Reissner's fiber,massa caudalis and ampulla caudalis in the spinal cord of lamprey larvae (Geotria australis)

Summary The subcommissural organ (SCO), Reissner's fiber (RF) and its massa caudalis of lamprey larvae (Geotria australis) were investigated immunocytochemically by use of an antiserum raised against bovine RF as primary antibody. The affinities of RF and massa caudalis for Ricinus communis agglutinin I (RCA) with and without previous acid hydrolysis, concanavalin A (Con A), wheat-germ agglutinin (WGA), aldehyde fuchsin, and PAS reaction were also studied.SCO and massa caudalis were strongly immunoreactive, whereas RF proper was distinctly negative. RF did not react with Con A and RCA. Only the periphery of RF was WGA-positive. RCA showed affinity for RF only after acid hydrolysis. RF was homogeneously stained by the aldehyde-fuchsin and PAS-methods. At variance with RF proper, the periphery of the massa caudalis reacted with RCA without previous acid hydrolysis, but its core was WGA-positive and reacted with RCA only after hydrolysis. It is suggested that (i) RF has a coat of glycoproteins containing sialic acid as terminal residue, whereas the massa caudalis possesses a coat with galactose as terminal residue; (ii) in RF proper and the massa caudalis the spatial arrangement of glycoproteins might be different.Routine transmission electron-microscopic observations indicate that in larvae of Geotria australis an open communication exists between the ampulla caudalis and blood capillaries via large cavities or lacunae.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, and Grant 6027 from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile     

The caudal spinal cord of coho salmon (Oncorhynchus kisutch): Immunocytochemical evidence of a “caudal serotoninergic system”

Summary The caudal spinal cord of the coho salmon was investigated by means of immunocytochemistry using antisera against serotonin, urotensin I, urotensin II, somatostatin and a urea-extract of bovine Reissner's fiber (AFRU). Populations of serotonin-immunoreactive (IR) neurons were found rostral and dorsal to the urophysis in close spatial association with caudal secretory neurons. Thick, smooth serotonin-IR processes extended toward the external surface of the spinal cord where they displayed conspicuous terminal dilatations. Thin, beaded serotonin-IR fibers appeared to innervate populations of caudal secretory and somatostatin-IR cerebrospinal fluid-contacting neurons. Most caudal neurosecretory cells displayed both urotensin I and urotensin II immunoreactivities; only a minority reacted exclusively with either urotensin I or urotensin II antisera. Urotensin II-IR and somatostatin-IR cerebrospinal fluid (CSF)-contacting neurons were found as an integral component of the central canal wall in the caudal spinal cord and filum terminale; their dendritic processes appeared to contact Reissner's fiber, which displayed a weak AFRU-immunoreactivity while inside the central canal, but became strongly reactive in the interior of the terminal ventricle as it formed the massa caudalis. The distribution of serotoninergic processes points to a regulatory role in the function of caudal secretory and CSF-contacting neurons and to a putative serotonin release into the subarachnoid space and/or meningeal vasculature. It is also suggested that the CSF-contacting neurons of the central canal may participate in a feedback mechanism controlling the secretory activity of the subcommissural organ.Supported by Grant A/1095-1 from the International Foundation for Science, Sweden, to C.Y.; Grant I/63-476 from Volkswagen-Stiftung to E.R.; and Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile     

Light- and electron-microscopic investigation of the rat subcommissural organ grafted under the kidney capsule,with particular reference to immunocytochemistry and lectin histochemistry

