Colocalization of p450 aromatase and oxytocin immunostaining in the rat hypothalamus.

With combined immunoperoxidase and immunofluorescence, we observed colocalization of cytochrome P450 aromatase with the posterior lobe peptide oxytocin and its associated neurophysin 1 in adult male rats. P450 was most abundant in the anterior hypothalamus. Colocalization of OT with P450 was observed in the preoptic region, the periventricular nucleus of the hypothalamus, the lateral subcommissural nucleus, and in the zona incerta. Magnocellular perikarya in the supraoptic and in the paraventricular nuclei contained only occasionally both antigens. P450 immunostaining overlapped to a great extent with known estrogen target regions. Oxytocinergic functions are controlled by estradiol while androgen receptors are mostly absent in neuroendocrine hypothalamic nuclei. Our findings suggest that systemic androgens may be aromatized to estrogens in male oxytocinergic neurons linked to the limbic system.

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.

Polarized expression of integrin beta1 in diencephalic roof plate during chick development, a possible receptor for SCO-spondin.

The roof plate of the caudal diencephalon is formed by the posterior commissure (PC) and the underlying secretory ependyma, the subcommissural organ (SCO). The SCO is composed by radial glial cells bearing processes that cross the PC and attach to the meningeal basement membrane. Since early development, the SCO synthesizes SCO-spondin, a glycoprotein that shares similarities to axonal guidance proteins. In vitro, SCO-spondin promotes neuritic outgrowth through a mechanism mediated by integrin beta1. However, the secretion of SCO-spondin toward the extracellular matrix that surrounds the PC axons and the expression of integrins throughout PC development have not been addressed. Here we provide immunohistochemical evidence to suggest that during chick development SCO cells secrete SCO-spondin through their basal domain, where it is deposited into the extracellular matrix in close contact with axons of the PC that express integrin beta1. Our results suggest that SCO-spondin has a role in the development of the PC through its interaction with integrin beta1.

Physiological response of bovine subcommissural organ to endothelin 1 and bradykinin.

The circumventricular organs (CVOs) regulate certain vegetative functions. Receptors for bradykinin (BDK) and endothelin (ET) have been found in some CVOs. The subcommissural organ (SCO) is a CVO expressing BDK-B2 receptors and secreting Reissner's fiber (RF) glycoproteins into the cerebrospinal fluid. This investigation was designed to search for ET receptors in the bovine SCO and, if found, to study the functional properties of this ET receptor and the BDK-B2 receptor. Cryostat sections exposed to (125)I ET1 showed dense labeling of secretory SCO cells, whereas the adjacent ciliated ependyma was devoid of radiolabel. The binding of (125)I ET1 was abolished by antagonists of ETA and ETB receptors. The intracellular calcium concentration ([Ca(2+)](i)) was measured in individual SCO cells prior to and after exposure to ET1, BDK, or RF glycoproteins. ET1 (100 nM) or BDK (100 nM) caused an increase in [Ca(2+)](i) in 48% or 53% of the analyzed SCO-cells, respectively. RF glycoproteins had no effect on [Ca(2+)](i) in SCO cells. ET and BDK evoked two types of calcium responses: prolonged and short responses. Prolonged responses included those with a constant slow decline of [Ca(2+)](i), biphasic responses, and responses with a plateau phase at the peak level of [Ca(2+)](i). ET1-treated SCO explants contained a reduced amount of intracytoplasmic AFRU (antiserum to RF glycoproteins)-immunoreactive material compared with sham-treated control explants. Our data suggest that ET1 and BDK regulate [Ca(2+)](i) in bovine SCO cells, and that the changes in [Ca(2+)](i) influence the secretory activity of these cells.

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.

Receptors for leptin and estrogen in the subcommissural organ of rabbits are differentially modulated by fasting.

