Subcommissural organ-Reissner's Fiber Research Articles

Calcitonin-like immunoreactivity in the subcommissural organ–Reissner's fiber complex of some freshwater and marine teleosts

The subcommissural organ (SCO) is a highly especialised circumventricular ependymal organ covering and penetrating the posterior commissure. The secretory products of the SCO condense to form Reissner's fiber (RF). Because of its extensive secretory activity and the chemical properties of its secretion, the organ functions as similar to the neurosecretory cells. Teleosts comprised of more than 20,000 extant species that show great diversity in terms of the form, habit and habitat. Affinity of calcitonin antibodies for the SCO–RF complex was used as a histochemical tool to study the morphology of some freshwater and seawater teleosts and its potential correlate to their osmotic environment. While intense to moderate calcitonin-like immunoreactivity was seen in the cells of the SCO of majority of the freshwater species viz., common carp, catfish, eel and perch; the SCO of goldfish revealed limited immunoreactivity. Like the SCO, the RF in all species was also immunostained with antibodies against calcitonin. It appeared as a single, continuous fiber that ran from SCO into the third ventricle and extended through the aqueduct, fourth ventricle and central canal of the spinal cord. In contrast to that in the freshwater fishes, the SCO–RF complex in majority of the seawater fishes, showed no calcitonin-like immunoreactivity. The data presented in this study described the comparative histomorphology of the SCO–RF complex and suggest a possiblity that the calcitonin-like immunoreactivity in the SCO–RF complex might be a feature correlated to the osmotic environment of the fish.

Neuropeptide signaling and hydrocephalus: SCO with the flow

Congenital hydrocephalus affects 0.1-0.3% of live births, with a high mortality rate (approximately 50%) in the absence of surgical intervention. Although the insertion of shunts alleviates the symptoms of the majority of congenital cases, the molecular basis of hydrocephalus and the mechanisms of cerebrospinal fluid (CSF) circulation remain largely unknown. Two important players are the subcommissural organ/Reissner's fiber (SCO/RF) complex and the ventricular ependymal (vel) cells that together facilitate the flow of the CSF through the narrow canals of the ventricular system. In this issue of the JCI, Lang et al. demonstrate that overexpression of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC1) receptor gene results in abnormal development of the SCO and vel cells, leading to congenital hydrocephalus (see the related article beginning on page 1924). The ligand for the PAC1 receptor is the neuropeptide PACAP, which uncovers what the authors believe to be a novel role for this signaling cascade in the regulation of CSF circulation.

Functional aspects of the subcommissural organ-Reissner's fiber complex with emphasis in the clearance of brain monoamines.

Reissner's fiber (RF) extends along the cerebral aqueduct, fourth ventricle, and the entire length of the central canal of the spinal cord. It grows continuously in the caudal direction by addition of newly released glycoproteins by the subcommissural organ (SCO) to its proximal end. Several hypotheses about RF function have been advanced. One of them postulates that RF binds biogenic amines present in the CSF and clears them away. In recent years, this hypothesis has been tested in our laboratory by using several experimental protocols. Firstly, the CSF concentration of monoamines was investigated in RF-deprived rats subjected to immunological neutralization of the SCO-RF complex. Secondly, the capacity of RF to bind monoamines in vivo was studied by injecting radiolabeled serotonin or noradrenaline into the rat CSF, and by perfusing them into the CSF, during one week, using an Alzet's osmotic pump. In vitro binding studies were performed using isolated bovine RF. All the findings obtained indicate that RF binds monoamines present in the ventricular CSF and then transports them along the central canal. In the absence of RF, the CSF concentration of monoamines increases sharply.

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

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

Subcommissural organ–Reissner's fiber complex of the teleost Clarias batrachus responds to GABA treatment

Subcommissural organ (SCO) is a highly specialized ependymal gland located in the roof of the third ventricle. The secretory products of the SCO, which condense to form Reissner's fiber (RF), were recently found to cross-react with the anti-calcitonin antibody. To understand the mechanisms regulating the formation of the RF and the possible function of these discrete structures, we studied the response of the SCO–RF complex to intracranially administered GABA, using immunocytochemical labeling with anti-calcitonin antibody. Although the SCO–RF complex of control fish was intensely immunostained, 1 h after GABA treatment, the ependymal cells revealed partial loss of immunoreactivity; the RF showed occasional loss of immunoreactivity with its diameter increased by about 56% of the control value. Following 2 h of GABA treatment, the SCO revealed dramatic loss of calcitonin-like immunoreactivity from the ependymal cells. The RF showed a dual response in this group, while in some segments the RF appeared conspicuously thick, elsewhere it appeared thin. The mean diameter was, however, not significantly different from the normal. Following 4 h of GABA treatment, while calcitonin-like immunoreactive material made its reappearance in the SCO, the RF diameter was uniformly reduced to about 35% of the control value. The responses by the RF as well as the SCO to intracranially administered GABA were blocked by pretreatment with bicuculline, a GABA A receptor antagonist. The results suggest that GABA, acting via GABA A receptors, may trigger the release of secretory material from the SCO and induce histomorphological changes in the RF indicative of discharge of stored material.

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

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

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.