Summary There is increasing evidence that, in the rat, a serotonin-mediated neural input may have an inhibitory influence on the secretory activity of the subcommissural organ (SCO). In the present investigation the rat SCO was studied 7, 30 and 90 days after transplantation under the kidney capsule, an area devoid of local serotonin-containing nerves. The grafted tissue was examined by use of immunocytochemistry employing a series of primary antisera, lectin histochemistry and transmission electron microscopy. The grafted SCO survived transplantation and contained, in addition to secretory ependymal and hypendymal SCO-cells, also elements immunoreactive with antisera against glial fibrillary acidic protein or S-100 protein. In transplants, SCO-cells produced a material displaying the characteristic immunocytochemical and lectin-binding properties of SCO-cells observed under in-situ conditions. The ependymal cells lined 1–3 small cavities, which contained secretory material. A fully developed structural equivalent of Reissner's fiber was, however, never found. The immunocytochemical and ultrastructural study of the grafted SCO showed an absence of nerve fibers within the graft and suggested a state of enhanced secretory activity. A network of protruding basal lamina structures connected the secretory cells to the newly formed capillaries revascularizing the SCO. One week after transplantation, long-spacing collagen started to appear in expanded areas of such laminar networks and also in the perivascular space. It is suggested (i) that the formation of long-spacing forms of collagen is triggered by factors provided by the SCO-secretory cells, and (ii) that secretory material of the ependymal and hypendymal cells may reach the reticular extensions of the basal lamina. In contrast to the SCO in situ, the grafted SCO-cells showed a positive immunoreaction for neuron-specific enolase. They became surrounded by a S-100-immunoreactive glial sheath that separated them from other transplanted cell types and the adjacent kidney tissue of the host.Supported by Grant I/63 476 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grants 187 and 0890/88 from Fondo Nacional de Desarrollo Cientifico y Tecnológico, Chile, and Grant S-85-39 from the Directión de Investigaciones, Universidad Austral de Chile. The authors wish to acknowledge the valuable help of Ms. Elizabeth Santibañez and Mr. Genaro Alvial (Valdivia) and Ms. Inge Lyncker (Giessen)     

Comparative immunocytochemical study of the subcommissural organ

Summary The subcommissural organs (SCO) of 76 specimens belonging to 25 vertebrate species (amphibians, reptiles, birds, mammals) were studied by use of the immunoperoxidase procedure. The primary antiserum was obtained by immunizing rabbits with bovine Reissner's fiber (RF) extracted in a medium containing EDTA, DTT and urea. Antiserum against an aqueous extract of RF was also produced. The presence of immunoreactive material in cell processes and endings was regarded as an indication of a possible route of passage. Special attention was paid to the relative development of the ventricular, leptomeningeal and vascular pathways established by immunoreactive structures.The SCO of submammalian species is characterized by (i) a conspicuous leptomeningeal connection established by ependymal cells, (ii) scarce or missing hypendymal cells, and (iii) a population of ependymal cells establishing close spatial contacts with blood vessels.The SCO of most mammalian species displays the following features: (i) ependymal cells lacking immunoreactive long basal processes, (ii) hypendymal secretory cells occurring either in a scattered arrangement or forming clusters, (iii) an occasional leptomeningeal connection provided by hypendymal cells, and (iv) in certain species numerous contacts of secretory cells with blood vessels. In the hedgehog immunoreactive material was missing in the ependymal formation of the SCO, but present in hypendymal cells and in the choroid plexuses. The SCO of several species of New-and Old-World monkeys displayed immunoreactive material, whereas that of anthropoid apes (chimpanzee, orangutan) and man was completely negative with the antisera used.Supported by Grant I/38 259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RR-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile.The authors wish to thank Mrs. Elizabeth Santibánez and Mr. Genaro Alvial for valuable technical cooperation, and Dr. P. Fernandez-Llebrez, University of Malaga, for providing the specimens of Natrix maura.     

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     

Immunocytochemical detection of Reissner's fiber-like glycoproteins in the subcommissural organ and the floor plate of wildtype and cyclops mutant zebrafish larvae

The subcommissural organ (SCO) and the floor plate (FP) secrete high molecular weight glycoproteins that polymerize in the form of the Reissner's fiber (RF). To study to what extent the absence of the FP affects the expression of these glycoproteins, we have investigated the brain and spinal cord of 48-h and 72-h wildtype and cyclops (cyc) mutant zebrafish larvae by using a polyclonal antiserum against bovine RF. Wildtype larvae showed immunoreactivity in the SCO at the dorsal forebrain-midbrain boundary. In the ventricle, over the SCO surface, thin immunoreactive fibers aggregated into an RF that ran along the third and fourth ventricles and the central canal of the spinal cord until, at its caudal end, the fiber disintegrated and formed a strongly immunoreactive massa caudalis that left the neural tube and invaded the surrounding tissues of the tail fin. The rostral end of the FP, lining the pontine flexure, was also strongly immunoreactive, as was the caudal third of the FP. Cyc mutants showed an immunoreactive SCO and fibrous material in the ventricle, but an RF was missing. There was no label in the ventral midline of the neural tube except in some specimens in which the caudal FP persisted and was immunoreactive. It is concluded that the product of the cyc gene is not required for the expression of SCO glycoproteins but for their polymerization into an RF in the brain ventricles.     

