortical cells markedly enhances neuronal survival. The responsive cells have been identified as neurons by labelling them with antibodies to neurofilament proteins. This neuronal survival effect is dose-dependent and does not require the presence of serum in the culture medium. Affinity-purified polyclonal antibodies raised against bovine Reissner's fibre partially block the effect of Reissner's fibre on neuronal survival. These results suggest that Reissner's fibre is involved in developmental processes of the central nervous system.     

The fine structure of the subcommissural cells and of Reissner's fibre in Myxine

Summary Reissner's fibre and the subcommisssural cells mainly from Myxine were investigated by light and electron microscope methods. The subcommissural cells carry several cilia and produce a chrome haematoxyphile secretion in the form of granules. It is probable that the nucleus as well as the mitochondria are involved in the synthesis of this material. The secretory release suggests an apocrine type in which the granules swell and form a fine-granulated, chrome haematoxyphile substance in the ventricle. Caudally in the subcommissural canal this material condenses to form Reissner's fibre, which in electron micrographs, except for a fine-granulated ground substance, does not show any structures, a limiting membrane or traces of cell organelles or remnants.Aided by grants from the Swedish Natural Science Research Council.     

Bovine Reissner’s fiber (RF) and the central canal of the spinal cord: an immunocytochemical study using a set of monoclonal antibodies against the RF-glycoproteins

The subcommissural organ secretes N-linked complex-type glycoproteins into the cerebrospinal fluid. These glycoproteins condense to form Reissner’s fiber (RF), which extends along the fourth ventricle and central canal of the spinal cord. A set of three monoclonal antibodies (Mabs 3E6, 3B1, and 2A5) has been obtained using these glycoproteins as immunogens. Competitive and sandwich enzyme-linked immunoassay methods have demonstrated that the three monoclonal antibodies are directed against different epitopes, and that there is no competition among them for their binding to glycoproteins of RF. Mab 3E6 displays immunoblotting properties that are similar to those of a polyclonal antibody against the pool of glycoproteins from RF, but that are different from those of Mabs 3B1 and 2A5. All three antibodies immunostain the bovine subcommissural organ and RF. A population of ependymal cells is stained by the polyclonal antibody, and Mabs 2A5 and 3E6, but not by Mab 3B1. The material present in a population of ependymal cells of the central canal, and the glycoproteins secreted by the subcommissural organ thus probably have partial chemical identity. Some evidence suggests that the immunoreactive ependymal cells are secretory cells. The luminal surface of the central canal is coated by a thin layer of material with immunocytochemical characteristics different from those of the ependymal cells; such a coat may correspond to material released from RF. Received: 19 December 1995 / Accepted: 30 April 1996     

Lectin histochemistry of the human fetal subcommissural organ

The subcommissural organ (SCO) of 7 human fetuses, 3 to 6.5 months old, was investigated by means of: (i) immunocytochemistry employing three different antisera against secretory products extracted from the bovine SCO and Reissner's fiber; (ii) lectin binding using concanavalin A (Con A; affinity: mannose, glucose), wheat-germ agglutinin (WGA; affinity: N-acetyl-glucosamine, sialic acid), and Limax flavus agglutinin (LFA; affinity: sialic acid). Sections of bovine SCO were processed simultaneously and examined for comparative purposes. The human fetal SCO displayed lectin-binding properties identical to those in the SCO of other mammals. Thus, Con A-binding sites were restricted to abundant supranuclear structures that most likely corresponded to the rough endoplasmic reticulum, but were missing from granules located in the apical cytoplasm. The latter secretory material was strongly WGA- and LFA-positive and formed a distinct zone in the most apical portion of the ependymal cells. In contrast, this type of reactivity was missing in the adjacent cells of ependyma proper. In the bovine SCO, LFA-positive granules were also aggregated in an apical layer. The secretory material in the bovine SCO, especially its apical granular component, was strongly immunoreactive with the three antisera used; the human fetal SCO, however, lacked this immunoreactivity. It is postulated that the SCO of human fetuses secretes glycoproteins with a carbohydrate chain similar to--and a protein backbone different from--the secretions elaborated by the SCO of other vertebrate species.     

Partial sequencing of Reissner’s fiber glycoprotein I (RF-Gly I)

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.     

Identification of Reissner's fiber-like glycoproteins in two species of freshwater planarians (Tricladida), by use of specific polyclonal and monoclonal antibodies

By using one polyclonal antiserum raised against bovine Reissner's fiber and seven monoclonal antibodies raised against bovine Reissner's fiber and against immunopurified bovine subcommissural organ glycoproteins, we have investigated two freshwater planarian species (Girardia tigrina, Schmidtea mediterranea) by light- and electron-microscopic immunocytochemistry. ELISA probes showed that the monoclonal antibodies recognized different, nonoverlapping, unrepeated, proteinaceous epitopes present in the same compounds of bovine Reissner's fiber. Cells immunoreactive to the polyclonal and monoclonal antibodies were found in the dorsal and ventral integument of both planarian species. Labeled cuboid epidermal cells bore cilia and displayed several types of secretory granules; they were covered by a film of immunoreactive material. Studies on adjacent thin and semithin sections revealed coexistence of label in the same regions and in the same cells when two different monoclonal antibodies were used. These results indicate that a secretory substance immunologically similar to the secretion of the vertebrate subcommissural organ is present in primitive tripoblasts such as planarians, suggesting that these secretions are ancient and well conserved in phylogeny.     

