Fine structural and cytochemical studies on the hamster subcommissural organ

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.     

Electron microscopic and cytochemical studies of the mouse subcommissural organ

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

Freeze-fracture replica studies of tight junctions in normal human bronchial epithelium

Using freeze-fracture techniques, tight junctional networks were observed in the human normal bronchial epithelium. They were morphologically classified into three types: type I was a loosely interconnected, most complicated network consisting of 7-11 roughly parallel wavy strands and situated between ciliated cells; type II was a randomly anastomosing, simple network made up of 2-4 strands and present between goblet cells; type III was an irregularly anastomosing network composed of 4-7 strands and located between a ciliated cell and a goblet cell. Type III junctions, when a goblet cell was strongly bulged, were located on the swollen ridge, the upper surface of which was separated by a deep groove from the bulged apical surface, around the lateral surface of the cell at the level of the luminal surface. The possible relation between the orientation of strands of these networks and extra- or intracellular stress was discussed.     

Studies on the subcommissural organ of Salmo gairdneri

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

The secretion of the subcommissural organ

Summary The secretion of the subcommissural organ (SCO) has been studied immunocytochemically by use of the following antisera: (1) antiserum against an aqueous extract of bovine Reissner's fiber (RF), (anti-RF-DC antiserum); (2) antiserum against the protein fraction F₁ obtained by gel electrophoresis of the aqueous RF-extract (RF-DF₁-antiserum); (3) antiserum against the protein fraction F₂ prepared in the same manner (RF-DF₂-antiserum). As shown by immunological and/or immunocytochemical experiments in bovines and rats, the three antisera are of high specificity, i.e., react exclusively with the secretion of the SCO, which appears to be a unique product of the vertebrate organism. Concerning the distribution of the reaction products within the SCO-cells, no differences were found light microscopically after use of the RF-DC-antiserum, the RF-DF₁-antiserum, or the RF-DF₂-antiserum.Comparative studies were carried out with the RF-DC-antiserum only. A positive immunocytochemical reaction of the SCO-secretion was obtained in many vertebrate species (mammals, birds, reptiles, amphibians, bony fishes, sharks, and cyclostomes). RF gives a positive reaction in mammals only; to date RF of non-mammalian vertebrates did not react immunocytochemically with the present antiserum. Comparative immunocytological studies have shown that (1) the SCO-cells of the ependymal layer as well as the SCO-cells of the hypendymal layer contain immunoreactive material, (2) in the majority of vertebrates hypendymal structures are more common than has been previously supposed, and (3) RF or constituents of this structure are produced by the SCO. The immunocytochemical studies have led to the impression that the SCO-secretion is not only discharged into the cerebrospinal fluid, but also in hypendymal vessels and/or leptomeningeal spaces, as has been postulated previously by Oksche and others (for review, see Oksche 1969).Supported by grants from the Ministry for Science and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. M. Eigenbrod, Mrs. D. Naumann and Mrs. B. Wolff is gratefully acknowledgedDedicated to Professor Berta Scharrer on the occasion of her 75th birthday     

Histochemical investigations on the human foetal subcommissural organ

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

Enzymatic organization of the subcommissural organ.

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...     

Vascular and leptomeningeal projections of the subcommissural organ in reptiles

Summary 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), periodicacid Schiff (PAS), periodic-acid silver-methenamine (PASM), 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 by 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.This work was partially supported by grants from the Stiftung Volkswagenwork, Federal Republic of Germany (1/38259), from the Dirección de Investigaciones, Universidad Austral de Chile (S-85-39), and from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile (6027; all to E.M.R.)     

Immunocytochemical investigation of the subcommissural organ in the rat

Summary [¹⁴C]2-deoxyglucose uptake by neurons located in the octavo-lateralis complex of adult flatfish is asymmetrical on the two sides of the brain. It appears that the neuronal activity on the side oriented upward is higher than that on the side of the brain facing downward. This finding may be significant with respect to the mechanisms of metamorphosis of flatfish and may account for the peculiar fact that these animals swim on one body side during adult live.     

Ontogenetical development of the chick and duck subcommissural organ

Summary The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day of incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used.In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 i) blood vessels penetrated the SCO, ii) scarce hypendymal cells appeared, iii) the first signs of ventricular release of IRM were noticed, iv) appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct; day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 i) hypendymal cells proliferated, developed processes and migrated dorsally, ii) ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. The duck SCO appears to follow a similar pattern of development.Supported by Grant I/60 935 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile. M.H. was recipient of a personal grant from JNO (29-5-54), which is gratefully acknowledged     

Freeze-fracture studies on tardigrade cuticle

The cuticles of the heterotardigrade Echiniscus testudo and the eutardigrades Macrobiotus hufelandi and Milnesium tardigradum have been studied using freeze-fracture technique. Most of the layers seen in conventional TEM micrographs can be visualized. There is no clear evidence that the trilaminar components of the cuticle such as the outer epicuticle and the tripartite layer separating epi- and intracuticle or procuticle (whose membranous origin has been suggested by previous authors) fracture like a lipid bilayer. Microfibres not resolved or only poorly resolved by TEM can be recognized in the procuticle of all three species. Obviously their visualization depends upon the fracture angle. In Echiniscus testudo and Milnesium tardigradum the intracuticle or at least parts of it show a wavy arrangement of microfibres. Parts of the ventral intracuticle of E. testudo fracture in an obviously non-random pattern revealing distinct sublayers.     

Complex-type glycoproteins synthesized in the subcommissural organ of mammals

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

Lectin histochemistry of the human fetal subcommissural organ

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

The subcommissural organ of Lacerta s. sicula Raf.: Functional studies

Summary 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.This work was presented at the First Colloquium of the European Pineal Study Group, November 20–24, 1978, in Amsterdam