Bovine Reissner Research Articles

The central nervous system of sea cucumbers (Echinodermata: Holothuroidea) shows positive immunostaining for a chordate glial secretion.

BackgroundEchinoderms and chordates belong to the same monophyletic taxon, the Deuterostomia. In spite of significant differences in body plan organization, the two phyla may share more common traits than was thought previously. Of particular interest are the common features in the organization of the central nervous system. The present study employs two polyclonal antisera raised against bovine Reissner's substance (RS), a secretory product produced by glial cells of the subcomissural organ, to study RS-like immunoreactivity in the central nervous system of sea cucumbers.ResultsIn the ectoneural division of the nervous system, both antisera recognize the content of secretory vacuoles in the apical cytoplasm of the radial glia-like cells of the neuroepithelium and in the flattened glial cells of the non-neural epineural roof epithelium. The secreted immunopositive material seems to form a thin layer covering the cell apices. There is no accumulation of the immunoreactive material on the apical surface of the hyponeural neuroepithelium or the hyponeural roof epithelium. Besides labelling the supporting cells and flattened glial cells of the epineural roof epithelium, both anti-RS antisera reveal a previously unknown putative glial cell type within the neural parenchyma of the holothurian nervous system.ConclusionOur results show that: a) the glial cells of the holothurian tubular nervous system produce a material similar to Reissner's substance known to be synthesized by secretory glial cells in all chordates studied so far; b) the nervous system of sea cucumbers shows a previously unrealized complexity of glial organization. Our findings also provide significant clues for interpretation of the evolution of the nervous system in the Deuterostomia. It is suggested that echinoderms and chordates might have inherited the RS-producing radial glial cell type from the central nervous system of their common ancestor, i.e., the last common ancestor of all the Deuterostomia.

Analysis and quantification of the secretory products of the subcommissural organ by use of monoclonal antibodies.

Bovine Reissner's fiber (RF) glycoproteins were used as antigen for the production of polyclonal and monoclonal antibodies (Mabs). We also produced Mabs against intracellular secretory glycoproteins of the bovine subcommissural organ (SCO). These Mabs were used for immunodetection of secretory proteins in situ (structural and ultrastructural immunocytochemistry), in blots, and in solutions. Three different antigen-mediated ELISA were designed to evaluate the affinity of the Mabs, to study the nature of the epitopes, and for competition test among Mabs. Two double antibody sandwich ELISA were designed to detect and quantify soluble secretory materials in different samples, to study coexistence of epitopes, and to elucidate whether epitopes for Mabs are repeated or not in the RF-glycoproteins. Twenty-three Mabs recognizing the bovine RF- and SCO-glycoproteins in solutions (ELISA) as well as in tissue sections, were obtained. Nineteen of these Mabs also recognized the pig SCO, 11 the rabbit SCO, 6 the dog SCO, and 5 the rat SCO. None of the Mabs recognized the SCO of non-mammalian species. The different types of ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moiety of the secretion, (2) they coexist in the same molecular forms and, with few exceptions, they did not overlap, (3) they were not repeated in the secretory molecule(s). Three Mabs were used for immunoblotting of RF; one of them revealed the same band pattern as that shown by an anti-RF serum. It is concluded that all Mabs raised in our laboratory are directed against non-repeated sequences of RF-glycoproteins that have not been conserved in vertebrate phylogeny.

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.

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA.

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.