In rabbits, the fasting-dependent reduction of LH secretion is likely mediated by leptin and estrogens via receptors in the brain. For the first time, using immunohistochemistry, the presence and regulation of receptors for leptin (Ob-R) and estradiol-17beta subtype alpha (ERalpha) were studied in the subcommissural organ (SCO) of rabbits, which were fed either ad libitum (control) or fasted for 48 h (treated) to verify whether this brain structure is a potential site of integration for metabolism and reproduction. In control rabbits, the cytoplasm of glial cells lining the SCO evidenced strong Ob-R immunoreactivity, whereas both ependymal and hypendymal cells of this glandular-like structure were negative. The Ob-R positive glial cells were identified as fibrous astrocytes using the phosphotungstic acid-hematoxylin histochemical (PTAH) and glial fibrillary acidic protein (GFAP) immunohistochemical techniques. ERalpha immunoreactive nuclei were detectable exclusively in the specialized cells forming the SCO, whereas surrounding astrocytes and neurons were negative. Compared to controls, in fasted rabbits, the staining of Ob-R immunoreaction was reduced in the cytoplasm of positive astrocytes, but greatly enhanced in plasma membranes, whereas the number of ERalpha immunoreactive SCO cells was increased (13.2+/-2.7 vs. 5.2+/-2.0, P<0.01). Ependymal cells lining the third ventricle were negative for both Ob-R and ERalpha. Our results indicate, although indirectly, that the SCO, together with the astrocytes in close contact with this structure, is a likely target for nutritional and gonadal signals carried by leptin and estrogens, suggesting that these specialized glial cells may regulate reproduction and metabolism through mechanisms still unknown.

The thrombospondin type 1 repeat (TSR) and neuronal differentiation: roles of SCO-spondin oligopeptides on neuronal cell types and cell lines.

SCO-spondin is a large glycoprotein secreted by ependymal cells of the subcommissural organ. It shares functional domains called thrombospondin type 1 repeats (TSRs) with a number of developmental proteins expressed in the central nervous system, and involved in axonal pathfinding. Also, SCO-spondin is highly conserved in the chordate phylum and its multiple domain organization is probably a chordate innovation. The putative involvement of SCO-spondin in neuron/glia interaction in the course of development is assessed in various cell culture systems. SCO-spondin interferes with several developmental processes, including neuronal survival, neurite extension, neuronal aggregation, and fasciculation. The TSR motifs, and especially the WSGWSSCSVSCG sequence, are most important in these neuronal responses. Integrins and growth factor receptors may cooperate as integrative signals. We discuss the putative involvement of the subcommissural organ/Reissner's fiber complex in developmental events, as a particular extracellular signaling system.

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

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

The bovine subcommissural organ: cytochemical and immunochemical characterization of the secretory process.

Specific glycoproteins of the bovine subcommissural organ (SCO) were studied by means of various techniques: light and electron microscopy, immunoaffinity chromatography, electrophoresis and Western blotting. Use of lectins (Con A, WGA, PHA-E and -L, LCA) allowed to specify the synthesis and release of complex-type glycoproteins that bear high-mannose-carbohydrate chains in their precursor forms and probably triantennary carbohydrate chains in their mature forms. Antibodies raised against SCO extracts were characterized by means of various tests and used to purify specific compounds. Immunopurified fractions using A99 polyclonal antibody contained numerous polypeptides reactive with Con A, their apparent molecular weight (MW) ranging from 240 to 50 kDa. Only two glycopeptides were strongly labeled with WGA (98 and 52/54 kDa MW). Immunopurified fractions using C1B8A8 monoclonal antibody, specific of the complex-type glycoproteins at different steps of glycosylation, showed three specific Con A-reactive polypeptides at 88, 54 and 34 kDa MW. Only the 34 kDa glycopeptide was strongly labeled with WGA. The latter could correspond to the monomeric form of the secreted compound. Electrophoretical analyses of Reissner's fiber material allowed the detection of a WGA-positive smear in the upper part of the blots, suggesting that the complex-type glycoproteins, when released into the CSF, constitute a stable polymer.

Developmental neuron-glia interactions: role of serotonin innervation upon the differentiation of the ependymocytes of the rat subcommissural organ.

