Electron microscopic and cytochemical studies of the mouse subcommissural organ

Summary In mice most of the ependymal cells of the subcommissural organ (SCO cells) are densely packed with dilated cisternae of the endoplasmic reticulum (ER) containing either finely granular or flocculent materials. The well developed supra-nuclear Golgi apparatus consists of stacks of flattened saccules and small vesicles; the two or three outer Golgi saccules are moderately dilated and exhibit numerous fenestrations; occasional profiles suggesting the budding of coated vesicles and formation of membrane-bound dense bodies from the ends of the innermost Golgi saccules are seen. A few coated vesicles and membrane-bound dense bodies of various sizes and shapes are also found in the Golgi region.The contents of the dilated ER cisternae are stained with periodic acid-silver methenamine techniques. In the Golgi complex the two or three inner saccules are stained as deeply as the dense bodies, and the outer saccules are only slightly stained. The stained contents of ER cisternae are more electron opaque than those of the outer but less opaque than those of the inner Golgi saccules and the dense bodies.Acid phosphatase activities are localized in the dense bodies, some of the coated vesicles in the Golgi region, and in the one or two inner Golgi saccules.On the basis of these results the following conclusions have been reached: (1) In mouse SCO cells the finely granular and the flocculent materials in the lumen of ER cisternae contain a complex carbohydrate(s) which is secreted into the ventricle to form Reissner's fiber; (2) the secretory substance is assumed to be synthesized by the ER and stored in its cisternae, and the Golgi apparatus might play only a minor role, if any, in the elaboration of the secretory material; (3) most of the dense bodies in the mouse SCO cells are lysosomal in nature instead of being so-called dark secretory granules.Sponsored by the National Science Council, Republic of China.     

Secretory activity of the subcommissural organ in Rana temporaria under osmotic stimulation

Summary The secretory activity of the subcommissural organ (SCO) in the frog Rana temporaria was studied under conditions of dehydration. After injection of a radioactive precursor the amount and concentration of radioactively labelled material in the SCO are smaller in dehydrated than in control animals. Concomitantly, the growth rate of the CSF-fibre (Reissner's fibre) increases in dehydrated animals. It follows that water deprivation enhances the secretory activity of the SCO.To investigate whether the SCO may be responsible for the secretion of an aldosteronotropic factor as suggested in the literature, brains were incubated in vitro with a radioactive precursor and with or without aldosterone. The SCO of the aldosterone-treated brains contains more radioactively labelled material than the SCO of the control brains. It is argued that this is indicative of a lower secretory activity. It means that aldosterone inhibits the secretory activity of the SCO, possibly by a process of negative feed-back regulation. The results of the present experiments can be interpreted in favour of an involvement of the SCO-Reissner's fibre complex in osmoregulation.     

Secretory activity of the subcommissural organ in Rana temporaria under osmotic stimulation

The secretory activity of the subcommissural organ (SCO) in the frog Rana temporaria was studied under conditions of dehydration. After injection of a radioactive precursor the amount and concentration of radioactively labelled material in the SCO are smaller in dehydrated than in control animals. Concomitantly, the growth rate of the CSF-fibre (Reissner's fibre) increases in dehydrated animals. It follows that water deprivation enhances the secretory activity of the SCO. To investigate whether the SCO may be responsible for the secretion of an aldosteronotropic factor as suggested in the literature, brains were incubated in vitro with a radioactive precursor and with or without aldosterone. The SCO of the aldosterone-treated brains contains more radioactively labelled material than the SCO of the control brains. It is argued that this is indicative of a lower secretory activity It means that aldosterone inhibits the secretory activity of the SCO, possibly by a process of negative feed-back regulation. The results of the present experiments can be interpreted in favour of an involvement of the SCO-Reissner's fibre complex in osmoregulation.     