The praeoptico-hypophysial system,Nucleus tuberis lateralis and the subcommissural organ of Gasterosteus aculeatus after changes in osmotic stimuli

Summary The histology and cytology of the praeoptico-hypophysial system, nucleus tuberis lateralis and subcommissural organ in Gasterosteus aculeatus were analyzed after the fishes had been put in waters of different salinity (see Tables 1–4).These three structures are all identical in fishes which are accustomed to fresh water and to 32 salt-water. A transference to hypertonic water causes changes in the neurosecretory system which suggest the existence of an antidiuretic principle in the neurosecretory substance. No such relationship is found when the fishes are put into hypotonic water. Further, no connection is found between either the secretion production in nucleus tuberis lateralis or in the subcommissural organ and variations in osmotic value.Aided by grants from the Swedish Natural Science Research Council.     

Complex-type glycoproteins synthesized in the subcommissural organ of mammals

Summary The secretory activity in the subcommissural organ (SCO) of the sheep and cow was examined by means of lectin histochemistry and cytochemistry. Among the various lectins tested, Concanavalin A (Con A) revealed glycoproteins rich in mannosyl residues in the rough endoplasmic reticulum of ependymal and hypendymal cells. One of these Con A-positive glycoproteins may represent the precursor of the specific secretory component elaborated in the SCO, giving rise to Reissner's fiber. Lens culinaris agglutinin (LCA) and Phaseolus vulgaris hemagglutinins (E-PHA and L-PHA), known to bind to oligosaccharides, as well as wheat-germ agglutinin (WGA) revealing neuraminic acid, labeled secretory granules located in the apical part of ependymal and hypendymal cells of ruminants, and also Reissner's fiber. Electron-microscopic visualization of WGA-positive material in the Golgi complex shows that complex-type glycoproteins are synthesized in the subcommissural organ of mammals. The electron-dense material is mainly secreted into the ventricular cavity and gives rise to Reissner's fiber. On the basis of lectin affinity for oligosaccharides, a structure of the complex-type oligosaccharide is proposed.     

Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal cord

Summary Reissner's fiber (RF) of the subcommissural organ (SCO), the central canal and its bordering structures, and the filum terminale were investigated in the bovine spinal cord by use of transmission electron microscopy, histochemical methods and light-microscopic immunocytochemistry. The primary antisera were raised against the bovine RF, or the SCO proper. Comparative immunocytochemical studies were also performed on the lumbo-sacral region of the rat, rabbit, dog and pig.At all levels of the bovine spinal cord, RF was strongly immunoreactive with both antisera. From cervical to upper sacral levels of the bovine spinal cord there was an increasing number of ependymal cells immunostainable with both antisera. The free surface of the central canal was covered by a layer of immunoreactive material. At sacral levels small subependymal immunoreactive cells were observed. From all these structures sharing the same immunoreactivity, only RF was stained by the paraldehyde-fuchsin and periodicacid-Schiff methods.At the ultrastructural level, ependymal cells with numerous protrusions extending into the central canal were seen in the lower lumbar segments, whereas cells displaying signs of secretory activity were principally found in the ependyma of the upper sacral levels. A few cerebrospinal fluid-contacting neurons were observed at all levels of the spinal cord; they were immunostained with an anti-tubulin serum.The lumbo-sacral segments of the dog, rat and rabbit, either fixed by vascular perfusion or in the same manner as the bovine material, did not show any immunoreactive structure other than RF.The possibilities that the immunoreactive ependymal cells might play a secretory or an absorptive role, or be the result of post-mortem events, are discussed.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de ChileThe authors wish to thank Dr. Enrique Romeny from the Valdivia abattoir for kindly providing the bovine spinal cords     

Distribution of LHRH in the rat and mouse brain with special reference to the tanycytes

Summary The distribution of luteinizing hormone-releasing hormone (LHRH) was studied in the rat and mouse brain by means of light and electron microscopic immunohistochemistry using the peroxidase-antiperoxidase method. An immunoreactive product to LHRH antiserum was found near the blood vessels of the vascular organ of the lamina terminalis. In the arcuate nucleus-median eminence region, an immunoreactive material occurred bilaterally in the hypothalamic tissue around the tuberoinfundibular sulci. Electron microscopy revealed that immunoreactive fibers observed light microscopically contain numerous granules 100–130 nm in diameter. No immunoreactive product was located in the tanycytes of the median eminence, the perikarya of hypothalamic neurons, and the parenchyma of several circumventricular organs (subfornical organ, subcommissural organ, pineal organ, area postrema).Supported by grants from the Ministry of Education of Japan and the Ford Foundation     

Immunohistochemical evidence for neuronal and non-neuronal synthesis of GABA in the rat subcommissural organ

In the past few years, several studies have demonstrated in the rat subcommissural organ the presence of nerve endings and modified ependymocytes showing an uptake of [³H]GABA. The present work was performed to demonstrate in this cerebral zone the possibility of a GABA synthesis by the immunohistochemical localization of glutamate decarboxylase (GAD). GAD-positive reaction was detected with unlabelled antibody-enzyme peroxidase anti-peroxidase. Some nerve terminals containing either clear round vesicles, or sometimes clear round vesicles and some large granular vesicles, exhibited a positive staining. These terminals could belong to GABAergic inputs in the subcommissural organ. The few reactive terminals containing some granular vesicles could be related to the serotoninergic input as suggested previously (Gamrani et al., 1981). Several ependymocytes of this structure contained GAD-like positive reaction; these cells are also capable of taking up [³H]GABA (Gamrani et al., 1981) and present neuronal properties with regard to GABA. However, the presence in their cytoplasm of enolase, a specific glial marker, related them to glial elements. The presence of GABA in these ependymocytes suggests a modulating function of GABA on the secretory activity of the subcommissural organ.