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The ependymal cells of the subcommissural organ (SCO) of the snake Natrix maura display long basal processes which terminate either on blood vessels or on the leptomeninges. The cell body and the basal processes contain a secretory material detectable immunocytochemically at the light-microscopic level using an antibody raised against bovine Reissner's fiber. The present investigation deals with the ultrastructural location in these cells of the (i) immunoreactive material; (ii) concanavalin A (Con A)- and wheat-germ agglutinin (WGA)-binding sites. In the subnuclear region the immunoreactive material was located within dilated cisternae of the rough endoplasmic reticulum and had affinity for Con A but not for WGA. In the supranuclear region the secretory material was exclusively located within numerous granules. Since all these granules showed affinity for WGA, they can be regarded as "post-Golgi" elements. Thus, at variance with the situation in the mammalian SCO, in the ophidian SCO most of the secretion is stored in secretory granules rather than in dilated cisternae of the rough endoplasmic reticulum. In the perivascular and leptomeningeal endings the immunoreactive material was located within granules which, because of their affinity for WGA, should also be regarded as true secretory granules derived from the Golgi apparatus. It is concluded that these granules are transported along the basal processes and accumulated in the perivascular and leptomeningeal endfeet. This observation favours the view of a local release of the content of these granules, since there is no evidence for a reverse transport of these granules all the way back from the distal termination to the apical pole, to be finally released into the ventricle.

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

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

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

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

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

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

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

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

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

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

The subcommissural organ (SCO) of the rat was investigated by use of histochemical and immunocytochemical methods at the light- and electron-microscopic levels. Consecutive thin methacrylate sections were stained with the pseudoisocyanin (Psi), immunoperoxidase (IMC; employing an antiserum against Reissner's fiber, AFRU), periodic acid-Schiff (PAS) and periodic acid-silver methenamine (SM) techniques, and reacted with six types of lectins. Psi, SM, concanavalin A (Con A) and IMC were also used for double and triple sequential staining of the same section. Increasing dilutions of AFRU (from 1:1000 to 1:200 000) were used for immunostaining of serial paraffin sections. In addition, ultrastructural localization of (i) Con A-binding sites and (ii) immunoreactive secretory material was performed. Some of these procedures were also applied to the ophidian and canine SCO. Con A-positive, Psi-positive and immunoreactive materials coexisted within the same cisternae of the rough endoplasmic reticulum. The Golgi apparatus lacked Con A-positive and immunoreactive substances. Apical secretory granules and secreted material lying on the surface of the SCO showed (i) the highest affinity for AFRU, but were (ii) Con A-negative, and (iii) wheat-germ agglutinin-, PAS- and SM-positive. Reissner's fiber displayed a low affinity for AFRU. It is suggested that the SCO secretes N-linked glycoproteins, the carbohydrate and protein moieties of which undergo (i) a maturation process before being released, and (ii) some kind of modification(s) after their release into the ventricle. The perivascular secretory cells of the dog SCO might secrete a material different from that secreted by the ependymal cells.

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

The subcommissural organ (SCO) of the snake Natrix maura was studied by use of the immunoperoxidase procedure. Primary antisera against bovine neurophysins (Nps I + II, OXY-Np), oxytocin (OXY), mesotocin (MST), arginine-vasotocin (AVT), somatostatin (SOM), beta-endorphin (END) and bovine Reissner's fiber were used. A conventional ultrastructural study, with special emphasis on the nerve fibers present in the SCO, was also performed. Nerve fibers containing immunoreactive OXY-Np and MST were seen to reach the SCO. The staining of adjacent sections with the anti-Reissner's fiber serum showed that the OXY-Np- and MST-immunoreactive fibers were distributed among the cell bodies and processes of the ependymal secretory cells. No fibers containing immunoreactive OXY, AVT, SOM or END were found in the SCO. The ultrastructural analysis revealed in the SCO the presence of nerve fibers filled with electron-dense granules, 170-210 nm in diameter. Although a direct apposition between these fibers and the SCO cells was frequently seen, no synaptic differentiations were identified. Structures identical to the Herring bodies (found in the neurohypophysis) were seen in the SCO.

Two kinds of secretion are formed in cells of the subcommissural organ (SCO) of Cebus apella. The "light secretion" is found in saccules originating from the endoplasmic reticulum. This secretion is stored in the peripheral portion of the cells and is not involved in formation of Reissner's fiber (RF). In close association with the Golgi complex, electron-dense granules are developed, containing a finely granular substance. These granules accumulate beneath the apical plasmalemma of the cell. Their content is discharged into the third ventricle, where it occurs in the form of a thin layer of secretion. This material appears to constitute the RF at the level of the entrance to the mesencephalic aqueduct.

