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.     

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.     

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 immunocyto-chemical 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.     

Reduced subcommissural organ glycoprotein immunoreactivity precedes aqueduct closure and ventricular dilatation in H-Tx rat hydrocephalus

The H-Tx rat has fetal-onset hydrocephalus associated with closure of the cerebral aqueduct and a reduction in the secretory cells of the subcommissural organ (SCO), a circumventricular organ situated in the dorsal wall of the cerebral aqueduct. The objective of this study was to determine the role of the SCO in hydrocephalus pathogenesis. Serial brain sections through aqueduct regions containing the SCO from H-Tx rats, together with non-hydrocephalic Fischer F344 rats, were studied at E16, before hydrocephalus onset, at E17, the beginning of onset, and at P0 when the hydrocephalus was overt. Tissues were immunostained by AFRU, an antibody against the SCO glycoprotein, and for the intermediate filament nestin. The area of SCO cells with AFRU immunostaining and the severity of lateral ventricle dilatation were quantified by image analysis. At E16 all fetuses had distinct SCO ependymal cells, open aqueducts and normal lateral ventricles. The H-Tx fetuses fell into two groups with large areas and small areas of AFRU immunoreactivity, all with a full complement of SCO cells. By E17, fetuses with small areas of immunoreactivity had reduced numbers of tall SCO secretory cells, and most had aqueducts closed posteriorly and dilated ventricles. Three additional fetuses with small areas of immunoreactivity had narrow but patent aqueducts and normal ventricles, and another had an open aqueduct and dilated ventricles. At P0, pups previously identified as hydrocephalic had small areas of AFRU immunoreactivity, an aqueduct that was closed anteriorly but open posteriorly, ventricular dilatation, and an absence of SCO secretory cells. The aqueduct even when closed was lined by typical ependymal cells throughout. Decreased nestin immunostaining accompanied the SCO changes. It is concluded that reduced SCO glycoprotein immunoreactivity precedes both aqueduct closure and expansion of the lateral ventricles in the H-Tx rat.Funding was provided by the National Institutes of Health (NS40359). K.C.S. was supported by the University of Florida Scholars Program and Sigma Xi Grants-in-Aid     

Synthesis of transthyretin by the ependymal cells of the subcommissural organ

Transthyretin (TTR) is a protein involved in the transport of thyroid hormones in blood and cerebrospinal fluid (CSF). The only known source of brain-produced TTR is the choroid plexus. In the present investigation, we have identified the subcommissural organ (SCO) as a new source of brain TTR. The SCO is an ependymal gland that secretes glycoproteins into the CSF, where they aggregate to form Reissners fibre (RF). Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. To investigate the CSF-soluble compounds secreted by the SCO further, antibodies were raised against polypeptides partially purified from fetal bovine CSF. One of these antibodies (against a 14-kDa compound) reacted with secretory granules in cells of fetal and adult bovine SCO, organ-cultured bovine SCO and the choroid plexus of several mammalian species but not with RF. Western blot analyses with this antibody revealed two polypeptides of 14 kDa and 40 kDa in the bovine SCO, in the conditioned medium of SCO explants, and in fetal and adult bovine CSF. Since the monomeric and tetrameric forms of TTR migrate as bands of 14 kDa and 40 kDa by SDS-polyacrylamide gel electrophoresis, a commercial preparation of human TTR was run, with both bands being reactive with this antibody. Bovine SCO was also shown to synthesise mRNA encoding TTR under in vivo and in vitro conditions. We conclude that the SCO synthesises TTR and secretes it into the CSF. Colocalisation studies demonstrated that the SCO possessed two populations of secretory cells, one secreting both RF glycoproteins and TTR and the other secreting only the former. TTR was also detected in the SCO of bovine embryos suggesting that this ependymal gland is an important source of TTR during brain development. Financial support was provided by grants 1030265 from Fondecyt, Chile, to E.M.R. and 201.035.002-1.0 DIUC to H.M.     

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.     

