Molecular Research 129 (2004) 151–162 www.elsevier.com/locate/molbrainres Research report Transcription of SCO-spondin in the subcommissural organ: evidence for down-regulation mediated by

Hans G. Richtera,*, Marı´a M. Tome´b, Carlos R. Yulisa, Karin J. Vı´oa, Antonio J. Jime´nezb, Jose´M.Pe´rez-Fı´garesb, Esteban M. Rodrı´gueza

aInstituto de Histologı´a y Patologı´a, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile bDepartamento de Biologı´a Celular y Gene´tica, Facultad de Ciencias, Universidad de Ma´laga, Spain Accepted 7 July 2004 Available online 13 August 2004

Abstract

The subcommissural organ (SCO) is a brain gland located in the roof of the that releases glycoproteins into the , where they form a structure known as Reissner’s fiber (RF). On the basis of SCO-spondin sequence (the major RF glycoprotein) and experimental findings, the SCO has been implicated in development; however, its function(s) after birth remain unclear. There is evidence suggesting that SCO activity in adult animals may be regulated by serotonin (5HT). The use of an anti-5HT serum showed that the bovine SCO is heterogeneously innervated with most part being poorly innervated, whereas the rat SCO is richly innervated throughout. Antibodies against serotonin receptor subtype 2A rendered a strong immunoreaction at the ventricular cell pole of the bovine SCO cells and revealed the expected polypeptides in blots of fresh and organ-cultured bovine SCO. Analyses of organ-cultured bovine SCO treated with 5HT revealed a twofold decrease of both SCO-spondin mRNA level and immunoreactive RF glycoproteins, whereas no effect on release of RF glycoproteins into the culture medium was detected. Rats subjected to pharmacological depletion of 5HT exhibited an SCO-spondin mRNA level twofold higher than untreated rats. These results indicate that 5HT down-regulates SCO-spondin biosynthesis but apparently not its release, and suggest that 5HT may exert the effect on the SCO via the cerebrospinal fluid. D 2004 Elsevier B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and regulators Topic: Serotonin

Keywords: Subcommissural organ; SCO-spondin; Serotonin; Serotonin receptors; Gene expression regulation; Bovine; Rat

1. Introduction threadlike structure known as Reissner’s fiber (RF) [17,33,35]. RF extends along the , fourth The subcommissural organ (SCO) is a highly differ- ventricle, and the central canal of the spinal cord. According entiated ependymal gland located in the roof of the third to Rodrı´guez et al. [34], a fraction of the secretory material ventricle, at the entrance of the Sylvian aqueduct. The SCO released at the apical pole of the SCO cells might remain differentiates at early stages of ontogenetic development soluble in the CSF. [24,38] and, with a few exceptions, it remains fully active The primary structure of the major bovine SCO secretory during the entire life span [17,33,35]. The SCO secretes high glycoprotein, SCO-spondin, has been fully established molecular weight glycoproteins [25], which are released into [8,11,18,26]; it is a large N-glycosylated protein (450 the cerebrospinal fluid (CSF) where they condense to form a kDa), encoded by a 14.5-kb mRNA. Several lines of evidence indicate that SCO-spondin plays a role in CNS development [11,12,18,21–23], but the function of the SCO * Corresponding author. Tel.: +56 63 29 3021; fax: +56 63 22 1604. in adulthood is not well understood. Nonetheless, the early E-mail address: [email protected] (H.G. Richter). view that the SCO–RF complex participates in the clearance