The rat subcommissural organ (SCO), which forms the roof of the third ventricle is an adequate model to study certain mechanisms of neuron-glia interactions in vivo. The ependymocytes, the main component of the SCO, have a glial origin. They possess particular phenotypic characteristics: they accumulate [3H]GABA by a specific uptake mechanism, contain transitory GFAP during ontogenesis and do not express PS100; on the other hand they receive a 5HT input which forms typical synaptic contacts. This innervation is of particular interest to approach neuron-glia interactions during the differentiation. Studies of GABA uptake carriers during ontogenesis in SCO ependymocytes show a correlation between the onset of the 5HT innervation and the advent of the GABA uptake. Moreover, destruction of the 5HT innervation by a neurotoxin (5-7-dihydroxytryptamine), before its arrival at the SCO in newborn rat, inhibits the formation of the GABA uptake system and causes the expression of PS100 in adult SCO cells. On the other hand, the SCO of newborn rats transplanted to the fourth ventricle of an adult host rat had no capacity to take up GABA and expressed PS100 3 months after its transplantation. Finally, the SCO ependymocytes of species devoid of 5HT innervation (rabbit, mice) were unable to take up GABA and contain PS100. These data suggest that neuron-glia interactions are necessary for the advent of GABA uptake carriers and can control the expression of glial markers during ontogenesis in SCO ependymocytes.

Transcription of SCO-spondin in the subcommissural organ: evidence for down-regulation mediated by serotonin.

The subcommissural organ (SCO) is a brain gland located in the roof of the third ventricle that releases glycoproteins into the cerebrospinal fluid, where they form a structure known as Reissner's fiber (RF). On the basis of SCO-spondin sequence (the major RF glycoprotein) and experimental findings, the SCO has been implicated in central nervous system development; however, its function(s) after birth remain unclear. There is evidence suggesting that SCO activity in adult animals may be regulated by serotonin (5HT). The use of an anti-5HT serum showed that the bovine SCO is heterogeneously innervated with most part being poorly innervated, whereas the rat SCO is richly innervated throughout. Antibodies against serotonin receptor subtype 2A rendered a strong immunoreaction at the ventricular cell pole of the bovine SCO cells and revealed the expected polypeptides in blots of fresh and organ-cultured bovine SCO. Analyses of organ-cultured bovine SCO treated with 5HT revealed a twofold decrease of both SCO-spondin mRNA level and immunoreactive RF glycoproteins, whereas no effect on release of RF glycoproteins into the culture medium was detected. Rats subjected to pharmacological depletion of 5HT exhibited an SCO-spondin mRNA level twofold higher than untreated rats. These results indicate that 5HT down-regulates SCO-spondin biosynthesis but apparently not its release, and suggest that 5HT may exert the effect on the SCO via the cerebrospinal fluid.

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.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell lineage of the subcommissural organ secretory ependymocytes: differentiating role of the environment.

SCO-ependymocytes have a secretory activity and a neural innervation relating them to neurosecretory nerve cells. To elucidate the cell lineage of the SCO-ependymocytes and emphasize the role of the neural innervation in their differentiation, in particular 5-HT innervation, we analyzed the developmental pattern of expression of several glial and neuronal markers: (1) in the SCO of mammals possessing (rat, cat) or devoid (mouse, rabbit) of 5-HT innervation, (2) in rat 5-HT deafferented SCO, and (3) in rat SCO transplanted in a foreign environment, the fourth ventricle. The ability of SCO-ependymocytes to transiently express GFAP during development and express the glial alpha alpha-enolase confirms the glial lineage of the SCO-ependymocytes. Synthesis of vimentin by SCO-ependymocytes relates them to the classical ependymocytes. The ability of mature SCO-ependymocytes to take up GABA only when they are innervated by 5-HT terminal underlines the role of the neural environment on the differentiation of these ependymocytes and suggests that differential maturation of the SCO according to its innervation, may lead to specific functional specialization of this organ in different species.

Presence and functional significance of neuropeptide and neurotransmitter receptors in subcommissural organ cells.

The subcommissural organ (SCO) of mammals is innervated by several neuropeptide and neurotransmitter systems. So far, substance P (SP), oxytocin (OXT), vasopressin (VP), somatostatin (SOM), thyrotropin-releasing factor (TRF), and angiotensin II (ANGII) were identified in neuropeptidergic input systems, and serotonin (5HT), gamma-amino butyric acid (GABA), noradrenaline (NA), dopamine (DA), and acetylcholine (Ach) were neurotransmitters observed in systems afferent to the SCO. In the present report, based on literature data and our own investigations, we describe the occurrence of peptide and transmitter receptors in the SCO by means of autoradiographic and biochemical studies. Further, we summarize aspects of the signal transduction cascades possibly linked to different receptor types of the SCO; these studies included the use of calcium imaging (FURA-2 technique), ELISA technique, and immunocytochemistry. Receptors were identified for adenosine, angiotensin II, imidazoline, glucocorticoids, mineralocorticoids, NA, and embryonic brain kinase. The studies on intracellular signal-transduction indicated receptors for tachykinins and for ATP. In SCO cells, Ca(++) and c-AMP were identified to act as second messengers. As important transcription factor, cAMP-/Ca(++)-response element binding protein (CREB) was observed. Ach and NA did not show a significant effect on the subcommissural signal transduction.