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 paical 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.

The nervous input to the subcommissural organ (SCO) of the rat has been investigated with Falck-Hillarp fluorescence histochemistry and electron microscopical techniques. Previous fluorescence histochemical observations of a dense plexus of serotoninergic nerve fibres in relation to the basal SCO were confirmed. Electron microscopically, unmyelinated fine varicose axons ranging in size from 0.1--0.6 micrometer were observed to penetrate into the SCO hypendyma. Boutons and presynaptic varicosities filled with a diversity of round and elongated clear vesicles, and occasional large dense cored vesicles establish asymmetric (Gray's type I) synaptic contacts with the basal processes and somata of the SCO ependymal and hypendymal cells. A typical varicosity in synaptic contact with an SCO cell contains a population of approximately 85% clear, elongated vesicles 45 X 60 nm in diameter, 15% clear, round vesicles 50 nm in diameter, and 1--2% large dense cored vesicles with a vesicle diameter of about 85 nm and a dense core diameter of 50--55 nm. The mean length of the postsynaptic membrane specialization was found to be 0.5 micrometer. Experiments with specific neurotoxic drugs revealed that the nerve terminals in synaptic contact with the SCO cells are identical to the fibres of the serotoninergic plexus identified fluorescence histochemically. Thus, an intraventricular injection of either 5,6-dihydroxytryptamine or 5,7-dihydroxytryptamine induced typical degenerative changes in most of the boutons in synaptic contact with the SCO cells, and also a disappearance of the yellow fluorescent nerve plexus. It is concluded that the SCO of the rat receives a dense plexus of serotonin-containing nerve fibres which form typical synaptic contacts with the specialized ependymal cells of the SCO and that these fibres may constitute the only direct nervous input to the organ. The degeneration of the serotoninergic synapses elicited a long-lasting, pronounced increase in the secretory activity of the SCO. Despite long survival times after the treatment with neurotoxic drugs, we found no evidence of regenerative restitution of the serotoninergic innervation nor normalization of the secretory activity of the SCO. The observed inverse relationship between secretory activity and serotoninergic innervation is in line with previous observations which indicate that the 5-hydroxytryptamine input to the SCO ependymal and hypendymal cells exerts a powerful inhibition on their protein synthetic machinary.

Antibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.

In the subcommissural organ (SCO) of the guinea pig, rat, golden hamster, and mouse the activity and distribution of enzymes related to the energy-supplying metabolism and of some marker enzymes of different cell organelles have been investigated by means of mostly modified histochemical methods. The results were compared with findings in the ciliated ependyma of the ventricular wall and with those in the ependyma of the choroid plexus of the third ventricle. In the ependymal part of the SCO only a moderate activity of hexokinase is observed in its specialized columnar cells whereas a high activity is present both in the ciliated ependyma and the choroid plexus. - The staining pattern of glucose-6-phosphatase is similar to that of hexokinase but this enzyme is found is the SCO only. - Likewise hexokinase, glycogen granules and enzymes related to glycogen metabolism (phosphoglucomutase, uridine-diphosphoglucose pyrophosphorylase, glycogen synthetase and phosphorylase) are regularly found most numerous and active in the nuclear and supra-nuclear area of the ependymal part. These enzymes are less active in both the other ependymal regions. - Uridine-diphosphoglucose dehydrogenase could not be demonstrated in the SCO. The NADP-linked enzymes of the pentose phosphate shunt, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, show a moderate activity which decreases also from the nuclear towards the apical area of the ependymal cells of the SCO. Enzymes of the glycolytic pathway, such as glucosephosphate isomerase, fructose-6-phosphate kinase, fructose-I,6-diphosphate aldolase, glyceraldehyde-3-phosphate and lactate dehydrogenase, are highly active in the SCO and are located mainly in the supranuclear area, too. Fructose-1,6-diphosphatase could not be demonstrated thus indicating that in the SCO the pathway is most probably only glycolytic but not gluconeogenetic. Compared to the ependyma of the ventricular wall and of the choroid plexus, in the SCO the M type subunits of lactate dehydrogenase predominate. Glycolytic enzymes are also very active in the choroid plexus but less in the ciliated ependyma. Compared to the ciliated ependyma and especially to the ependyma of the choroid plexus, the activities of enzymes which are only present in mitochondria (NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, NAD-linked malate dehydrogenase after preextraction, cytochrome oxidase, 3-hydroxybutyrate and glycerolphosphate and glutamate dehydrogenase) are relatively low. Mitochondria are accumulated near the superior pole of the nuclei as well as in the most apical part of the ependymal cells. - The staining pattern of NADP-linked isocitrate and malate dehydrogenase as well as of NADH dehydrogenase suggests that these enzymes are localized both in and out of mitochondria. The extramitochondrial activity of the first two enzymes might be localized in the cytosol. The extramitochondrial activity of NADH dehydrogenase might be localized in the endoplasmic reticulum...