SCO-spondin is evolutionarily conserved in the central nervous system of the chordate phylum

Bovine subcommissural organ-spondin was shown to be a brain-secreted glycoprotein specifically expressed in the subcommissural organ, an ependymal differentiation located in the roof of the Sylvian aqueduct. Also, subcommissural organ-spondin makes part of Reissner's fiber, a phylogenetically and ontogenetically conserved structure present in the central canal of the spinal cord of chordates. This secretion is a large multidomain protein probably involved in axonal growth and/or guidance. As Reissner's fiber is highly conserved in the chordate central nervous system, we sought genes orthologous to the bovine subcommissural organ-spondin gene by Southern blot analysis in several members of the chordate phylum: urochordates, cephalochordates, cyclostomes, and lower and higher vertebrates, including humans. In addition, conserved glycoproteins present in the subcommissural organ and Reissner's fiber were revealed by immunohistochemistry using antibodies raised against bovine Reissner's fiber. Variation in the sites of Reissner's fiber production according to chordate subphylum, presence of this structure in the spinal cord, and conservation of the subcommissural organ-spondin gene are discussed in the context of chordate central nervous system development. These results indicate that subcommissural organ-spondin is an ancient ependymal secretion, making part of Reissner's fiber, that may have had an important function during the evolution of the central nervous system in chordates, including that of the spinal cord.

Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner's fiber.

The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner's fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.

Rostral floor plate (flexural organ) secretes glycoproteins immunologically similar to subcommissural organ glycoproteins in dogfish ( Scyliorhinus canicula) embryos

The subcommissural organ of vertebrates secretes glycoproteins into the cerebrospinal fluid of the third cerebral ventricle. This material polymerizes in Reissner's fiber. During ontogenetic development, besides the subcommissural organ, the ependyma lining the pontine flexure constitutes an additional Reissner's fiber-secreting gland named flexural organ. We have studied the secretion of the flexural organ and the subcommissural organ in dogfish ( Scyliorhinus canicula) embryos using three different antisera and the lectins concanavalin A and wheat germ agglutinin. AFRU is an antiserum against the bovine Reissner's fiber; Ab-600 is an antiserum against 600 kDa dogfish subcommissural organ glycoproteins; and APSO is an antiserum against immunoaffinity purified bovine subcommissural organ secretory glycoproteins. These three antisera immunostained the flexural organ indicating that it contains epitopes similar to those present in bovine and dogfish subcommissural organ glycoproteins. It seems highly probable that the flexural organ and the subcommissural organ of dogfish embryos secrete similar compound(s). Other ependymal regions were also immunostained with Ab-600 and APSO antisera. Then, Reissner's fiber-like glycoproteins were transiently expressed by most embryonary ependymal cells. These glycoproteins might play a role in the development of the central nervous system of vertebrates.

Identification of a high molecular weight polypeptide in the subcommissural organ of the chick embryo.

The subcommissural organ is an ependymal brain gland that secretes, into the ventricular cerebrospinal fluid, high molecular weight glycoproteins that form Reissner's fiber. Precursor and processed forms of secretion have been demonstrated by immunoblotting in the subcommissural organ of mammals and fish. In the chicken only a processed form has as yet been identified. In the present report, we have studied the subcommissural organ of 13-day-old chick embryos using (1) an antiserum against bovine Reissner's fiber, and (2) the lectins, concanavalin A and Limax flavus agglutinin. Paraffin sections of the subcommissural organ and blots of subcommissural organ extracts have been analyzed. The ependymal cells of sectioned subcommissural organ are strongly stained with the antiserum. Concanavalin A binds to materials in all cytoplasmatic regions, whereas Limax flavus agglutinin identifies materials confined to the apex of the ependymal cells. In the blots, a band of 540 kDa is immunostained. This band is positive for concanavalin A positive but negative for Limax flavus agglutinin and is thereby regarded as representing a precursor form of the secretion.

Bovine Reissner's fiber (RF) and the central canal of the spinal cord: an immunocytochemical study using a set of monoclonal antibodies against the RF-glycoproteins.