0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.07.003 152 H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 of CSF monoamines [13] has gained strong support from 5HT during the whole culture period (n=8 groups of recent investigations [5,30,36]. explants). The incubation medium together with 5HT was To understand how the SCO secretory activity is changed every 3 days. At the end of the culture period, the regulated may contribute to further elucidate its function(s). explants were subjected to total RNA isolation as described Several neural systems apparently involved in the control of below. the SCO secretory activity have been described [14,27]. The serotonergic input to the SCO originated in is 2.1.2.2. Effect of 5HT on the expression of RF glycoproteins prominent and has been consistently found in several (SCO-spondin included). Twogroupsof50explants species. According to Le´ger et al. [16] and Mbllgard et al. cultured during 15 days in the absence or presence of 2 [19], serotonin (5HT) participates in the regulation of the AM 5HT were fixed and processed for immunocytochem- SCO secretory activity. istry to demonstrate RF glycoproteins (SCO-spondin The aim of the present investigation was twofold: to included; see below). Two other groups of explants cultured immunodetect 5HT receptors in the SCO and to analyze the during 15 days in the absence or presence of 2 AM 5HT effect of 5HT on SCO-spondin transcription and translation were processed for immunoblotting for the demonstration of under in vivo and in vitro conditions. We found that the RF glycoproteins (see below). bovine SCO expresses 5HT2A receptors that are preferen- tially located at the apical cell pole. Using cultured bovine 2.1.2.3. Effect of 5HT on the release of RF glycoproteins by SCO, it was determined that 5HT down-regulates tran- SCO explants. Fifteen-day-old SCO explants cultured in scription of the SCO-spondin gene and decreases the the absence or presence of 2 AM 5HT were further cultured amount of its translation products, while release of for 1 h in the presence of an antibody against the RF secretory products to the incubation medium was appa- glycoproteins (AFRU, see below), conjugated with fluo- rently not affected. Rats subjected to pharmacological rescein isothiocyanate (40 Al/ml). After washing the living depletion of 5HT exhibited a SCO-spondin mRNA level explants with antiserum-free culture medium, they were about twofold higher than untreated rats. These results observed under a fluorescence microscope to visualize the indicate that 5HT down-regulates SCO-spondin biosyn- immunofluorescent secretion aggregated on the surface of thesis but not release, and suggest that 5HT may exert its the SCO ependymal cells (cf. Ref. [39]). Control explants effect on the SCO via the CSF. The pharmacological and were incubated with FITC-conjugated rabbit IgG. After molecular characterization of the 5HT receptors operating microscopic analysis and photography, all the samples in the SCO needs to be investigated. were fixed in Bouin’s fixative and processed for immuno- cytochemistry as described below, using anti-rabbit IgG as primary antibody. This allowed to visualize, in tissue 2. Material and methods sections, the binding of AFRU to the extracellular secretory material released by the living SCO explants 2.1. In vitro experiments (cf. Ref. [39]).

2.1.1. Tissue sampling 2.1.3. Immunocytochemistry SCOs from 153 adult bovine (about 10 min Bovine explants (see above), were fixed by immersion postmortem) were obtained at the local slaughterhouse in Bouin’s fluid for 5 days, and embedded in Paraplast. (FRIVAL, Valdivia). Serial sections, 8-Am thick, were obtained and mounted on gelatin-coated slides. Fresh SCOs, obtained from adult 2.1.2. Organ culture of bovine SCO bovine brains (n=4), were processed in the same way. The immunoperoxidase method of Sternberger et al. [41] was 2.1.2.1. Effect of 5HT on the transcription of SCO-spondin applied using rabbit anti-RF glycoproteins (AFRU, gene. To determine the effect of 5HT (creatinine sulfate A=antibody, FR=fiber of Reissner, U=urea [31])as complex; Sigma, St. Louis, MO, USA) on the expression of primary antibody. The following treatments were sequen- the SCO-spondin gene in the bovine SCO maintained in tially applied to the sections: (1) AFRU (1:10,000) for 18 vitro, about 2400 explants were obtained as described by h; (2) antirabbit IgG developed in goat (E.M. Rodrı´guez, Schoebitz et al. [39]. Twenty-four groups of explants were Instituto de Histologı´a y Patologı´a, Valdivia, Chile), prepared; each experimental group included 100 explants diluted 1:15, 30 min; (3) peroxidase–anti-peroxidase obtained from five SCOs (20 explants/SCO); 15 ml of (PAP; Sigma), diluted 1:75, 30 min; (4) 0.02% 3.3V- serum-free culture medium, prepared according to Schoe- diaminobenzidine tetrahydrochloride (DAB, Sigma) con- bitz et al. [39], were used per culture dish containing 100 taining 0.002% H2O2 for 15 min. All antisera were explants. The following conditions were compared: (1) applied at room temperature, diluted in 0.041 M TRIS freshly prepared noncultured explants (n=8 groups of 100 buffer, pH 7.8, containing 0.7% nongelling seaweed explants each); (2) 15 days of culture, without 5HT (n=8 lambda carrageenan (Sigma) as saturating agent and groups of explants); and (3) 15 days of culture with 2 AM 0.5% Triton X-100. H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 153

