Morphological evidence for the presence of two cell types in the ependyma of the subcommissural organ of the snake,Natrix maura

Summary 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.     

Immunocytochemical study of the hypothalamic magnocellular neurosecretory nuclei of the snake Natrix maura and the turtle Mauremys caspica

Summary An immunocytochemical study of the magnocellular neurosecretory nuclei was performed in the snake Natrix maura and the turtle Mauremys caspica by use of antisera against: (1) a mixture of both bovine neurophysins, (2) bovine oxytocin-neurophysin, (3) arginine vasotocin, and (4) mesotocin. Arginine vasotocin- and mesotocin-immunoreactivities were localized in individual neurons of the supraoptic and paraventricular nuclei, with a distinct pattern of distribution in both species. The same cells appeared to be stained by the anti-oxytocin-neurophysin and anti-mesotocin sera. The supraoptic nucleus can be subdivided into rostral medial and caudal portions. In N. maura, but not in M. caspica, neurophysin-immunoreactive neurons were found in the retrochiasmatic nucleus. No immunoreactive elements were seen in the suprachiasmatic nucleus of both species after the use of any of the antisera. A dorsolateral aggregation of neurophysin-containing cells, localized over the lateral forebrain bundle, was present in both species. Magnocellular and parvocellular neurophysin-immunoreactive neurons were present in the paraventricular nucleus of both species. In the turtle, the paraventricular neurons were arranged into four distinct layers parallel to the ependyma; these neurons were bipolar with the major axis perpendicular to the ventricle, and many of them projected processes toward the cerebrospinal-fluid compartment. In N. maura a group of large neurons of the paraventricular nucleus was found in a very lateral position. The posterior lobe of the hypophysis and the external zone of the median eminence contained arginine vasotocin- and mesotocin-immunoreactive nerve fibers. The lamina terminalis of both species was supplied with a dense bundle of fibers containing immunoreactive neurophysin. Neurophysin-immunore-active fibers were also present in the septum, some telencephalic regions, including the cortex and the olfactory tubercule, in the paraventricular organ, and the periventricular and periaqueductal gray of the brainstem.This work was partially supported by a Grant S-85-39 from the Direccion de Investigaciones, Universidad Austral de Chile to E.M. Rodriguez     

The distribution of corticotropin-releasing factor-immunoreactive neurons and nerve fibers in the brain of the snake,Natrix maura

Summary The anatomical distribution of neurons and nerve fibers containing corticotropin-releasing factor (CRF) has been studied in the brain of the snake, Natrix maura, by means of immunocytochemistry using an antiserum against rat CRF. To test the possible coexistence of CRF with the neurohypophysial peptides arginine vasotocin (AVT) and mesotocin (MST) adjacent sections were stained with antisera against the two latter peptides. CRF-immunoreactive (CRF-IR) neurons exist in the paraventricular nucleus (PVN). In some neurons of the PVN, coexistence of CRF with MST or of CRF with AVT has been shown. Numerous CRF-IR fibers run along the hypothalamo-hypophysial tract and end in the outer layer of the median eminence. In addition, some fibers reach the neural lobe of the hypophysis. CRF-IR perikarya have also been identified in the following locations: dorsal cortex, nucleus accumbens, amygdala, subfornical organ, lamina terminalis, nucleus of the paraventricular organ, nucleus of the oculomotor nerve, nucleus of the trigeminal nerve, and reticular formation. In addition to all these locations CRF-IR fibers were also observed in the lateral septum, supraoptic nucleus, habenula, lateral forebrain bundle, paraventricular organ, hypothalamic ventromedial nucleus, raphe and interpeduncular nuclei.     