Immunochemical analysis of the subcommissural organ-Reissner's fiber complex using antibodies against alkylated and deglycosylated glycoproteins of the bovine Reissner's fiber

Reissner's fiber (RF) has been isolated, solubilized, and used to raise polyclonal antibodies. The present investigation has been designed: (1) to obtain antibodies against RF-glycoproteins in their native form (anti-RF-BI), after irreversible denaturation by alkylation (anti-RF-A), and after alkylation and deglycosylation by using endoglycosidase F (anti-RF-DE); (2) to use these antisera for a comparative immunocytochemical study of the subcommissural organ (SCO)-RF complex; (3) to establish the molecular mass of the deglycosylated RF-glycoproteins. Anti-RF-BI reacts with the SCO of all the species investigated. Anti-RF-A and anti-RF-DE only react with bovine SCO and RF. The three antisera stain the same bands in immunoblots of extracts of bovine SCO and RF, but anti-RF-A and anti-RF-DE reveal additional bands. The epitope common to all species reacting with anti-RF-BI is thus probably conformational in nature and associated with the integrity of the disulfide bonds. The lack of antibodies against conserved sequential epitopes in anti-RF-A does not support previous assumptions on the conserved nature of the SCO secretion. After deglycosylation by using endoglycosidase F, the RF-glycoproteins present a reduction in their molecular mass ranging between 10% and 25%. The three larger compounds (450, 300, and 230 kDa) lose their affinity for Limax flavus agglutinin (affinity = sialic acid), whereas the 190-kDa compound (170 kDa after deglycosylation) keeps its affinity for this lectin suggesting that it has N-linked and O-linked carbohydrate moieties, the three larger proteins probably having only N-linked carbohydrates.

Identification and partial characterization of the secretory glycoproteins of the bovine subcommissural organ-Reissner's fiber complex. Evidence for the existence of two precursor forms

The subcommissural organ (SCO) is a brain gland whose secretory material is released into the cerebrospinal fluid where it condenses into a thread-like structure known as Reissner's fiber (RF). This fiber extends along the aqueduct, fourth ventricle and central canal of the spinal cord. The present investigation was designed to identify and partially characterize the secretory products of the bovine SCO in their intracellular location and after they have been released and packed into RF form. 5,000 SCOs were dissected out under a microscope, whereas RF of 30,000 cows were collected by perfusing the central canal of the spinal cord with artificial cerebrospinal fluid. Extracts of SCO and RF were used for (i) raising polyclonal antibodies; (ii) immunoblotting; (iii) lectin binding on electrotransfers: concanavalin A (affinity = mannose, glucose) and Limax flavus agglutinin (affinity = sialic acid); (iv) immunoaffinity chromatography; (v) preparative SDS-PAGE and raising of polyclonal antibodies against each of the secretory glycoproteins identified in the immunoblots. All antibodies and the two lectins were also applied to tissue sections of the SCO and RF of several species. The immunocytochemical study of the bovine SCO using an anti-RF serum showed that the secretory material present in the rough endoplasmic reticulum (RER), secretory granules and in RF is strongly immunoreactive. Con A binding sites were only found in the endoplasmic reticulum, whereas Limax flavus agglutinin revealed secretory granules and RF, only. In the blots the immunostaining was used to identify secretory polypeptides. The glycosylated nature of the latter was established by their affinity for Con A and/or Limax flavus agglutinin. Furthermore, this latter lectin allowed us to distinguish whether the intracellular source of a secretory glycoprotein is from a pre-Golgi (RER) or a post-Golgi (secretory granules) compartment. Four glycoproteins were identified in the SCO with apparent molecular weights of 540, 450, 320 and 190 kDa. The three former were also purified by immunoaffinity chromatography. The 540 and 320 kDa forms are present in the SCO but missing in RF, have affinity for Con A, but not for LFA. It is suggested that these two compounds correspond to two precursor forms. The 450 and 190 kDa glycoproteins are present in both, the SCO and RF, and have affinity for Con A and Limax flavus agglutinin. These most likely correspond to processed forms. The presence of more than one precursor was further substantiated by immunocytochemical findings using antisera against the 540, 450 and 320 kDa forms. Only one of the two precursors found in the bovine SCO could be seen in the rat SCO, and neither of them could be detected in the snake SCO.

Ultrastructural immunocytochemical study of the massa caudalis of the subcommissural organ-Reissner's fiber complex in lamprey larvae (Geotria australis): Evidence for a terminal vascular route of secretory material

The massa caudalis of the subcommissural organ-Reissner's fiber complex of lamprey larvae (Geotria australis) was studied immunocytochemically at the ultrastructural level by use of the immunoperoxidase-silver methenamine procedure. An antiserum raised against bovine Reissner's fiber was utilized as primary antibody. The caudalmost portion of the central canal and its ampulla caudalis communicate, via wide intercellular spaces in their dorsal wall, with large cavities or lacunae. In addition, distinct openings in the dorsal wall of the ampulla establish an open communication between the latter and the lacunae. The lacunae are lined by slender processes of cells of unknown nature. No junctional complexes can be observed between these cells, which lack a basal lamina. The lacunae communicate with structures resembling blood capillaries, however, they are devoid of a basal lamina. These peculiar vessels, in turn, are in direct communication with characteristic blood capillaries. Reissner's fiber (RF) and its massa caudalis are strongly immunoreactive with the antiserum used. The wide intercellular spaces in the dorsal wall of the central canal and the ampulla, as well as the lumina of the (i) lacunae, (ii) modified vessels and (iii) blood capillaries are filled with a flocculent, strongly immunoreactive material. No immunoreactive material was found outside these structures. Thus, the blood capillaries appear to represent the only final target of RF-material arriving at the ampulla caudalis.