The subcommissural organ secretes N-linked complex-type glycoproteins into the cerebrospinal fluid. These glycoproteins condense to form Reissner's fiber (RF), which extends along the fourth ventricle and central canal of the spinal cord. A set of three monoclonal antibodies (Mabs 3E6, 3B1, and 2A5) has been obtained using these glycoproteins as immunogens. Competitive and sandwich enzyme-linked immunoassay methods have demonstrated that the three monoclonal antibodies are directed against different epitopes, and that there is no competition among them for their binding to glycoproteins of RF. Mab 3E6 displays immunoblotting properties that are similar to those of a polyclonal antibody against the pool of glycoproteins from RF, but that are different from those of Mabs 3B1 and 2A5. All three antibodies immunostain the bovine subcommissural organ and RF. A population of ependymal cells is stained by the polyclonal antibody, and Mabs 2A5 and 3E6, but not by Mab 3B1. The material present in a population of ependymal cells of the central canal, and the glycoproteins secreted by the subcommissural organ thus probably have partial chemical identity. Some evidence suggests that the immunoreactive ependymal cells are secretory cells. The luminal surface of the central canal is coated by a thin layer of material with immunocytochemical characteristics different from those of the ependymal cells; such a coat may correspond to material released from RF.

Immunochemical analysis of the subcommissural organ-Reissner's fiber complex using antibodies against alkylated and deglycosylated glycoproteins of the bovine Reissner's fiber

Reissner's fiber (RF) has been isolated, solubilized, and used to raise polyclonal antibodies. The present investigation has been designed: (1) to obtain antibodies against RF-glycoproteins in their native form (anti-RF-BI), after irreversible denaturation by alkylation (anti-RF-A), and after alkylation and deglycosylation by using endoglycosidase F (anti-RF-DE); (2) to use these antisera for a comparative immunocytochemical study of the subcommissural organ (SCO)-RF complex; (3) to establish the molecular mass of the deglycosylated RF-glycoproteins. Anti-RF-BI reacts with the SCO of all the species investigated. Anti-RF-A and anti-RF-DE only react with bovine SCO and RF. The three antisera stain the same bands in immunoblots of extracts of bovine SCO and RF, but anti-RF-A and anti-RF-DE reveal additional bands. The epitope common to all species reacting with anti-RF-BI is thus probably conformational in nature and associated with the integrity of the disulfide bonds. The lack of antibodies against conserved sequential epitopes in anti-RF-A does not support previous assumptions on the conserved nature of the SCO secretion. After deglycosylation by using endoglycosidase F, the RF-glycoproteins present a reduction in their molecular mass ranging between 10% and 25%. The three larger compounds (450, 300, and 230 kDa) lose their affinity for Limax flavus agglutinin (affinity = sialic acid), whereas the 190-kDa compound (170 kDa after deglycosylation) keeps its affinity for this lectin suggesting that it has N-linked and O-linked carbohydrate moieties, the three larger proteins probably having only N-linked carbohydrates.

Reissner?s fibre supports the survival of chick cortical neurons in primary mixed cultures

Reissner's fibre, a thread-like structure present in the central canal of the spinal cord, is a product of the condensation of specific glycoproteins that are released by specialized ependymal cells into the cerebrospinal fluid. These secretory ependymocytes constitute the subcommissural organ, a circumventricular organ that lines the roof of the third ventricle of the brain. The subcommissural organ/Reissner's fibre complex is a permanent structure in the vertebrate central nervous system. The addition of bovine Reissner's fibre itself or of soluble material released by Reissner's fibre to primary mixed cultures of chick cerebral cortical cells markedly enhances neuronal survival. The responsive cells have been identified as neurons by labelling them with antibodies to neurofilament proteins. This neuronal survival effect is dose-dependent and does not require the presence of serum in the culture medium. Affinity-purified polyclonal antibodies raised against bovine Reissner's fibre partially block the effect of Reissner's fibre on neuronal survival. These results suggest that Reissner's fibre is involved in developmental processes of the central nervous system.