Immuno- and lectin-electron-microscopic investigation of the neural lobe of the hypophysis in the snake Natrix maura

Summary By the use of lectin histochemistry, and immunocytochemistry with antisera against bovine neurophysins I and II (NPs), arginine vasotocin (AVT) and mesotocin (MST), the neural lobe of the hypophysis in the snake Natrix maura was investigated at the light- and electron-microscopic levels. While paraldehyde-fuchsin stained virtually all neurosecretory endings, the periodic acid-Schiff reaction revealed only a portion of these elements. Furthermore, concanavalin-A and wheat-germ agglutinin lectins reacted with some but not all terminals. While in electron micrographs lectin-positive neurosecretory endings displayed medium-sized, pale neurosecretory granules, those from lectinnegative endings were larger and denser. The antiserum against the two NPs revealed the entire population of neurosecretory endings. The antiserum to AVT stained more numerous fiber elements than the antiserum to MST. Ultrastructurally, correlations concerning size and electron density can be found, on the one hand, between AVT-immunoreactive and lectin-positive neurosecretory granules and, on the other hand, between MST-immunoreactive and lectinnegative granules. The use of immuno-electron microscopy for the characterization of the different endings in the neural lobe and the presence of carbohydrates in some of them is discussed.This work was supported by the Directión General de Universidades e Investigación de la Junta de Andalucía (Grant BOJA 27/9/88) and the Direction General de Investigación Científica y Técnica (DGICYT Grant PB87 0710) Espaa     

Secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula): evidence for the existence of precursor and processed forms

The probable presence of oxytocin in the hypothalamo-hypophysial system of two reptilian species, the snake Natrix maura and the turtle Mauremys caspica, was re-investigated. A high-pressure liquid chromatographic analysis of the turtle neural lobe revealed the existence of vasotocin, mesotocin, and a third compound co-eluting with oxytocin. Brains from both species were fixed by vascular perfusion with Bouin's fluid. Adjacent paraffin sections were immunostained using antisera against the following substances: (1) bovine oxytocin-neurophysin; (2) a mixture of bovine oxytocin-neurophysin and vasopressin-neurophysin; (3) dogfish neurophysins; (4) oxytocin; (5) arginine-vasotocin; (6) mesotocin; (7) somatostatin. Immunoreactivity against oxytocin was found in parvocellular neurons of the snake suprachiasmatic nucleus and cerebrospinal-fluid contacting neurons of the medial nucleus of the infundibular recess of both species, the latter immunoreactivity being much more conspicuous in the turtle. Numerous fibers containing immunoreactive oxytocin extended between the medial nucleus of the infundibular recess, and the internal region of the medium eminence and the neural lobe. The oxytocin-immunoreactivity in all locations was completely abolished by preabsorption of the anti-oxytocin serum with three different oxytocin preparations. None of the neurons of the suprachiasmatic and medial nucleus of the infundibular recess, including the oxytocin-immunoreactive elements, reacted with either the antineurophysin sera used, or the anti-vasotocin or anti-mesotocin antibodies. The possible existence of a reptilian oxytocin-neurophysin is discussed. The alternative that, in the reptilian hypothalamus, neurons synthesize a compound closely related to, but different from oxytocin is also considered.     

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.     

Evidence for basal secretion in the subcommissural organ of the adult rabbit

Summary Morphological evidence is presented supporting the possibility of basal secretion into hypendymal capillaries of the adult rabbit subcommissural organ (SCO). The synthetic apparatus of the SCO cell is described as well as the heterogeneous granules and vesicles which are concentrated in the basal processes bordering a widened perivascular space. The origin of the electron dense granules, of which two fairly distinct subgroups are found, is discussed.A binding of secretory sacs to the lateral plasma membrane is seen. The possibility of a lateral secretion is supported by the presence of a system of extracellular channels between SCO cells which are filled with a flocculent material resembling that of the secretory sacs.Nerve perikarya which are separated from the SCO by only a few glial fibers are demonstrated. Synapses are described in nerve fascicles bordering on the hypendymal capillaries. The possibility of an innervation of the hypendymal region is discussed as well as possible nervous connections with the pineal gland.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen.     