Pinealocytes immunoreactive with antisera against secretory glycoproteins of the subcommissural organ: A comparative study

Summary By means of light-microscopic immunocyto-chemistry two polyclonal antibodies (AFRU, ASO; see p. 470) directed against secretory glycoproteins of the subcom-missural organ were shown to cross-react with cells in the pineal organ of lamprey larvae, coho salmon, a toad, two species of lizards, domestic fowl, albino rat and bovine (taxonomic details, see below). The AFRU-immunoreactive cells were identified as pinealocytes of the receptor line (pineal photoreceptors, modified photoreceptors or classical pinealocytes, respectively) either due to their characteristic structural features or by combining AFRU-immunoreaction with S-antigen and opsin immunocytochemistry in the same or adjacent sections. Depending on the species, AFRU- or ASO-immunoreactions were found in the entire perikaryon, inner segments, perinuclear area, and in basal processes facing capillaries or the basal lamina. In most cases, only certain populations of pinealocytes were immunolabeled; these cells were arranged in a peculiar topographical pattern. In lamprey larvae, immunoreactive pinealocytes were observed only in the pineal organ, but not in the parapineal organ. In coho salmon, the immunoreaction occurred in S-antigen-positive pinealocytes of the pineal end-vesicle, but was absent from S-antigen-immunoreactive pinealocytes of the stalk region. In the rat, AFRU-immunoreaction was restricted to S-antigen-immunoreactive pinealocytes found in the deep portion of the pineal organ and the habenular region. These findings support the concept that several types of pinealocytes exist, which differ in their molecular, biochemical and functional features. They also indicate the possibility that the AFRU- and ASO-immunoreactive material found in certain pinealocytes might represent a proteinaceous or peptidic compound, which is synthesized and released from a specialized type of pinealocyte in a hormone-like fashion. This cell type may share functional characteristics with peptidergic neurons or paraneurons.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, to E.M.R. and A.O.; Grant S-85-39 from the Direccion de Investigaciones, Universidad Austral de Chile, to E.M.R.; Grant 187 from FONDECYT, Chile, to C.R.Y.; and Grant Ko 758/3-1 from the Deutsche Forschungsgemeinschaft, Federal Republic of Germany, to H.W.K.     

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     

The terminal organ of the subcommissural complex of chordates: definition and perspectives

The subcommissural organ (SCO) exhibits anatomical characteristics of an endocrine organ: The secretion is released either into the blood (hypendymal capillaries) or the CSF of the 3rd ventricle; excretory ducts are absent; the active secretory activity of the ependymal cells can be regulated by humorally transmitted messages or by neural input. The rate of production of the Reissner's fibre (RF) by the SCO is rather fast, and the secretory material is stored in the ampulla caudalis (AC) and must be continuously discharged accordingly. Structures jointly involved in depletion of the AC and the decomposition and removal of the massa caudalis (MC) are collectively called the terminal organ (TO). The TO of the SCO-complex is formed by an assemblage of different structures in the caudal segment of the spinal cord (neurogenic part) and in the tissues (non-neurogenic part) which encompass this part of the cord. The different parts of the TO are characterized, even at the cellular level, by specializations which support the discharge as well as the dissolution of the material of the MC. The RF may be a detoxicator for the CSF, but also a carrier of hormonally active substances. In this case the TO is a site of release of hormones. The function of the entire complex is still under discussion, particularly its role in endocrine integration.     