Histochemical investigations on the human foetal subcommissural organ

Summary A histochemical investigation was carried out on subcommissural organs from 28 human foetuses with crown-rump lenths ranging from 28 mm to 167 mm. The human foetal subcommissural organ (SCO) consists of a characteristic, high columnar epithelium covering the anterior-inferior surface of the posterior commissure.Histochemical reactions provide clear cut evidence that the cells of SCO contain glycoproteins as well as abundant amounts of glycogen. A very strong activity was found for cystine, tyrosine, tryptophan and arginine. Based on reactions for nucleoproteins different cell types were described.A remarkable activity of alkaline phosphatase clearly delineated the borders of the SCO. The localization of the reaction products corresponded to the outlines of the plasma membranes. A strong nodular cytoplasmic activity of acid phosphatase and of AS-esterase was found.Special attention was paid to an accumulation of hyaluronic acid and chondroitin-4-sulphate and/or chondroitin-6-sulphate in a perivascular position. The extended perivascular space was discussed in relation to the blood-brain barrier. It was suggested that the human foetal SCO is very active and that this activity has to do with an exchange of neurohormones from the SCO to the blood and in addition that absorptive and secretory functions might be carried out between the cerebro-spinal fluid and the SCO.     

The ependymal secretion of the fetal and adult rat subcommissural organ. morphological aspects linked to the synthesis,storage and release of the secretory products

Different localizations of secretory material are noted in adult and fetal subcommissural organ (SCO) in light microscopy. At the electron microscope level, the secretory ependymocytes reveal frequent associations among mitochondria and ribosomes of the endoplasmic reticulum (ER). In the SCO ependymocytes of the adult rat, the relationship between mitochondria and ribosomes of the ER is observed in the subgolgian zone, the ER cisternal profiles are smooth except where they face the mitochondria. Here, a constant interval of 40-45 nm separates the ribosome-coated ER membrane from the external membrane of the mitochondria. This association evidences a functional cooperation between mitochondria and ER, at least in some phases of the synthesis of the organ's gliosecretory material. By contrast, in the fetus (17-21 fetal day), the synthetic apparatus displays an entirely granular ER. The secretory products are stored as flocculent material which fills the ER cisternae. In the apical zone of the ependymocytes, as the membrane of the dense secretory granules fuses with the apical plasmalemma, the granules release their contents into the ventricular cavity. A possible link between the releasing process and the coated vesicles is discussed.     

The ependymal secretion of the fetal and adult rat subcommissural organ. morphological aspects linked to the synthesis, storage and release of the secretory products

Different localizations of secretory material are noted in adult and fetal subcommissural organ (SCO) in light microscopy. At the electron microscope level, the secretory ependymocytes reveal frequent associations among mitochondria and ribosomes of the endoplasmic reticulum (ER). In the SCO ependymocytes of the adult rat, the relationship between mitochondria and ribosomes of the ER is observed in the subgolgian zone, the ER cisternal profiles are smooth except where they face the mitochondria. Here, a constant interval of 40-45 nm separates the ribosome-coated ER membrane from the external membrane of the mitochondria. This association evidences a functional cooperation between mitochondria and ER, at least in some phases of the synthesis of the organ's gliosecretory material. By contrast, in the fetus (17-21 fetal day), the synthetic apparatus displays an entirely granular ER. The secretory products are stored as flocculent material which fills the ER cisternae. In the apical zone of the ependymocytes, as the membrane of the dense secretory granules fuses with the apical plasmalemma, the granules release their contents into the ventricular cavity. A possible link between the releasing process and the coated vesicles is discussed.     