Secretory activity and serotonin innervation of lizard's subcommissural organ.

We investigated immunohistochemically the subcommissural organ (SCO) glycoprotein secretion, its serotoninergic (5-HT) innervation and the possible control of this innervation upon the SCO activity in lizards (Agama impalearis, Saurodactylus mauritanicus and Eumeces algeriensis). Inside the SCO, interspecific differences in the intensity and the distribution of both secretory product and 5-HT nerve fibers were observed. Compared with Agama and Eumeces, the SCO of Saurodactylus displayed intense secretory products and several 5-HT fibers. In Saurodactylus, i.p. injection of parachlorophenylalanine, a potent inhibitor of 5-HT synthesis, produced a marked decrease of SCO secretory product. We report in this study species differences of the lizard SCO secretory activity and its possible physiological control by 5-HT innervation, as previously demonstrated in mammals.

The dispersed cell culture as model for functional studies of the subcommissural organ: preparation and characterization of the culture system.

The subcommissural organ (SCO) is an enigmatic secretory gland of the brain, which is believed to be derived from ependymal (glial) precursor cells. We here developed a dispersed cell culture system of the bovine SCO as an approach to functional analyses of this brain gland. Tissue of the bovine SCO obtained from the slaughterhouse was papain dissociated either directly after dissection or after preparation of SCO explants. The latter had been maintained for 4-6 weeks in organ culture. The dispersed cells were cultured for up to 14 days and continuously tested for their secretory state by immunostaining of their secretory product. With respect to the morphology of the SCO cells (shape, processes, nucleus), no difference was found between the culture of freshly dissociated SCOs and that of dissociated SCO explants. In all cases, the dissociation caused a dedifferentiation; typical elongated cells were formed increasingly after 1 day of culture. Thereafter, only the cellular size increased, whereas the shape and the viability of the cells remained unchanged. Proliferating SCO cells were never observed. The culture obtained from fresh SCO tissue contained more glia cells and fibrocytes than the culture prepared from SCO explants. The proliferation of glia cells and fibrocytes was suppressed by blocking the mitotic activity with cytosine-beta-D-arabino furanoside (CAF). The cytophysiological features of the cultured dispersed cells of both origins did not differ as demonstrated by classical histology, by immunocytochemistry for the secretory products of the SCO, by the characteristics of calcium influx into the cytoplasm ([Ca2+]i) and cyclic adenosine monophosphate (cAMP) after stimulation with adenosine-5-triphosphate, substance P or serotonin, and by the activation of the transcription factor cAMP-responsive element-binding protein. Because of the maintenance of their viability, their capacity to release the secretory product into the culture medium, their receptive capacity, and their signal transduction pathways, we conclude that the dispersed cell culture system, especially that obtained from SCO explants, represents an appropriate and useful model for functional studies of the mammalian SCO.

Neuronal control of [3H]GABA uptake in the ependymocytes of the subcommissural organ: an in vivo model of neuron-glia interaction.

The rat subcommissural organ (SCO) is a particular but adequate paradigm for the approach, in vivo, to some aspects of neuron-glia interaction in gamma-aminobutyric acid (GABA) uptake. The rat SCO ependymocytes (the main component of this structure lying at the junction of the aqueduct and the third ventricle) accumulate [3H]GABA by a highly specific uptake mechanism and receive a serotoninergic input forming typical synaptic contacts. It seems that there is a correlation between the capacity of the rat SCO ependymocytes to take up [3H]GABA and the presence of a serotonin (5-HT) innervation. Indeed, in the newborn rat, no uptake of [3H]GABA was observed before the onset of this innervation and the increased [3H]GABA accumulation in the SCO was correlated with the appearance of the 5-HT terminals in the SCO. Moreover, in the mouse, whose SCO is devoid of a 5-HT innervation, no accumulation of [3H]GABA was observed in the SCO ependymocytes. Thus, the 5-HT innervation could be involved directly or indirectly in the onset of the GABA uptake carriers. On the other hand, in adult rats parachlorophenylalanine (pCPA) treatment decreased the 5-HT content of the SCO, and increased [3H]GABA accumulation; such an augmentation was not observed when rats were treated with pCPA plus 5-hydroxytryptophan to restore the 5-HT content. However, an increase in 5-HT content of the SCO by pargyline treatment appeared to have no effect on [3H]GABA uptake. Control of GABA uptake activity by 5-HT in the SCO ependymocytes could be an interesting model for the study of a possible interaction between amino-acids and other neurotransmitters by terminating their action in the extracellular space.