The secretory activity of the subcommissural organ (SCO) is affected by adrenalectomy, adrenalectomy + castration, and by an increase in ambient temperature in adrenalectomized and adrenalectomized + castrated animals. Adrenalectomy inhibits the activity of the SCO. After adrenalectomy + castration the decrease in the secretory activity of the SCO is more rapid. In contrast, an increase in the ambient temperature in adrenalectomized animals induces a recrudescence of the activity of the SCO. The increase in temperature in castrated + adrenalectomized lizards does not affect the inhibition produced by this type of surgical treatment. The histological changes are discussed on the basis of results obtained in the present study and in previous experiments.

In the snake, Natrix maura, and the turtle, Mauremys caspica, the basal processes of the ependymal cells of the subcommissural organ project toward the local blood vessels and the leptomeninges. These processes and their endings were studied using aldehyde-fuchsin (AF), periodic-acid Schiff (PAS), periodic-acid silver-methenamine (PA-SM), concanavalin A (ConA), wheat germ agglutinin (WGA), immunoperoxidase staining (employing an antiserum against bovine Reissner's fiber; AFRU), and conventional transmission electron microscopy. For the purposes of comparison, the ventricular cell pole was also analyzed. The secretory material located in the ventricular cell pole and that present in ependymal endings had only a few staining properties in common, i.e., affinity for AF, ConA, and AFRU at a dilution of 1:1000. On the other hand, PAS, PA-SM, WGA, and AFRU at a dilution of 1:200,000 stained the apical (ventricular) secretory material but not the secretory material of the ependymal processes. The histochemical features of the secretory material located in the terminals of ependymal processes, as well as the presence at these sites of numerous rough-endoplasmic-reticulum cisternae and secretory granules, suggest that secretory material may be synthesized in these terminals. The probable fate of this material, i.e., release to the perivascular and leptomeningeal spaces or transport to the ventricular cell pole, is discussed.

Under the scanning electron microscope, the rat subcommissural organ (SCO) appears as an oval zone, rich in kinocilia and well delimited from the non-specific ependymal epithelium. This zone surrounds the cranial and posterior part of the mesencephalic canal's entrance. The ependymal cells of the SCO show coniform processes with microvilli and kinocilia. In contact with the apical pole of the peripheric SCO-ependymocytes lie scarce supraependymal axons. The Reissner's fiber is composed by the twining of the fibrillary structures emerging from the area of the SCO.

Synapse formation and maturation in the subcommissural organ (SCO) of Wistar rats were studied from birth to the end of the first month. Modifications of the secretory ependyma were analyzed over the same period. On the 1st postnatal day, the large varicosities in contact with the SCO ependymocytes appeared immature (absence or low density of vesicular population, no synaptic membrane differentiation). The synaptic contacts were formed from the 3rd postnatal day, near the glandular cell nuclei (0.1 micron distance); progressively, the content of the axonal boutons and the pre- and post-synaptic specializations became similar to those of adults. From the 21st day on, the axo-glandular innervation was considered analogous to that in the adult. Using immunocytochemistry, it was found that the increase in the serotonin-immunoreactive fiber density in the whole organ was time-dependent. Light and electron microscopy demonstrated changes in the morphology of SCO ependymocytes during the first postnatal weeks, notably in the endoplasmic reticulum and content ot apical protrusions. On postnatal day 14, two types of ependymal cells, neonatal-like and adult-like, coexisted. The evolution of SCO ependymocytes coincided with the progressive onset and maturation of axo-glandular innervation taking place after birth.

The secretory activity of the SCO cells of Lacerta s. sicula Raf. is strongly reduced during the winter. Such reduction is documented by the decrease of the decrease of the number of secretory granules type A and B described in previous papers in the summer SCO cells. Also the sacks of RER filled with electron-dense material (type C secretion) are very few; in their place there are, in the basal region of the cells, large vacuoles. In the distal region of the cells, at the free cell surface, a pronounced increase in the number of microvilli is noticed.