Synthesis of transthyretin by the ependymal cells of the subcommissural organ

Transthyretin (TTR) is a protein involved in the transport of thyroid hormones in blood and cerebrospinal fluid (CSF). The only known source of brain-produced TTR is the choroid plexus. In the present investigation, we have identified the subcommissural organ (SCO) as a new source of brain TTR. The SCO is an ependymal gland that secretes glycoproteins into the CSF, where they aggregate to form Reissners fibre (RF). Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. To investigate the CSF-soluble compounds secreted by the SCO further, antibodies were raised against polypeptides partially purified from fetal bovine CSF. One of these antibodies (against a 14-kDa compound) reacted with secretory granules in cells of fetal and adult bovine SCO, organ-cultured bovine SCO and the choroid plexus of several mammalian species but not with RF. Western blot analyses with this antibody revealed two polypeptides of 14 kDa and 40 kDa in the bovine SCO, in the conditioned medium of SCO explants, and in fetal and adult bovine CSF. Since the monomeric and tetrameric forms of TTR migrate as bands of 14 kDa and 40 kDa by SDS-polyacrylamide gel electrophoresis, a commercial preparation of human TTR was run, with both bands being reactive with this antibody. Bovine SCO was also shown to synthesise mRNA encoding TTR under in vivo and in vitro conditions. We conclude that the SCO synthesises TTR and secretes it into the CSF. Colocalisation studies demonstrated that the SCO possessed two populations of secretory cells, one secreting both RF glycoproteins and TTR and the other secreting only the former. TTR was also detected in the SCO of bovine embryos suggesting that this ependymal gland is an important source of TTR during brain development. Financial support was provided by grants 1030265 from Fondecyt, Chile, to E.M.R. and 201.035.002-1.0 DIUC to H.M.     

Studies on the subcommissural organ of Salmo gairdneri

The light microscopic analysis of serial sections of the subcommissural organ (SCO) of the rainbow trout (Salmo gairdneri) shows that the form of the groove-like (in cross section) organ varies over its total length. Its rostral origin is a tunnel-like structure anterior to the orifice of the hollow pineal stalk. The SCO forms the dorsal wall of the brain. Caudally the SCO is increasingly displaced from the surface of the brain by the fibers of the posterior commissure; the organ ends in a tabular area beyond the latter. The orifice of the pineal stalk is surrounded by the ependyma of the SCO that invaginates like a funnel and joins with the ependyma of the pineal stalk after a considerable narrowing. The rudimentary parapineal organ is located on the left side of the brain and is connected with the left habenular ganglion through the parapineal tract. It contacts the third ventricle with a short channel within the ependyma of the SCO. The histological organization of the ependymal and hypendymal cells of the SCO is typical of teleosts. Secretory material is located basally and apically in relation to the nucleus, but there is no indication of a basal secretory release. Basal ependymal processes terminate with broadened endings at the membrana limitans externa. The apical product is discharged into the third ventricle, where it aggregates into the thread-like structure of Reissner's fibre. The SCO cells have no direct contact with cerebral or meningeal blood vessels.     

The subcommissural organ and the development of the posterior commissure in chick embryos

The subcommissural organ (SCO) is an ependymal differentiation located in the diencephalon under the posterior commissure (PC). SCO-spondin, a glycoprotein released by the SCO, belongs to the thrombospondin superfamily and shares molecular domains with axonal pathfinding molecules. Several lines of evidence suggest a relationship between the SCO and the development of the PC in the chick: (1) their close location to each other, (2) their differentiation at the same developmental stage in the chick, (3) the abnormal PC found in null mutants lacking an SCO and (4) the release by the SCO of SCO-spondin. By application of DiI crystals in the PC of chick embryos, we have identified the neurons that give rise to the PC. Labelling is confined to the magnocellular nucleus of the PC (MNPC). To gain insight into the role of the SCO in PC development, coculture experiments of explants of the MNPC region (MNPCr) from embryos at embryonic day 4 (E4) with SCO explants from E4 or E13 embryos have been performed and the neurite outgrowth from the MNPCr explants has been analysed. In the case of coculture of E4 MNPCr with E4 SCO, the number of neurites growing from the MNPCr is higher at the side facing the SCO. However, when E4 MNPCr and E13 SCO are cocultured, the neurites grow mostly at the side opposite to the SCO. These data suggest that, at early stages of development, the SCO releases some attractive or permissive molecule(s) for the growing of the PC, whereas at later stages, the SCO has a repulsive effect over neurites arising from MNPCr.     