Synapse-like contacts between axons of the pineal tract and the subcommissural organ in Rana perezi (Anra) and their absence in Carassius auratus (Teleostei): ultrastructural tracer studies

The present study was designed to investigate the controversial subject of the existence of a neural input from the pineal organ via the pineal tract to the subcommissural organ (SCO) in teleosts and anurans. Horseradish peroxidase was injected into the pineal organ and pineal tract of Carassius auratus and Rana perezi. Within the pinealofugal fibers the tracer was visualized at the light-and electron-microscopic levels either by immunocytochemistry using an anti-peroxidase serum, or by revealing the enzymatic activity of peroxidase. In both species, labeled myelinated and unmyelinated fibers of the pineal tract were readily traced by means of electron microscopy. In R. perezi, numerous terminals contacting the SCO cells in a synapse-like (synaptoid, hemisynaptic) manner bore the label, whereas a different population of endings was devoid of the tracer, indicating that in this species the SCO receives a dual neural input, one of pineal origin, the other of unknown source and nature. In the SCO of C. auratus, neither labeled nor unlabeled synapse-like contacts were found. Thus, in this latter species, a direct neural input to the SCO is missing. It is concluded that the secretory activity of the SCO can be controlled by different mechanisms in different species, and that more than one neural input mechanism may operate in the same species.     

Spatial and structural interrelationships between secretory cells of the subcommissural organ and blood vessels

Summary In 76 specimens (amphibians, reptilians, mammals) belonging to 25 different vertebrate species, the region of the subcommissural organ (SCO) was investigated with the use of a primary antiserum raised against an extract of bovine Reissner's fiber+the immunoperoxidase procedure according to Sternberger et al. (1970).In the SCO of a toad (Bufo arenarum) and several species of reptiles (lacertilians, ophidians, crocodilians), the ependymal cells were the only type of secretory cell displaying vascular contacts, whereas in mammals ependymal and hypendymal cells established intimate spatial contacts with blood vessels. In Bufo arenarum, but especially in the reptilian species examined, the ependymo-vascular relationship was exerted by a population of ependymal cells having a rather constant location within the SCO and projecting to capillaries that showed a remarkably constant pattern of anatomical distribution. In the SCO of mammals the modality and degree of the structural relationships between secretory cells and blood vessels varied greatly from species to species. In the SCO of the armadillo and dog the secretory tissue was organized as a thick, highly vascularized layer with most of the cells oriented toward the capillaries. A rather opposite situation was found in the SCO of New-and Old-World monkeys, where vascular contacts were restricted to a few ependymal cells.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidal Austral de Chile     

Light and electron-microscopic immunocytochemistry and lectin histochemistry of the subcommissural organ: Evidence for processing of the secretory material

Summary 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 11000 to 1200 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 moeities 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.Supported by Grant I/38 259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Direction de Investigaciones, Universidad Austral de Chile. The authors wish to thank Mrs. Elizabeth Santibáñez, Mr. Genaro Alvial and Mr. Luis Delannoy (Valdivia), and Mrs. Ragnhild Momberger (Giessen) for valuable technical cooperation     

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species

Summary The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.     

Colloid droplets in the magnocellular secretory neurons of the reptilian hypothalamus: An immunocytochemical and lectin-histochemical study