Immunocytochemical and ultrastructural evidence for a neurophysinergic innervation of the subcommissural organ of the snake Natrix maura

Summary 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), -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.This work was partially supported by Grants 1/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, conceded to Esteban M. Rodríguez     

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     

Preliminary electronmicroscopical observations on the ampulla caudalis and the discharge of the material of Reissner's fibre into the capillary system of the terminal part of the tail of Ammocoetes (Agnathi)

The discharge of the material of the Reissner's fibre (RF) and the massa caudalis (MC) into the "meningeal spaces" has until now not been studied in detail and with the aid of the electron-microscope. It was generally assumed that the material of the MC disintegrates in the "meningeal spaces", but the nature and function of those spaces have not been established. The CNS of Ammocoetes ist encompassed by the meninx primitiva, which is the meningeal tissue of the lower vertebrates; the meninx does not differentiate during ontogeny into the pia mater and the arachnoides. In contrast, the meninx primitiva of mammals is the anlage in the ontogenetical sense of the leptomeninges in adult individuals. The results presented in this paper are valid for Ammocoetes only; Petromyzon and Myxine must be studied anew from a perspective which may be the result of our study. Of particular interest would be an anatomical analysis of these caudalmost structures of the CNS (Ampulla caudalis [AC], MC, neuropori, lacunae etc.) in Branchiostoma. The diameter of the RF of Ammocoetes is approximately 1.7 micron; this is not different from the diameter of the RF in adult individuals. In the AC the RF divides into several smaller fibres which eventually disintegrate and form the amorphous MC. In sagittal sections, the fibrillary structure of the RF is distinct. In cross sections the small fibres may appear as globules. The terminal part of the RF is very often coiled. The ependyma of the caudal part of the canalis centralis (CC) ist not remarkable; liquor contacting neurons are frequently found with their dendrites touching the RF. Liquor contacting neurons are never found in the ependyma of the AC. The ependymal cells of the dorsal part of the AC possess neither kinocilia or microvilli. The disappearance of these organelles begins in the dorsal part of the caudalmost segment of the CC. Most surprising is the distribution of the attachment devices which are probably desmosomes. In the ependyma of the caudal part of the spinal cord the attachment devices are typically situated; in the ependyma of the AC these devices are absent. We use the general term "attachment devices" until the nature of these devices has been clearly determined. In the caudalmost part of the spinal cord as well as in the AC, the intercellular spaces between the ependymal cells communicate, forming a 3-dimensional labyrinth. If there are no attachment devices between the proximal parts of the ependymal cells, the intercellular spaces may provide a passageway between the CC and the loose tissue of the meninx primitiva.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.)     

Secretory glycoproteins of the rat subcommissural organ are N-linked complex-type glycoproteins. Demonstration by combined use of lectins and specific glycosidases,and by the administration of tunicamycin

Summary Two experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO). Protocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouin's fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, -galactosidase, -mannosidase, -glucosidase and -N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissner's fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissner's fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunicamycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).Supported by Grant I/63-476 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-89-01 from the Dirección de Ivestigaciones, Universidad Austral de Chile, and Grant 0890/88 from FONDECYT, Chile     

Identification of Reissner's fiber-like glycoproteins in two species of freshwater planarians (Tricladida), by use of specific polyclonal and monoclonal antibodies

By using one polyclonal antiserum raised against bovine Reissner's fiber and seven monoclonal antibodies raised against bovine Reissner's fiber and against immunopurified bovine subcommissural organ glycoproteins, we have investigated two freshwater planarian species (Girardia tigrina, Schmidtea mediterranea) by light- and electron-microscopic immunocytochemistry. ELISA probes showed that the monoclonal antibodies recognized different, nonoverlapping, unrepeated, proteinaceous epitopes present in the same compounds of bovine Reissner's fiber. Cells immunoreactive to the polyclonal and monoclonal antibodies were found in the dorsal and ventral integument of both planarian species. Labeled cuboid epidermal cells bore cilia and displayed several types of secretory granules; they were covered by a film of immunoreactive material. Studies on adjacent thin and semithin sections revealed coexistence of label in the same regions and in the same cells when two different monoclonal antibodies were used. These results indicate that a secretory substance immunologically similar to the secretion of the vertebrate subcommissural organ is present in primitive tripoblasts such as planarians, suggesting that these secretions are ancient and well conserved in phylogeny.     