2.1.4. Western blot of bovine SCO explants using anti-RF 2.2. Serotonin fibers and 5HT2A receptor in the bovine and glycoproteins and anti-5HT2A receptor rat SCO

2.1.4.1. Experimental groups, treatments, and protein The SCO and the region of the brain cortex adjacent to extraction. Seven pools (each pool containing the the ventricle (5-mm distance to the ventricular surface) were explants from four SCOs) were cultured in the presence dissected out from six bovine brains in a local slaughter- (four pools) or absence (three pools) of 2 AM 5HT house (Malaga, Spain; maximum postmortem time period during 15 days, under the conditions described by about 10 min), and immersed in the periodate–lysine– Schoebitz et al. [39]. The incubation medium together paraformaldehyde fixative mixture (PLP) during 24–48 h at with 5HT was changed every 2 days. Two additional 4 8C. Additionally, two Wistar rats (females, 80 days old) pools consisted of 50 tissue samples obtained from fresh were anesthetized with ether and transcardially perfused SCOs in the same way as if they were to be used as with PLP. Subsequently, bovine SCOs and brain cortex explants (i.e., freshly prepared noncultured explants). samples, and rat brains, were transferred to a 30% sucrose Ammonium bicarbonate protein extracts were prepared solution before obtaining frozen sections (20-Am thick). from each one of the nine pools, as described by Nualart After washing in phosphate-buffered saline (PBS), sections et al. [25], and their protein content was determined were immunostained during 72 h at 4 8C using polyclonal according to Bradford [4]. This experiment was carried antibodies raised in rabbits against: (1) 5HT (Sigma, S- out three times. From each pool, 20 Al (corresponding to 5545; dilution 1:2000); and (2) the 5HT2A receptor (see 0.1 SCO=10% of a protein extract obtained from 1 above; dilution 1:500). Detection of primary antibody was SCO) were used for Western blot analyses with anti- carried out by means of biotinylated goat anti-rabbit bodies against 5HT2A receptor or RF glycoproteins, as immunoglobulins (Dako, Denmark, E-0432; dilution follows. 1:500; 1-h incubation at 22 8C), and ExtrAvidin-peroxidase (Sigma, E2886; dilution 1:2000; 1-h incubation at 22 8C). 2.1.4.2. Western blot of RF glycoproteins. Samples were Sections were then transferred to a 0.05% DAB solution subjected to SDS-PAGE, using a 5–15% polyacrylamide containing 0.002% H2O2. Nickel ammonium sulfate hex- linear gradient, and then blotted into nitrocellulose sheets ahydrate (0.03%) was added to the DAB solution in some [43]. Immunoreactive polypeptides were detected, after immunostaining sessions to intensify the labeling [6]. blocking nonspecific protein binding sites, using an Antibodies were diluted in PBS, pH 7.3, containing 1% enhanced chemiluminescence (ECL) system (Super Sig- bovine serum albumin, 5% goat normal serum, and 0.5% nal, Pierce [44]), as previously described [29], but using Triton X-100 (Sigma). Before immunostaining, sections AFRU as primary antibody (see above; dilution were treated with (1) H2O2 to block endogenous peroxidase 1:25,000), for 3 h, followed by goat anti-rabbit IgG activity and (2) an avidin solution followed of a biotin conjugated with horseradish peroxidase (HRP), dilution solution (avidin/biotin blocking kit, Vector, CA, USA. SP- 1:12,500, for 90 min, in darkness. After being developed 2001) to block binding of ExtrAvidin or biotinylated and fixed, the autoradiographic films were digitalized antibodies with endogenous biotin, biotin binding proteins, and the relative band density was analyzed with the and other nonspecific binding substances that could be software Scion Image Beta 4.0.2 (available at Scion: present in the sections. Omission of the primary antibody http://www.scioncorp.com). was used as negative control of the