Analysis of the efferent projections of the lateral geniculate nucleus with special reference to the innervation of the subcommissural organ and related areas

In order to define central neurons projecting to the subcommissural organ (SCO) and to related areas in the postero-medial diencephalon, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the lateral geniculate nucleus of the rat. PHA-L-labelled neurons send axonal processes medially through the posterior thalamic nuclei and the posterior commissure to the other hemisphere. Branches of fibres originating from this projection form a plexus of nerve terminals in the underlying precommissural nucleus and in the nucleus of the posterior commissure. A small number of PHA-L-immunoreactive nerve fibres penetrate from the precommissural nucleus into the lateral part of the SCO. A few labelled fibres penetrate directly from the posterior commissure into the medial part of the caudal SCO. Most of the PHA-L-immunoreactive fibres occur in the hypendymal layer, although a few terminate near the ependymal cells of the organ. Many labelled fibres are found in the ventricular ependyma adjacent to the SCO, some fibres lying close to the ventricular lumen. These results were obtained only if the tracer was delivered into the intergeniculate leaflet of the lateral geniculate nucleus (IGL). The IGL innervates both the suprachiasmatic nucleus and the pineal organ; the connections between the IGL and the midline structures, including the SCO, suggest that these areas are influenced by the circadian system.     

Isograft and xenograft of the subcommissural organ into the lateral ventricle of the rat and the formation of Reissner’s fiber

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.     

Immunohistochemical demonstration of serotonergic and peptidergic nerve fibers in the subcommissural organ of the dog

Summary The distributional patterns of serotonin-, luteinizing hormone-releasing hormone (LHRH)-, oxytocin (OXT)- and vasopressin (VP)-immunoreactive nerve fibers were studied in the subcommissural organ (SCO) of the dog by use of the peroxidase-antiperoxidase technique.Abundant serotonergic and moderate numbers of peptidergic nerve fibers running toward the ventricular surface were observed among the cylindrical ependymal cells in the SCO of the dog. Concerning the distributional density of the peptidergic nerve fibers, VP-immunoreactive fibers displayed the highest and LHRH-immunoreactive fibers the lowest values. Most serotonergic and peptidergic fibers returned to the basal portion of the SCO after forming loops immediately beneath the ventricular surface of the ependymal layer. Serotonin-immunoreactive fibers often established a perivascular plexus around the blood vessels in the SCO.At the electron-microscopic level, after use of antiserum to serotonin dark immunoprecipitate was observed in large granular vesicles and the matrix surrounding small and large, clear vesicles and mitochondria; VP immunoreactivity was localized in the large granular vesicles.Serotonergic nerve fibers could be detected in the SCO of the newborn dog. Although the distributional density was in principle not different from that in the adult animal, individual fibers showed immature features such as growth cones and insufficiently swollen varicosities. After penetrating into the ventricle, in the newborn dog, a few serotonin-immunoreactive fibers ran for a relatively long distance on the ependymal surface.     

Immunohistochemical localization of glial fibrillary acidic protein (GFAP) and vimentin in the subcommissural organ of the Mongolian gerbil (Meriones unguiculatus)

Summary The chemical composition of intermediate filaments (IF's) in the ependyma of the subcommissural organ (SCO) of the Mongolian gerbil (Meriones unguiculatus) was investigated immunohistochemically in paraffin-embedded tissue. Antibodies against glial fibrillary acidic protein (GFAP), vimentin, neurofilament proteins and cytokeratins were used. Only GFAP and vimentin were detected in the non-specialized diencephalic ependyma and in the ependymocytes of the SCO. Staining could be observed in apical and basal processes of the SCO-cells. The latter processes extended into the posterior commissure up to the subpial surface, thus establishing a well-developed leptomeningeal route of ependymal projections. In contrast to the homogeneous vimentin-labeling, the SCO was particularly immunoreactive for GFAP in its lateral aspects and in the supraand precommissural parts. The coexpression of GFAP and vimentin in a subclass of SCO-ependymocytes was demonstrated on differentially immunostained semithin sections. The present study confirms the glial nature of the SCO-ependyma, which has been a matter of debate recently. It appears from this investigation that the high degree of secretory activity in the SCO does not necessarily lead to the disappearance of glial IF proteins. Moreover, the SCO-cells belong to the expanding group of mature astroglia, which is characterized by coexpression of GFAP and vimentin. The morphological similarity between SCO-ependymocytes and tanycytes is underscored by their common immunoreactivity against these two IF proteins. In view of the absence of GFAP from the rat SCO, interspecific differences must be considered in the evaluation of the IF protein composition.     