Light and electron microscopic immunocytochemical localization of thyroglobulin in the thyroid gland of the anadromous sea lamprey,Petromyzon marinus L., during its upstream migration

Summary Antibodies made against thyroglobulin (TG) were used in an immunocytochemical study for the light and electron microscopic localization of TG in the thyroid gland of the anadromous sea lamprey, Petromyzon marinus, during its upstream migration. TG was found in the follicular lumen and in some colloid droplets within the follicular cells. Except for an immunoreactive product observed in a small portion of the interstitial connective tissue, the location of TG in the lamprey was similar to that in the thyroid of the rat.Supported by National Research Council of Canada Grant no. A5945 to J.H.Y. We thank Dr. F.W.W. Beamish and Mr. R. Robinson who helped in the capture of the lamprey     

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.     

Intercellular channels in the pars tuberalis of the rat hypophysis and their relationship to the subarachnoid space

Summary A system of intercellular channels is described in the pars tuberalis (PT) of the female rat. These spaces are lined by all types of cells found in the PT and are not sealed off by tight junctions. Ventrally and dorsally, the intercellular spaces open toward the basement membranes separating the PT from (i) the subarachnoid space, and (ii) the perivascular space of the portal capillaries, respectively. These intercellular channels differ from the follicles, which are also found in the PT, being lined by a particular type of cell.In a second group of female rats an epoxy mixture was injected into the third ventricle; 10 min thereafter horseradish peroxidase was infused into the cisterna magna. After processing the brain for the demonstration of exogenous peroxidase, it was found that the tracer had reached the subarachnoid space adjacent to the hypothalamus and entered into all ventricular cavities with the exception of the infundibular recess. Under these experimental conditions it was found that the tracer fills all intercellular channels of the PT, thus indicating that there is no barrier between the subarachnoid space and the PT. It is suggested that the subarachnoid space should be regarded as a probable route for the transport of trophic factor(s) and/or secretory product(s) of the PT.Supported by Grant S-80-13 from Directión de Investigaciones, Universidad Austral de Chile     

Immuno-electron-microscopic analysis of the basal route of secretion in the subcommissural organ of the rabbit

Summary 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.Supported by Grants from the Ministry for Sciences and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. S. Mehnert, Mrs. E. Siebert, Mrs. Ch. Schneider, Mrs. I. Seifert and Mr. H. Wolf is gratefully acknowledgedDedicated to Prof. Dr.Dr.h.c. Andreas Oksche on the occasion of his 60th birthday     

Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal cord

Summary Reissner's fiber (RF) of the subcommissural organ (SCO), the central canal and its bordering structures, and the filum terminale were investigated in the bovine spinal cord by use of transmission electron microscopy, histochemical methods and light-microscopic immunocytochemistry. The primary antisera were raised against the bovine RF, or the SCO proper. Comparative immunocytochemical studies were also performed on the lumbo-sacral region of the rat, rabbit, dog and pig.At all levels of the bovine spinal cord, RF was strongly immunoreactive with both antisera. From cervical to upper sacral levels of the bovine spinal cord there was an increasing number of ependymal cells immunostainable with both antisera. The free surface of the central canal was covered by a layer of immunoreactive material. At sacral levels small subependymal immunoreactive cells were observed. From all these structures sharing the same immunoreactivity, only RF was stained by the paraldehyde-fuchsin and periodicacid-Schiff methods.At the ultrastructural level, ependymal cells with numerous protrusions extending into the central canal were seen in the lower lumbar segments, whereas cells displaying signs of secretory activity were principally found in the ependyma of the upper sacral levels. A few cerebrospinal fluid-contacting neurons were observed at all levels of the spinal cord; they were immunostained with an anti-tubulin serum.The lumbo-sacral segments of the dog, rat and rabbit, either fixed by vascular perfusion or in the same manner as the bovine material, did not show any immunoreactive structure other than RF.The possibilities that the immunoreactive ependymal cells might play a secretory or an absorptive role, or be the result of post-mortem events, are discussed.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de ChileThe authors wish to thank Dr. Enrique Romeny from the Valdivia abattoir for kindly providing the bovine spinal cords