immunoreaction. Immunostained sections were mounted on gelatin-coated 2.1.4.3. Western blot of 5HT2A receptor protein. Samples slides. without heating were subjected to electrophoresis on 10% SDS-PAGE (without h-mercaptoethanol) followed by blot- 2.3. In vivo experiments ting to PVDF membranes (Bio-Rad Laboratories, CA, USA). Blots were saturated with 0.01 M PBS containing 2.3.1. Animals 5% nonfat dry milk and 0.1% Tween-20 (Sigma) for 90 Sprague–Dawley adult male rats (about 200 g body min, followed by incubation in an antibody against a weight; n=30) were used. The rats were caged together in synthetic peptide corresponding to amino acids 22–41 of groups of five and provided with food and water ad the rat 5HT2A receptor (Oncogene, MA, USA, PC176L; libitum, under constant temperature (25 8C) and a light/ dilution 1:250), overnight. Goat anti-rabbit IgG-HRP dark cycle of 12:12 (L12:D12; lights on at 8 a.m.). The (Sigma, A6154) was used as secondary antibody, diluted condition light on consisted of indirect illumination with 1:5000 in the saturation solution, incubation was for 1 h, in white fluorescent light. darkness. Immunoreactive bands were visualized by using an ECL system (ECL Western Blotting Detection Reagents 2.3.2. Inhibition of serotonin synthesis RPN2109, Amersham, UK) as instructed by the manufac- PCPA (dl-p-chlorophenylalanine, Sigma; a potent inhib- turer. The whole procedure was performed at room itor of 5HT synthesis) was administered to three experimen- temperature. tal groups, five rats each; 400 mg/kg of the drug, diluted in 154 H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 sterile saline, were delivered by a single i.p. injection (2 GACTCTGTGCCACATACCTG-3V.Basenumbers p.m.). Three groups of five rats were injected with vehicle according to the 15,441-bp linear mRNA sequence and used as control. Twenty-four hours after the injection, defined as Bos taurus SCO-spondin (LOC282659) at the animals were sacrificed by decapitation and their SCOs GenBank (accession number NM_174706). The expected quickly dissected out and subjected to total RNA extraction amplification product has a size of 233 bp [29] (Fig. 1A); as described below. (2) rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene, defined by the following 2.4. Expression, sequencing, and semiquantitative analysis cDNA sequences: reverse, bases 794–813, sequence, 5V- of specific mRNAs by means of reverse transcriptase- AGATCCACAACGGATACATT-3V and forward, bases polymerase chain reaction (RT-PCR) 506–525, sequence, 5V-TCCCTCAAGATTGTCAGCAA- 3V. The expected amplification product has a size of 309 2.4.1. Materials and probes bp [42]. The following primer pair was designed with the Restriction enzymes, SuperScript II RNaseHÀ reverse assistance of OLIGO 4.1 (Primer Analysis Software, Ply- transcriptase, Taq DNA polymerase, Concert PCR-purifi- mouth, MN, USA) and BLASTN 2.2.1 tools [2] (available cation system, DNase I, PCRx enhancer system, 500-bp at http://www.ncbi.nlm.nih.gov), and synthesized by Gibco DNA ladder, and Trizol reagent were all purchased from BRL Custom Primers: rat SCO-spondin, defined by the Gibco BRL (Life Technologies, Rockville, MD, USA). following cDNA sequences, reverse, bases 149–168, DNA size marker for 1 kb was purchased from Sigma. sequence, 5V-TAGGGTCACCCTAGCTCCCT-3V and for- The following primers available in our laboratory were ward, bases 44–63, sequence, 5V-ACCTGCCACCTTGC- used: (1) bovine SCO-spondin, defined by the following CAGCCT-3V. Base numbers according to the partial cDNA cDNA sequences: reverse, bases 13,503–13,526, se- sequence from rat SCO-spondin obtained in the present quence, 5V-CACTGTAGCTCCCCGCGCTGGCAT-3V and report (GenBank accession number AY227384). The forward, bases 13,294–13,317, sequence, 5V-ACTG- expected amplification product has a size of 127 bp (Fig. 1A).