Light- and electron-microscopic immunocytochemical investigation of the subcommissural organ using a set of monoclonal antibodies against the bovine Reissner's fiber

Ten monoclonal antibodies (Mabs) against glycoproteins of the bovine Reissner's fiber (RF) have been used in a structural and ultrastructural immunocytochemical investigation of the bovine subcommissural organ (SCO) and RF. The SCO of other vertebrate species has also been studied. For comparison, polyclonal antibodies against bovine RF (AFRU) were used. The SCO and RF of ox, pig and dogfish and the SCO of dog, rabbit, rat and frog were submitted to light-microscopic immunocytochemistry using AFRU and Mabs. Postembedding ultrastructural immunocytochemistry was applied to sections of bovine SCO using AFRU and Mabs. Bovine SCO consists of ependymal and hypendymal cell layers, the latter being arranged as cell strands across the posterior commissure, or as hypendymal rosette-like structures. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with AFRU. Six Mabs showed the same staining pattern as AFRU, one Mab stained RF strongly and SCO weakly, two Mabs stained RF but not SCO, and, finally, one Mab (3B1) exclusively stained the apices of the ependymal and hypendymal cells. All Mabs recognized the SCO and RF of the pig. Two Mabs bound to the SCO of the dog. One Mab stained the SCO of the rabbit and another the SCO of the rat. The SCO of frog and dogfish were totally negative. Bovine SCO stained with AFRU, showed label in the rough endoplasmic reticulum (RER) and the secretory granules (SG) of the ependymal and hypendymal cells. The former, in the form of parallel cisternae, reticulum or concentric rings, was seen throughout all cytoplasmic regions. SG were abundant in the apical pole of the ependymal and hypendymal cells. Only one Mab showed a staining pattern similar to AFRU. Five Mabs showed strong reactions in the SG but weak labeling of the RER. Mab 3B1 showed the label confined to the SG only. Our results suggest that: (i) in the bovine tissue, some epitopes are present in both precursor and processed materials, whereas others are characteristic of mature glycoproteins present in SG and the RF; (ii) the bovine SCO secretes at least two different compounds present in ependymal and hypendymal cells; (iii) both compounds coexist in the same secretory granule; (iv) there are conserved, class-specific, and species-specific epitopes in the glycoproteins secreted by the SCO of vertebrates.

Class-specific epitopes detected by polyclonal antibodies against the secretory products of the subcommissural organ of the dogfish Scyliorhinus canicula

We have raised antisera against extracts of the subcommissural organ (SCO) of the dogfish, Scyliorhinus canicula L. Brains of 2900 specimens were collected in acetone, and the region containing the SCO and posterior commissure was removed and extracted in three different media. Antisera against these crude extracts were raised in rats and rabbits. Sequential absorptions of the antisera with extracts from different regions of the dogfish brain were performed to eliminate unwanted antibodies. When used to immunostain sections of the whole central nervous system of the dogfish, these purified antisera reacted selectively with the SCO-Reissner's fiber complex. An antiserum against bovine Reissner's fiber was also used. The antisera against the dogfish SCO and bovine Reissner's fiber showed the same staining pattern in the SCO and the Reissner's fiber of the dogfish. For comparative purposes, the brains of 15 vertebrate species from all vertebrate classes were immunostained with both antisera. The anti-dogfish SCO serum reacted with the SCO of the dogfish and that of other phylogenetically related elasmobranch species. Neither the SCO of a primitive elasmobranch species, Heptranchias perlo, nor the SCO of the other classes of vertebrates reacted with the anti-dogfish SCO serum. However, the antiserum against bovine Reissner's fiber reacted with the SCO of all the investigated species. It is concluded that some epitopes (or compounds) in the secretory material of the SCO are class-specific, whereas others are conserved and are synthesized by the SCO in most vertebrate species.

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

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.