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.

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.

The subcommissural organ of the rat has been studied by a combination of light, scanning and transmission electron microscopy. Special attention has been paid to the appearance of the ventricular surface of the organ. The ependyma-derived cells on the surface of the organ are extremely long with a width which diminishes steadily from the base, where the nucleus is situated, to the ventricular surface. This results in a fan-shaped arrangement of cells, the cytoplasm of each cell reaching the surface of the ventricular system. The apical surface of the cell has small ciliated extensions 1-1.5 micrometers in diameter protruding into the ventricular lumen. With conspicuous rough endoplasmic reticulum and Golgi apparatus, and microtubules in profusion, the subcommissural organ cells possess the hallmarks of active secreting cells. The surface of the cells shows pinocytic vesicles-suggesting exchange of materials with the cerebrospinal fluid.

The results of a preliminary immunocytochemical investigation on the subcommissural organ (SCO) in rats show that (1) Reissner's fiber (RF) or essential compounds of the RF are produced by the SCO, (2) the immunoreactive material is produced in the epithelial cells of the SCO as well as in the hypendymal cells, and (3) the immunoreactive material of the SCO belongs to a category of endogenous peptides to date not demonstrable immunocytochemically in other brain structures.

A karyometric study of the ependymal cells of the subcommissural organ (SCO) was made in male albino mice castrated at 20 days and examined at 25, 35, 45, 55 and 85 days of life, and in control groups of the respective ages. The values of the nuclear areas and perimeters of SCO cells as well as those of the global volume of the SCO are significantly lower in the experimental groups, and the nuclear membrane shows less invaginations. No changes are observed after gonadectomy in the subjacent thalamic ependyma.

On a study of the most rostral part of the optic tectum of Clemmys caspica leprosa a periodically striated structure was detected at the subcommissural organ (SCO). It has been made a detailed analysis about its topographical relationship with vessels and with hypendyma, and also about its own periodical pattern of structure. This finding was also considered on a comparative point of view, keeping in mind the previous observations by some authors upon the rat's SCO and other related findings outside the central nervous system. Finally a functional interpretation is discussed.

The architecture of the basal region of the subcommissural organ (SCO) and the subjacent neuropil was studied in the brush-tailed possum, Trichosurus vulpecula (Marsupialia). Several structural features suggest that the basal mode of SCO-secretion may be as prominent as the well-established apical secretion. Some of the features that speak in favour of basal secretion are: (1) the existence of deep processes of secretion-laden SCO cells which reach and surround the capillaries in the hypendyma and the subjacent neuropil; (2) the presence of perivascular spaces, some of which may contain a material that resembles the secretory product of the SCO; (3) positive staining by means of paraldehyde-fuchsin of secretory material in the pericapillary zone of vessels in the hypendyma and its corresponding neuropil; (4) presence of labyrinths of the basal lamina of capillaries and associated nerves. The presence of nerve and other cell processes adjacent to the perivascular space, labyrinths and capillary wall suggests discharge into the capillaries of material such as the basal secretory product of the SCO. However, the absence of fenestrae in capillary endothelium in the context of the foregoing observations is enigmatic and speaks against the possibility of a conventional release of secretion. Nevertheless, it is possible that a secretory product containing particles of low molecular weight combined with some specific features of the local capillary endothelium, which facilitate transport, may be critical factors making possible transfer of such product(s) from the SCO to the capillaries.

The basal region of the subcommissural organ (SCO) was studied in the chicken Gallus gallus. The presence of concentric rough endoplasmic reticulum associated with lipid droplets in the basal processes of the ependymal cells, along with the increase of the lysosomal population, can be interpreted as evident signs of degeneration of the basal processes. The nearly absence of secretory processes ending on the meningeal blood vessels, suggests that the leptomeningeal route is missing. On the contrary, the presence of basal ependymal processes in contact with the hypendymal vessels could indicate that the ependymo-vascular pathway is well developed. In this sense, several structural features suggest a basal secretory mechanism from the SCO-cells: 1) Positive staining with paraldehyde-fuchsin of secretory material in the pericapillar region of hypendymal vessels and in the basal processes which reach the capillary wall; 2) presence of secretory material within the process endfeet in contact with hypendymal capillaries, and 3) existence of labyrinthis of the basal lamina of the capillaries.