Fig. 1. Similarity analysis of a rat SCO-spondin partial sequence and the bovine SCO-spondin mRNA. The sequence of the bovine 233-bp fragment [29] was chosen between TSRs 23 and 24 of the SCO-spondin glycoprotein [18]. (A) Nucleotide sequence. (B) Amino acid sequence. Base/amino acid numbers are indicated at both sides of the sequence. Mismatches are indicated by bold font. In the upper panel, the upstream and downstream primers used in the present report are underlined. The sequence of the purified PCR product obtained from rat SCO RNA using the bovine amplimer pair showed 86% identity to that of the corresponding bovine SCO-spondin region. The 233-bp rat SCO-spondin–cDNA partial sequence obtained in the present report has been deposited in GenBank (accession number AY227384). Bov: bovine SCO-spondin; Rat: rat SCO-spondin; Asterisk: from Ref. [18]. H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 155

2.4.2. RNA isolation, RT-PCR amplification, and 3. Results sequencing All samples were subjected to tissue homogenization 3.1. Immunodetection of 5HT2A receptor in tissue sections with Trizol reagent for total RNA purification, according to and blots of bovine SCO the manufacturer’s instructions. One microgram of each RNA sample was digested with DNase I, reverse tran- A polyclonal antibody against a synthetic peptide scribed, subjected to PCR amplification, and analyzed on corresponding to amino acids 22–41 of the rat 5HT2A agarose gels as described by Richter et al. [29]. The 233-bp receptor was used to investigate the expression of this PCR product generated from rat SCO RNA, using the receptor subtype in the bovine and rat SCO. The ependymal primer pair specific for bovine SCO-spondin (see Section and hypendymal secretory cells of the bovine SCO showed 2.4.1 above), was pooled and run on a preparative agarose a strong immunoreaction, whereas the neighboring ciliated gel. The band corresponding to the expected 233-bp product was immunonegative (Fig. 2A,B and D). In the was cut off the gel and purified using a Concert Kit (Gibco SCO ependymal cells, the 5HT2A receptor immunoreactivity BRL). The purified 233-bp PCR product was quantified by was very strong at the apical cell pole (Fig. 2A,B); a specific digital densitometry after electrophoresis using HindIII- but weak immunoreaction was evident throughout the digested E-DNA as standard and by absorbance at 260 nm. cytoplasm of the ependymal and hypendymal cells (Fig. Double-strand sequencing of the PCR product was per- 2A,D). In the cerebral cortex, 5HT2A receptor immunor- formed by a custom sequencing service (Universidad de eactivity was detected in the perikaryon and dendrites of Concepcio´n, Chile). Subsequently, the partial cDNA pyramidal and nonpyramidal neurons (Fig. 2B, insert). sequence from rat SCO-spondin was used to design specific The Western blot analysis using the anti-5HT2A receptor primers (see Section 2.4.1 above). All primer pairs used in antibody revealed the expected 98-kDa 5HT2A receptor the present investigation rendered consistent results under polypeptide in extracts of both, fresh bovine SCO, and brain the PCR conditions described by Richter et al. [29], but the cortex (Fig. 2C, left panel). The 98-kDa 5HT2A receptor amplification yield of the rat SCO-spondin PCR fragment polypeptide continued to be expressed by the bovine SCO (127 bp) had to be improved by using PCRx enhancer cells after 15 days of organ culture, either in presence or system (1Â; Gibco BRL) and an annealing temperature of absence of 2 AM 5HT (Fig. 2C, right panel). 60 8C. 3.2. Serotonergic innervation of rat and bovine SCO 2.4.3. Semiquantitative analysis of specific mRNAs by RT- PCR The serotonergic innervation pattern of the bovine and rat To evaluate the effect of each treatment in a semi- SCO was investigated using a rabbit polyclonal antibody quantitative manner, we generated PCR efficiency curves against 5HT. In the two caudal thirds of the bovine SCO, based on template limit dilution. Appropriate dilutions were very few serotonergic fibers reached the SCO; some of them empirically determined for cDNA pools (containing cDNA innervated the hypendymal cells (Fig. 2E), while others aliquots from each experimental group), to ensure that the reached the basal region of the secretory ependymal cells resulting PCR products were derived only from the (Fig. 2E,H). In the cephalic third, close to the , exponential phase of the amplification. Under these 5HT fibers reaching the SCO cells were abundant. At conditions, the yield of PCR product was found to be variance, in the rat SCO, the immunolabeled serotonergic proportional to the input cDNA for both, SCO-spondin (Fig. fibers formed a dense basal plexus (Fig. 2F,G; cf. Ref. [20]). 3A) and GAPDH (not shown). For quantitative analysis, In this species, the serotonergic supraependymal plexus was aliquots of the PCR reaction were subjected to electro- prominent; the fibers of this plexus established contact with phoresis on a 1.5% agarose gel and the products were the free surface of the ciliated ependyma but the ventricular visualized by ethidium bromide staining. The gel image was surface of the SCO was free of serotonin fibers (Fig. 2F,G). digitalized and the band intensity quantified by means of digital densitometry (Scion Image Beta 4.0.2; available at 3.3. The SCO-spondin mRNA expression in cultured bovine Scion: http://www.scioncorp.com). SCO is down-regulated by 5HT