Light- and electron-microscopic investigation of the rat subcommissural organ grafted under the kidney capsule,with particular reference to immunocytochemistry and lectin histochemistry

Summary There is increasing evidence that, in the rat, a serotonin-mediated neural input may have an inhibitory influence on the secretory activity of the subcommissural organ (SCO). In the present investigation the rat SCO was studied 7, 30 and 90 days after transplantation under the kidney capsule, an area devoid of local serotonin-containing nerves. The grafted tissue was examined by use of immunocytochemistry employing a series of primary antisera, lectin histochemistry and transmission electron microscopy. The grafted SCO survived transplantation and contained, in addition to secretory ependymal and hypendymal SCO-cells, also elements immunoreactive with antisera against glial fibrillary acidic protein or S-100 protein. In transplants, SCO-cells produced a material displaying the characteristic immunocytochemical and lectin-binding properties of SCO-cells observed under in-situ conditions. The ependymal cells lined 1–3 small cavities, which contained secretory material. A fully developed structural equivalent of Reissner's fiber was, however, never found. The immunocytochemical and ultrastructural study of the grafted SCO showed an absence of nerve fibers within the graft and suggested a state of enhanced secretory activity. A network of protruding basal lamina structures connected the secretory cells to the newly formed capillaries revascularizing the SCO. One week after transplantation, long-spacing collagen started to appear in expanded areas of such laminar networks and also in the perivascular space. It is suggested (i) that the formation of long-spacing forms of collagen is triggered by factors provided by the SCO-secretory cells, and (ii) that secretory material of the ependymal and hypendymal cells may reach the reticular extensions of the basal lamina. In contrast to the SCO in situ, the grafted SCO-cells showed a positive immunoreaction for neuron-specific enolase. They became surrounded by a S-100-immunoreactive glial sheath that separated them from other transplanted cell types and the adjacent kidney tissue of the host.Supported by Grant I/63 476 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grants 187 and 0890/88 from Fondo Nacional de Desarrollo Cientifico y Tecnológico, Chile, and Grant S-85-39 from the Directión de Investigaciones, Universidad Austral de Chile. The authors wish to acknowledge the valuable help of Ms. Elizabeth Santibañez and Mr. Genaro Alvial (Valdivia) and Ms. Inge Lyncker (Giessen)     

Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures

The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca(2+)](i)), and its precise role remains to be clarified. SP elicited rises in [Ca(2+)](i) in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK(3) and P2Y(2) receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca(2+)](i) comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca(2+)](i) was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca(2+)](i) involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.     

Class-specific epitopes detected by polyclonal antibodies against the secretory products of the subcommissural organ of the dogfish Scyliorhinus canicula

We have raised antisera against extracts of the subcommissural organ (SCO) of the dogfish, Scyliorhinus canicula L. Brains of 2900 specimens were collected in acetone, and the region containing the SCO and posterior commissure was removed and extracted in three different media. Antisera against these crude extracts were raised in rats and rabbits. Sequential absorptions of the antisera with extracts from different regions of the dogfish brain were performed to eliminate unwanted antibodies. When used to immunostain sections of the whole central nervous system of the dogfish, these purified antisera reacted selectively with the SCO-Reissner's fiber complex. An antiserum against bovine Reissner's fiber was also used. The antisera against the dogfish SCO and bovine Reissner's fiber showed the same staining pattern in the SCO and the Reissner's fiber of the dogfish. For comparative purposes, the brains of 15 vertebrate species from all vertebrate classes were immunostained with both antisera. The anti-dogfish SCO serum reacted with the SCO of the dogfish and that of other phylogenetically related elasmobranch species. Neither the SCO of a primitive elasmobranch species, Heptranchias perlo, nor the SCO of the other classes of vertebrates reacted with the anti-dogfish SCO serum. However, the antiserum against bovine Reissner's fiber reacted with the SCO of all the investigated species. It is concluded that some epitopes (or compounds) in the secretory material of the SCO are class-specific, whereas others are conserved and are synthesized by the SCO in most vertebrate species.     

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.