Ontogenesis of the secretory epithelium of the bovine subcommissural organ. A histofluorescence study using lectins and monoclonal antibodies.

A spatio-temporal analysis of the differentiation of a group of specialized (secretory) ependymal cells in the subcommissural organ (SCO) of the brain was undertaken in the bovine using a monoclonal antibody (C1B8A8) which is specific of the secretory process in this organ. In addition, lectins (concanavalin agglutinin (Con A), Lens culinaris agglutinin (LCA), wheat germ agglutinin (WGA), and Phaseolus vulgaris agglutinin (PHA] were used to analyse the maturation of the carbohydrate moieties of the secretory product (subcommissuralin). Monoclonal antibody NC-1 specific to a complex carbohydrate epitope including a terminal 3-sulfoglucuronyl residue similar to HNK-1 was also tested to compare the reactivity of the SCO with that of other brain structures. These cells express a specific antigen related to the known secretory activity of the SCO during early embryogenesis (2 months). This antigen is recognized by C1B8A8 antibody and by Con A suggesting that high mannose-type glycoproteins are synthesized at this stage. Later on (approximately 3.5 months), appearance of C1B8A8, WGA, LCA, L- and E-PHA-positive material in the apical lining of the ependymal cells, close to the ventricular cavity, suggests that maturation of the complex-type glycoproteins (Asn-linked) occurs at this stage. Presence of secretory material in the CSF and Reissner's fibre could be detected using the same probes at a stage of 4 months. As early as 2 months NC-1-positive material was detected in the ependyma of the mesencephalic roof, while no reaction occurred in the SCO epithelium. This suggests that the carbohydrate moieties of subcommissuralin is different from that of ependymins beta and gamma. Using specific monoclonal antibodies, molecular characterization of subcommissuralin and experimental analyses on its accurate role in brain development will further our tentative comparison with ependymins. The secretory ependymal cells in the SCO express a particular phenotype and could represent an increasing model to study cell differentiation in the brain.

Postnatal secretion of the subcommissural organ of the Meriones shawi: control of serotonin innervation.

The postnatal development of the subcommissural organ (SCO) glycoprotein secretion in form of Reissner's fiber and the putative control of the serotonin innervation upon the SCO activity were examined by immunohistochemistry in the semi-desert rodent, Meriones shawi. Abundant SCO secretory material and numerous serotoninergic fibers reaching the SCO were observed in newborns meriones. An increase of both secretory material and serotonin fibres density inside the SCO was observed during postnatal period and into adulthood. Neurotoxic destruction with 5,7-dihydroxytryptamine of the SCO serotonin input in the adult or the inhibition of serotonin synthesis by para-chlorophenylalanine at different postnatal ages, resulted in a decrease of the intensity of SCO Reissner's fiber immunolabelling suggesting a reduction in the SCO secretory material. This result might reflect either an inhibition of the synthesis or a stimulation of release of secretory material. These data suggest that serotonin innervation could be precociously involved in the regulation of the merione SCO secretion.

Developmental neuron-glia interaction: role of the serotonin innervation upon the onset of GABA uptake into the ependymocytes of the rat subcommissural organ.

The subcommissural organ (SCO) of the rat allows the analysis of neuron-glia interactions, in vivo, during the maturation of the brain. The SCO contains a single glial cell type which receives a homogeneous serotonin (5-HT) innervation. The onset of gamma-aminobutyric acid (GABA) uptake transport into the SCO ependymocytes is dependent on the 5-HT innervation since destruction of this innervation, at birth, or transplantation of newborn rat SCO ependymocytes to the fourth ventricle of adult host rats prevented the appearance of [3H]GABA uptake as visualized by autoradiography.