Single injection into the cerebrospinal fluid of antibodies against the secretory material of the subcommissural organ reversibly blocks formation of Reissner's fiber: immunocytochemical investigations in the rat

An antibody (cf. Rodríguez et al. 1984b) raised in rabbits against the glycoproteins of the bovine Reissner's fiber (RF) was injected into the lateral brain ventricle of 38 rats with the aim to interfere with RF formation. The rats were killed 20 min; 1, 4, 8, 12 h; and 1, 2, 3, 5, and 8 days after the injection. Based on the fact that the material secreted by the subcommissural organ (SCO) into the cerebrospinal fluid (CSF) first condenses on the organ surface as a distinct layer (pre-RF material) and then becomes assembled to form RF and that both structures are distinguishable in tissue sections, three immunostaining procedures were applied. They served to visualize: (i) secretory material that had not bound the injected antibody; (ii) secretory material-antibody complexes formed in vivo; and (iii) antibody not bound to its antigen and present in the ventricles and the subarachnoid space. After a single injection of the above-mentioned antibody the following events were observed: (1) The antibody was present in the brain cavities for at least 8 h. (2) The injected antibody bound selectively to the pre-RF and RF. (3) Pre-RF displayed antibody binding during the 24 h following the injection. During the 2nd and 3rd post-injection days, the pre-RF was free of antibody, indicating that it was formed by newly released secretory material. (4) Approximately 4 h after the injection, the RF detached from the SCO and underwent fragmentation. Clusters of these fragments were found in the Sylvian aqueduct and fourth ventricle. (5) In the fragmented original RF the injected antibody against Reissner's fiber remained bound throughout the entire period of observation, i.e. for 8 days. (6) In rats of the 1-, 3-, 5- and 8-day-groups, RF was missing from the central canal of the spinal cord. (7) One day after the injection, a new RF structure started to grow from the rostral end of the SCO. This newly formed fiber could be distinguished from the original RF because of (i) its normal appearance; (ii) it did not display binding of the injected antibody. (8) At day 3, the growing RF had not yet extended to the Sylvian aqueduct. (9) At day 8, the new RF reached the fourth ventricle. Control experiments involved the intraventricular administration of (i) an antibody against the secretory material extracted from the entire bovine SCO; (ii) antivasopressin; and (iii) rabbit IgG. From these only antibody (i) bound to pre-RF and RF.

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

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, beta-galactosidase, alpha-mannosidase, alpha-glucosidase and beta-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 Tunica-mycin 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).

Ultrastructural immunocytochemistry and lectin histochemistry of the subcommissural organ in the snake Natrix maura with particular emphasis on its vascular and leptomeningeal projections

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.

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

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.

Ultrastructural immunocytochemical study of the massa caudalis of the subcommissural organ-Reissner's fiber complex in lamprey larvae (Geotria australis): Evidence for a terminal vascular route of secretory material

The massa caudalis of the subcommissural organ-Reissner's fiber complex of lamprey larvae (Geotria australis) was studied immunocytochemically at the ultrastructural level by use of the immunoperoxidase-silver methenamine procedure. An antiserum raised against bovine Reissner's fiber was utilized as primary antibody. The caudalmost portion of the central canal and its ampulla caudalis communicate, via wide intercellular spaces in their dorsal wall, with large cavities or lacunae. In addition, distinct openings in the dorsal wall of the ampulla establish an open communication between the latter and the lacunae. The lacunae are lined by slender processes of cells of unknown nature. No junctional complexes can be observed between these cells, which lack a basal lamina. The lacunae communicate with structures resembling blood capillaries, however, they are devoid of a basal lamina. These peculiar vessels, in turn, are in direct communication with characteristic blood capillaries. Reissner's fiber (RF) and its massa caudalis are strongly immunoreactive with the antiserum used. The wide intercellular spaces in the dorsal wall of the central canal and the ampulla, as well as the lumina of the (i) lacunae, (ii) modified vessels and (iii) blood capillaries are filled with a flocculent, strongly immunoreactive material. No immunoreactive material was found outside these structures. Thus, the blood capillaries appear to represent the only final target of RF-material arriving at the ampulla caudalis.

Vascular and leptomeningeal projections of the subcommissural organ in reptiles. Lectin-histochemical, immunocytochemical, and ultrastructural studies.

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.