The subcommissural organ expresses D<Subscript>2</Subscript>, D<Subscript>3</Subscript>, D<Subscript>4</Subscript>, and D<Subscript>5</Subscript> dopamine receptors

Dopamine receptors have been found in certain populations of non-neuronal cells in the brain, viz., discrete areas of ciliated ependyma and the ependymal cells of the choroid plexus. We have studied the presence of both tyrosine-hydroxylase-immunoreactive nerve fibers and dopamine receptors in the subcommissural organ (SCO), an ependymal brain gland that is located in the roof of the third ventricle and that secretes, into the cerebrospinal fluid, glycoproteins that aggregate to form Reissners fiber (RF). Antibodies against D₂, D₃, D₄, and D₅ dopamine receptors were used in immunoblots of bovine striatum, fresh SCO, and organ-cultured SCO, and in immunocytochemistry of the bovine, rat, and mouse SCO. Only a few tyrosine-hydroxylase fibers appeared to reach the SCO. However, virtually all the secretory ependymal and hypendymal cells of the SCO immunoreacted with antibodies against D₂, D₄, and D₅ receptors, with the last-mentioned rendering the strongest reaction, especially at the ventricular cell pole of the secretory ependymocytes, suggesting that dopamine might reach the SCO via the cerebrospinal fluid. The antibodies against the four subtypes of receptors revealed corresponding bands in immunoblots of striatum and fresh SCO. Although the cultured SCO displayed dopamine receptors, dopamine had no apparent effect on the expression of the SCO-spondin gene/protein or on the release of RF-glycoproteins (SCO-spondin included) by SCO explants, suggesting that dopamine affects the function(s) of the SCO differently from the secretion of RF-glycoproteins.Financial support was provided by grants PI 030756 and Red CIEN, Instituto de Salud Carlos III, Spain (to J.M.P.F.), and 1030265 from Fondecyt, Chile (to E.M.R.)     

Immunohistochemical and ultrastructural localisation of peptide-containing nerves and myocardial cells in the human atrial appendage

Summary The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptidecontaining nerves and myocardial cells producing ANP.     

Beobachtungen am Subcommissuralorgan und Reissnerschen Faden der Schildkröte unter osmotischer Belastung

Zusammenfassung Subcommissuralorgan (SCO) und Reissnerscher Faden (RF) von drei verschiedenen Schildkrötenarten wurden unter osmotischer Belastung lichtmikroskopisch untersucht. Ergänzend zu einer früheren Ependymstudie von Fleischhauer (1957) wird ein rostral im Recessus mesocoelicus gelegener Anteil des SCO beschrieben, der sich durch Vakuolen und Ganglienzellen im Hypendym, sowie durch sein flaches, kubisches Ependym vom typischen Organaufbau abhebt. Der Reissnersche Faden schließt sich unter osmotischer Belastung der Tiere nicht zu einem kompakten Sekretfaden, sondern gestaltet sich im Bereich des 3. Ventrikels zu einem reusenähnlichen Netzwerk mit je einem basalen und dorsalen Verdichtungsstrang. Es fallen deutliche Unterschiede in der Struktur des geronnenen Liquors innerhalb und außerhalb des Netzwerkes auf. Die lagemäßige Beziehung des dorsalen Zentrums dieses Systems zu einem Sulcus medialis tecti schließt Ependym und Hypendym des Ventrikeldaches in die Untersuchung mit ein. Lage und Ausdehnung einer hypendymalen Ganglienzelleiste mit sekretorischer Potenz und Beziehung zu Kapillaren regen zu der Annahme an, daß auch die basalen Ependymanteile des SCO von Ganglienzellen abstammen. Entsprechend dem hohen Gehalt an Acetylneuraminsäure (Sialinsäure) und biogenen Aminen wird vermutet, daß dem RF im Liquor eine Bedeutung als organischer Ionenaustauscher zukommt (SteRBA,1969). Die beobachtete Netzbildung bietet eine Oberflächenvergrößerung, die für einen Ionenaustauscher eine günstige Voraussetzung darstellt.

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Subcommissural organ and Reissner's fibre of the tortoise under conditions of osmotic stress