Low-temperature-embedded tissue of the subcommissural organ (SCO) of the rabbit was analyzed for the basal route of secretory product by means of indirect immuno-metal cytochemistry (protein A-gold technique) at the electron-microscopic level. By use of (1) an antiserum against bovine Reissner's fibre (see Sterba et al. 1981) and, thereafter, (2) particulate gold-marker solution, immunoreactive sites could be clearly visualized within the extracellular matrix of both (a) the basal part of the ependymal cell layer, and (b) the hypendyma proper. Abundant secretory material was identified within (i) dilated intercellular spaces (a + b) as well as (ii) branching basal lamina labyrinths and distinct perivascular spaces (b). All these compartments are thought to belong to a system of extracellular channels, which may function in secretion directed toward hypendymal blood vessels.

Examination of neonatal Mongolian gerbil subcommissural organ (SCO) by freeze-fracture electron microscopy reveals that the epithelial layer (the ependyma) possesses distinctive and very complex tight junctions which connect the apical portions of adjacent ependymal cells. The tight junction network of the Mongolian gerbil is more extensive neonatally than in the adult, and is in general, more conspicuous than in other animal species. The consistent finding of such very complex tight junctions at the interface between the C.S.F. and the SCO suggests that the SCO ependyma, at least in neonatal Monglian gerbils, might function as a tight epithelium with an ability to maintain steep solute gradients and high potential differences. The sealing-off of the SCO towards the C.S.F. (the C.S.F.--SCO barrier) and the effective blood-brain barrier in the organ (the blood-SCO barrier) might well be of functional significance, perhaps in relation to mechanisms involved in the control and/or detection of the stability of the internal environment of the brain.

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 postnatal development of the global volume of the subcommissural organ (SCO) and of the karyometric changes of the ependymocytes in the SCO and the adjacent ventricle is studied in male albino mice aged from 25 to 160 days, and in a hypothyroid group treated with propylthiouracil with and without interruption of the treatment at 35 days. Hypothyroidism produces a decrease of the global volume of the SCO and of the nuclear size of the ependymocytes in the SCO and the adjacent ventricle.

Specific neurotoxic destruction of the serotoninergic innervation of the subcommissural organ of the rat is followed by an efficient reinnervation by collateral sprouting of non-monoaminergic axons, which normally do not innervate the SCO cells. Morphologically, the reinnervating fibres totally replace the serotoninergic synapses lost by the lesion, but, functionally, they fail to substitute for the potent inhibitory control of secretory activity normally exerted by the serotoninergic innervation. It is possible that the observed reinnervation by foreign synapses explains why the regrowing serotoninergic neurons fail to re-establish their connections with the SCO.

The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30 degrees C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981). Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The "periodically striated bodies", which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.

Secretory functions of the subcommissural organ (SCO) were investigated using mainly transmission and scanning electron microscopes, in 40 adult (19 males, 21 females) Trichosurus vulpecula which were caught in different seasons over several years. There is clear evidence of apical secretion, Reissner's fiber (RF), a presumptive basal secretory material with associated perivascular space and labyrinthine basal lamina. Secretory function does not appear to be related to the sex of the animal or to the season when they were investigated. Radioimmunoassay of blood, SCO and pineal gland (PIN) for melatonin (MEL) was carried out in 18 adult animals in summer and winter. Gonads were also histologically examined. The SCO showed very little MEL. Findings suggest that the PIN of this animal has a low capacity to store and secrete MEL. Further, there is no evidence for the increased storage or secretion of MEL during darkness, and more specifically at midnight. The present investigators support the view that other indoles such as methoxytryptophol or pineal peptides may elicit the physiological effects of PIN.

The subcommissural organ (SCO) of the golden hamster (Mesocricetus auratus) was studied by conventional electron microscopy, freeze-fracture technique, zinc-iodide-osmium (ZIO) and acid phosphatase cytochemical reactions. The ultrastructure of hamster SCO cells shows a few flattened cisternae of rough endoplasmic reticulum (ER) without dilated ones in the cytoplasm. The Golgi apparatus is very well developed. Freeze-fracture studies also indicate only short profiles of flattened ER in the cytoplasm endorsing the absence of dilated ER cisternae. After the treatment with ZIO mixture, reaction products were observed over flattened cisternae of the ER and the nuclear envelope. The Golgi apparatus was also reactive toward the ZIO mixture. Acid phosphatase activities are localized in the inner one or two saccules of the Golgi apparatus and dense bodies. From these results we suggest that (1) hamster SCO cells do not accumulate secretory material in the cytoplasm in the form of discrete secretory granules or dilated cisternae of ER, and (2) hamster SCO cells may possess extremely high secretory activity or may not be actively involved in secretory function at all as in rats or other rodents.