2.5. Data analysis After trying several concentrations of 5HT and periods of culture, it was determined that 2 AM 5HT has the highest The data were subjected to one-way ANOVA followed effect/dosage, and 15-day-old SCO explants as good by the post hoc Tukey test (SigmaStat, version 2.03; Access responsive ones. Having established the optimal cDNA Softek Inc) to determine the statistical significance of the working dilution (1:10; Fig. 3A), three types of SCO tissue relative values obtained for both, SCO-spondin mRNA, and samples were processed to analyze the SCO-spondin RF glycoproteins expression levels. Finally, the results were mRNA transcription rates: Group I, 800 SCO explants (8 plotted using GraphPad Prism (version 3.02; GraphPad lots of 100 explants each) were cultured in the absence of Software). 5HT; Group II, 800 SCO explants (8 lots of 100 explants 156 H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 each) were cultured in the presence of 2 AM 5HT for the overall total RNA extraction yield was 187F35 ng/explant whole culture period; Group III, 800 SCO tissue samples (8 (meanFS.D.). lots of 100 samples each) were obtained from fresh SCOs in The highest level of SCO-spondin mRNA was observed the same way as if they were used as explants for organ in explants cultured in the absence of 5HT (Group I; Fig. culture. All samples were subjected to RNA isolation. The 3B, lane 1; Fig. 3C); for the statistical analysis, this level H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 157