Summary The subcommissural organ (SCO) and Reissner's fibre of three species of tortoises living under conditions of osmotic stress was investigated with the light microscope. Complementary to a study of the reptilian ependyma by Fleischhauer (1957) a particular rostral part of the SCO was found, situated in the recessus mesocoelicus just in front of the posterior commissure. This part differs from the surrounding ependyma by its subependymal layer of nerve cells and vacuoles, and by its flat, cuboidal ependymal cells.Under osmotic stress the structure of Reissner's fibre in the course of its passage through the 3rd ventricle is not that of a compact fibre, as it is well known, but it consists of a spongy network of many single filaments. There are obvious differences in the appearance of the clotted cerebrospinal fluid within and outside this network. At the end of the 3rd ventricle the fibers lie closely together and form a dorsal and basal dense layer. The spatial relation of the dorsal layer of this network to the sulcus medialis tecti includes ependymal and subependymal layers of the roof of the 3rd ventricle into this study. Location and extention of this subependymal layer of nerve cells with secretory activity and close relation to the capillaries suggest, that they have the same origin from nerve cells as the basal parts of the ependyma of the SCO.According to the high content of sialic acid and biogenic amines one can imagine, that Reissner's fibre has some importance as a cation-exchanger in the cerebrospinal fluid. The observed formation of a filamentous network by which its surface is largely increased, is in favour of this assumption.

     

Secretory activity of the subcommissural organ in Rana temporaria under osmotic stimulation

Summary The secretory activity of the subcommissural organ (SCO) in the frog Rana temporaria was studied under conditions of dehydration. After injection of a radioactive precursor the amount and concentration of radioactively labelled material in the SCO are smaller in dehydrated than in control animals. Concomitantly, the growth rate of the CSF-fibre (Reissner's fibre) increases in dehydrated animals. It follows that water deprivation enhances the secretory activity of the SCO.To investigate whether the SCO may be responsible for the secretion of an aldosteronotropic factor as suggested in the literature, brains were incubated in vitro with a radioactive precursor and with or without aldosterone. The SCO of the aldosterone-treated brains contains more radioactively labelled material than the SCO of the control brains. It is argued that this is indicative of a lower secretory activity. It means that aldosterone inhibits the secretory activity of the SCO, possibly by a process of negative feed-back regulation. The results of the present experiments can be interpreted in favour of an involvement of the SCO-Reissner's fibre complex in osmoregulation.     

Secretory activity of the subcommissural organ in Rana temporaria under osmotic stimulation

The secretory activity of the subcommissural organ (SCO) in the frog Rana temporaria was studied under conditions of dehydration. After injection of a radioactive precursor the amount and concentration of radioactively labelled material in the SCO are smaller in dehydrated than in control animals. Concomitantly, the growth rate of the CSF-fibre (Reissner's fibre) increases in dehydrated animals. It follows that water deprivation enhances the secretory activity of the SCO. To investigate whether the SCO may be responsible for the secretion of an aldosteronotropic factor as suggested in the literature, brains were incubated in vitro with a radioactive precursor and with or without aldosterone. The SCO of the aldosterone-treated brains contains more radioactively labelled material than the SCO of the control brains. It is argued that this is indicative of a lower secretory activity It means that aldosterone inhibits the secretory activity of the SCO, possibly by a process of negative feed-back regulation. The results of the present experiments can be interpreted in favour of an involvement of the SCO-Reissner's fibre complex in osmoregulation.     

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.     

Role of the subcommissural organ in the pathogenesis of congenital hydrocephalus in the HTx rat

The present investigation was designed to clarify the role of the subcommissural organ (SCO) in the pathogenesis of hydrocephalus occurring in the HTx rat. The brains of non-affected and hydrocephalic HTx rats from embryonic day 15 (E15) to postnatal day 10 (PN10) were processed for electron microscopy, lectin binding and immunocytochemistry by using a series of antibodies. Cerebrospinal fluid (CSF) samples of non-affected and hydrocephalic HTx rats were collected at PN1, PN7 and PN30 and analysed by one- and two-dimensional electrophoresis, immunoblotting and nanoLC-ESI-MS/MS. A distinct malformation of the SCO is present as early as E15. Since stenosis of the Sylvius aqueduct (SA) occurs at E18 and dilation of the lateral ventricles starts at E19, the malformation of the SCO clearly precedes the onset of hydrocephalus. In the affected rats, the cephalic and caudal thirds of the SCO showed high secretory activity with all methods used, whereas the middle third showed no signs of secretion. At E18, the middle non-secretory third of the SCO progressively fused with the ventral wall of SA, resulting in marked aqueduct stenosis and severe hydrocephalus. The abnormal development of the SCO resulted in the permanent absence of Reissner’s fibre (RF) and led to changes in the protein composition of the CSF. Since the SCO is the source of a large mass of sialilated glycoproteins that form the RF and of those that remain CSF-soluble, we hypothesize that the absence of this large mass of negatively charged molecules from the SA domain results in SA stenosis and impairs the bulk flow of CSF through the aqueduct.     