Summary The immunocytochemical and lectin-binding properties of the magnocellular neurosecretory neurons in the hypothalamus of 2 reptilian species, the snake Natrix maura and the lizard Liolaemus cyanogaster, were investigated. Particular attention was paid to the secretory droplets present in these neurons. Antisera against bovine neurophysins I+II, arginine-vasotocin, and mesotocin were used. The following lectins were applied: concanavalin A (Con A), wheat-germ agglutinin (WGA), and Limax flavus agglutinin (LFA). Adjacent 1-m-thick methacrylate sections were used to investigate the same secretory neuron and the same colloid droplets with all three antisera and all three lectins. Several sections were treated with trypsin and urea before immunostaining or lectin binding. Con A bound to both vasotocin- and mesotocin-immunoreactive neurons, WGA exclusively to vasotocin neurons; neither of these neurons reacted with LFA. The colloid droplets were present in vasotocin neurons but absent in the mesotocin neurons. These secretory droplets showed an affinity for Con A but not for WGA, and reacted with antisera against neurophysins and vasotocin. In Natrix maura, the colloid droplets became reactive with Con A and the antisera used only after pretreatment of the sections with trypsin and urea. Within the hypothalamo-neurohypophyseal system, antiserum against vasotocin and WGA revealed the same fiber bundles. It is concluded (i) that in reptiles the vasotocin-neurophysin precursor is glycosylated, (ii) that vasotocin neurons have the exclusive capacity to form colloid droplets, and (iii) that these droplets are an intracisternal (RER) storage form of the vasotocin-neurophysin precursor.This work was partially supported by Grants BOJA 27/9/88 from the Dirección General de Universidades e Investigación de Junta de Andalucía and DGICYT PB87-0710 from the Comisión Interministerial de Ciencia y Tecnología, Madrid, to P.F.-LL.; and Grant 89-01 from the Dirección de Investigaciones, Universidad Austral de Chile, to E.M.R.     

Electron microscopic study of the origin and formation of Reissner's fiber in the subcommissural organ of Cebus apella (Primates,Platyrrhini)

Summary 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.     

The subcommissural organ of the adult and pouch-young brush-tailed possum (Trichosurus vulpecula)

Summary Scanning electron microscopy (SEM) was used to examine the surface features of the subcommissural organ (SCO) and the roof of the cerebral aqueduct of 35 adult and 33 pouch-young Trichosurus vulpecula. The animals were at various developmental stages and of both sexes. In the adult animals, the surfaces of the groove and the adjacent medial walls of the ridges of the SCO were characterized primarily by microvilli. Typically, Reissner's fibre was associated closely with the median groove of the SCO. The ridges and the paramedian grooves of the SCO were often heavily ciliated displaying many cerebrospinal fluid-contacting nerve processes. These processes were of varying lengths with terminal and preterminal varicosities. The observed morphology supports a hypothesis suggesting that under certain physiological and pathological conditions the flow of CSF may be directed away from the heavily ciliated ridges to the poorly ciliated groove containing the Reissner's fibre. In the youngest pouch-young animals there were no cilia, CSF-contacting nerve processes, nor supraependymal cells. Also the surface features of the SCO in the young assumed adult appearance before the adjacent roof of the cerebral aqueduct. These findings suggest the possibility that the SCO begins to function early in ontogenetic development. Acknowledgement. Technical assistance of Mrs. G. Hermanis is gratefully acknowledged.The author thanks the Director of Wild Life and National Parks, South Australia, for permission to use brush-tailed possums     

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.     

Comparison of several parameters related to the secretory activity of the subcommissural organ in European green frogs

Summary In European green frogs the secretory activity of the subcommissural organ (SCO) was investigated and quantified measuring three parameters considered to be closely related to the cellular processes of synthesis and release of secretory material by the cells of the SCO: (1) the amount of stained secretory material in the SCO; (2) the amount of secretory material in the SCO labelled by a radioactive precursor; and (3) the growth rate of the liquor (cerebrospinalis) fibre (LF). A significant negative linear correlation appears to exist between the growth rate of the LF, on the one hand, and the amount of stained secretory material as well as the amount of radioactively labelled secretory material, on the other hand. A significant positive linear correlation exists between the amounts of stained material and radioactively labelled secretory material. The occurrence in the SCO of European green frogs of a larger amount of stained and/or of radioactively labelled secretory material is probably an expression of a lower (LF-producing) secretory activity. In the light of these observations the suitability of the three parameters as a measure of the secretory activity of the SCO is discussed.     

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.