Fig. 3. Effect of 5HT on SCO-spondin mRNA transcription in bovine SCO explants. (A) Relationship between cDNA amplification (233-bp SCO-spondin PCR product using the bovine amplimer pair shown in Fig. 1A) and template dilution of a cDNA pool of each experiment from bovine SCO explants. The agarose gel band is shown under the corresponding point in the PCR efficiency curve. (B) Representative electrophoresis of PCR products showing the effect of 5HT on SCO-spondin mRNA transcription in fresh and cultured bovine SCO. The 233-bp RT-PCR product was amplified from DNase I-digested RNA (1 Ag) obtained from pools of 100 explants. Lanes 1, no 5HT 15d-explants; 2, fresh SCO; 3, 2 AM 5HT 15d-explants; 4, DNA size marker (1 kb, Sigma). (C) Semiquantitative densitometric analysis. There is an increase in the expression of SCO-spondin mRNA under conditions in which 5HT input to the SCO is missing or reduced (*Pb0.05). For each one of the conditions, each point represents the meanFS.E.M. of results from eight independent experiments of 100 explants each. Data are expressed as a percentage of SCO-spondin mRNA content measured after culture without 5HT, which was considered as 100%. One- way ANOVA followed by the post hoc Tukey test. was considered as 100% of transcriptional activity. The (out of three experiments) of these 5HT-treated explants, SCO-spondin mRNA expression in fresh samples of SCO control explants, and fresh SCO samples is shown in Fig. (Group III) was moderately but significantly lower as 4A. Note that we included the two fresh samples displaying compared to that of Group I (Fig. 3B, lane 2; Fig. 3C), the highest and lowest signals that better reflect the whereas in the SCO explants cultured in the presence of 2 dispersion shown in the middle column of Fig. 4B. AM 5HT during 15 days (Group II), the SCO-spondin The Western blot signal obtained for RF glycoproteins in transcriptional rate decreased to about half of that of Group I SCO explants cultured in absence of 5HT, being the highest, (Fig. 3B, lane 3; Fig. 3C). was considered as 100% expression for the statistical analysis. As compared to this value, the expression of RF 3.4. A significant decrease in the amount of immunoreactive glycoproteins in the fresh SCO was equivalent to 85%, and RF glycoproteins, SCO-spondin included, was detected in in the SCO explants cultured in the presence of 2 AM 5HT the bovine SCO cultured in the presence of 5HT was equivalent to 58% (Fig. 4B). Thus, there is a significant increase ( Pb0.001) in the expression of RF glycoproteins The impact of the decrease in the expression level of under conditions in which 5HT input to the SCO is missing SCO-spondin mRNA observed in SCO explants treated with or low. Worth noticing is the fact that in the three groups of 5HT on the translation rate of RF glycoproteins (SCO- explants, the relative amounts of immunoreactive RF spondin included) was investigated by immunoblotting, glycoproteins and of SCO-spondin mRNA correlate well; using an ECL system with antibodies against RF glyco- thus, as compared to the SCO cultured in the absence of proteins (AFRU). Seven pools (each pool containing the 5HT, the fresh SCO had a 15% decrease in the expression of explants from four SCOs) were cultured in the presence FR-glycoproteins and a 26% decrease in the amount of (four pools) or absence (three pools) of 2 AM 5HT during 15 SCO-spondin mRNA, whereas the explants cultured in the days. Two additional pools consisted of 50 tissue samples presence of 5HT had a 42% decrease in the expression of obtained from fresh SCOs in the same way as if they were to FR-glycoproteins and a 44% decrease in the amount of be used as explants. A representative Western blot analysis SCO-spondin mRNA.

Fig. 2. (A–D) Immunostaining of 5HT2A receptors in bovine SCO. (A) Frontal section through the bovine SCO showing the 5HT2A receptor immunoreactivity of the ependymal (E) and hypendymal (Hy) secretory cells. The immunostaining is strong at the apical pole of the SCO ependymocytes (arrow). The neighboring ependyma remains immunonegative (arrowhead). (B) Higher magnification of the SCO ependymal cells (E). The label appears mostly concentrated at the apical cell pole (thick arrow). A less intense 5HT2A receptor immunoreactivity is seen in the basal region of the secretory ependymal cells (thin arrow). (Insert) Control section of the bovine cerebral cortex displaying positive immunostaining reaction in the perikaryon (arrow) and dendrites of neurons. (C) Immunoblots of protein extracts obtained from fresh bovine SCO, brain cortex (SCO, CTX; left panel) and SCO explants cultured for 15 days in the presence or absence of 2 AM 5HT (right panel). The expected 98-kDa 5HT2A receptor polypeptide was detected in all samples. (D) Detail of 5HT2A receptor immunoreactive hypendymal SCO cells (Hy) surrounded by immunonegative fibers of the (PC). (E–H) Immunostaining of bovine and rat SCO with anti-5HT serum. (E) Only a few serotonergic fibers (arrows) reach the bovine SCO. Some of them appear to be innervating hypendymal cells (Hy) while others continue their course among secretory ependymal cells (E). The area framed by rectangle is shown at higher magnification in Fig. 2H. (F,G) The serotonergic fibers form a dense plexus located at the basal region of the ependymal layer of the rat SCO (thick arrow). Thin arrows point to the supraependymal serotonergic fibers contacting the ciliated ependyma. PC, posterior commissure. A: Â32, B: Â500 (insert: Â250), D: Â250, E: Â250, F: Â125; G: Â250; H: Â1200. 158 H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162