Confocal imaging and phylogenetic considerations of the subcutaneous neurons in the Atlantic hagfish Myxine glutinosa

We used confocal microscopy and immunohistochemistry to characterize the morphology of the subcutaneous neurons and the innervation of the slime glands and striated muscles in the hagfish Myxine glutinosa. A rich plexus of 5HT‐, ChAT‐ and TH‐positive neurons is described in the capsule of the slime glands. These neurons, like those of the subcutaneous plexus, receive pericellular terminations from the axons of central cells. Capsular neurons receive innervation from 5HT‐positive and nNOS‐positive nerve fibres. Other nerve endings belonging to two separate nerve populations are identified in the striated muscles. They contain TH and nNOS immunoreactivity. Due to the lack of any topographical labelling, the cell origin and the projections of the neurons into the cranial and spinal nerves are unknown. This study provides anatomical evidence of multiple (5HT and nNOS) peripheral innervation of the neurons. However, it does not provide information about the function of these neurons in the hagfish. We suggest that hagfish neurons have a phylogenetic relationship with the spinal group of the dorsal cells of lampreys and the supramedullary cells of teleosts.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

RT97: a marker for capsaicin-insensitive sensory endings in the rat skin

The mouse monoclonal antibody RT97, which recognises the 200-kDa neurofilament subunit in its phosphorylated form, selectively labels the somata of sensory A-fibres (large light cells) in the dorsal root ganglion of the rat. We have tested the hypothesis that this antibody also visualises large diameter sensory fibres and their end structures in peripheral tissue, in particular in the skin. RT97 immunoreactivity is found in endings that are known to be served by myelinated afferent fibres, including Meissner-like endings, Merkel discs, hair follicle receptors, Pacinian corpuscles and free nerve endings. RT97 immunoreactivity has not, however, been observed in endings of presumably unmyelinated sensory fibres (intraepidermal fibres immunoreactive for substance P and calcitonin gene-related peptide) or in sympathetic fibres innervating sweat glands and blood vessels. In addition, neither systemic (100–150 mg/kg as adults) nor perineural capsaicin pre-treatment affects RT97 immunoreactivity in the skin. The data indicate that RT97 is a useful marker in the study of the capsaicin-insensitive sensory innervation of the skin and possibly other peripheral organs.     

Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures

The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca(2+)](i)), and its precise role remains to be clarified. SP elicited rises in [Ca(2+)](i) in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK(3) and P2Y(2) receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca(2+)](i) comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca(2+)](i) was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca(2+)](i) involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.     

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.     

Immunohistochemical localization of glial fibrillary acidic protein (GFAP) and vimentin in the subcommissural organ of the Mongolian gerbil (Meriones unguiculatus)

Summary The chemical composition of intermediate filaments (IF's) in the ependyma of the subcommissural organ (SCO) of the Mongolian gerbil (Meriones unguiculatus) was investigated immunohistochemically in paraffin-embedded tissue. Antibodies against glial fibrillary acidic protein (GFAP), vimentin, neurofilament proteins and cytokeratins were used. Only GFAP and vimentin were detected in the non-specialized diencephalic ependyma and in the ependymocytes of the SCO. Staining could be observed in apical and basal processes of the SCO-cells. The latter processes extended into the posterior commissure up to the subpial surface, thus establishing a well-developed leptomeningeal route of ependymal projections. In contrast to the homogeneous vimentin-labeling, the SCO was particularly immunoreactive for GFAP in its lateral aspects and in the supraand precommissural parts. The coexpression of GFAP and vimentin in a subclass of SCO-ependymocytes was demonstrated on differentially immunostained semithin sections. The present study confirms the glial nature of the SCO-ependyma, which has been a matter of debate recently. It appears from this investigation that the high degree of secretory activity in the SCO does not necessarily lead to the disappearance of glial IF proteins. Moreover, the SCO-cells belong to the expanding group of mature astroglia, which is characterized by coexpression of GFAP and vimentin. The morphological similarity between SCO-ependymocytes and tanycytes is underscored by their common immunoreactivity against these two IF proteins. In view of the absence of GFAP from the rat SCO, interspecific differences must be considered in the evaluation of the IF protein composition.     

Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig

The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.