Fig. 4. Translation of RF-glycoproteins in fresh and cultured bovine SCO. (A) Representative Western blot of 5HT free (-5HT) and 5HT-treated explants. An enhanced chemiluminescence system with anti-RF-glycoproteins serum (AFRU) as first antibody was used. Each lane was loaded with an amount of protein equivalent to 0.1 SCO (i.e., 10% of a protein extract obtained from 1 SCO). (B) Semiquantitative densitometric analysis. There is an increase in the expression of RF-glycoproteins under conditions in which 5HT input to the SCO is missing or low (*, Pb0.001). For each one of the conditions, each point represents the meanFS.D. of results from four independent measurements for 5HT treatment, and from three independent measurements for untreated explants (this experiment was carried out three times). Data are expressed as a percentage of immunoreactive RF-glycoproteins content measured after culture without 5HT, which was considered as 100%. One-way ANOVA followed by the post hoc Tukey test.

3.5. 5HT does not impair release of RF glycoproteins by located at the 3V-end of the full-length bovine SCO-spondin cultured bovine SCO mRNA. Both sequences have a significant similarity at the nucleotide and amino acid level (86% identical; Fig. 1A,B, The observation under a fluorescence microscope of 15- respectively). Besides, bases 33–216 of the 233-bp rat day-old living explants that had been previously incubated SCO–spondin–cDNA partial sequence obtained in the in culture medium containing FITC-AFRU revealed a high present report (GenBank accession number AY227384) number of fluorescent clusters of secretory immunoreactive were found to be identical to bases 5316–5499 of the material on top of the moving cilia covering the explant 11,130-bp linear mRNA sequence defined as Rattus surface (cf. Ref. [39]). When the same experiment was norvegicus similar to SCO-spondin B. taurus mRNA at performed using explants cultured for 15 days in the GenBank (accession number XM_231721). Based on the rat presence of 2AM 5HT, fluorescent clusters on the surface SCO-spondin partial sequence obtained in the present of the explants were also observed. After taking photo- report, we designed an amplimer pair (rat SCO-spondin graphs, these living explants were fixed, embedded, and cut. primers; Fig. 1A) that amplifies a 127-bp PCR product. Adjacent paraffin sections were processed for (1) complete Despite the moderate size of this PCR fragment, its high GC immunostaining using AFRU as primary antibodies; this content (60%) demanded the use of dimethyl sulfoxide as allowed to visualize intracellular and extracellular SCO cosolvent (1Â Enhancer PCRx, Gibco BRL) to generate secretory material (Fig. 5B); (2) incomplete immunostaining efficient amplification conditions. using anti-rabbit IgG as primary antibody; this was used to A representative experiment comparing the SCO-spondin detect the AFRU antibodies added to the culture and that mRNA transcriptional activity in the SCO of PCPA-treated had reacted with aggregated extracellular secretory material (i.e., 5HT-depleted) and control rats is shown in Fig. 6. The released by the SCO (Fig. 5C,D). Both the SCO explants results obtained from three pools (five SCOs per pool) of cultured in the presence (Fig. 5C,D) or absence (not shown) PCPA-treated rats and three pools (five SCOs per pool) of of 5HT displayed extracellular secretory material released control rats, expressed as coefficients SCO-spondin/ by the ependymocytes. This nonquantitative experimental GAPDH (PCPA) and SCO-spondin/GAPDH (PBS), were approach provides new evidence clearly indicating that 2.90F0.14 and 1.55F1.06 (meansFS.D.), respectively. under the effect of 5HT, the explants of SCO continue to Hence, the SCO-spondin mRNA expression level was release RF glycoproteins. 1.87-fold higher in 5HT-depleted animals. The immunostaining of SCO ependymocytes using To exclude the possibility of genomic DNA amplifica- antibodies against RF glycoproteins rendered a similar tion, all the RNA templates (1 Ag) were predigested with labeling pattern and intensity under both in vivo (Fig. 5A) DNase I. Finally, omission of either RNA template or and in vitro (Fig. 5B) conditions. reverse transcriptase rendered no signal (not shown).

3.6. The SCO-spondin mRNA expression in the in vivo rat SCO is down-regulated by 5HT 4. Discussion

Next, we investigated the SCO-spondin mRNA expres- The regulatory role of 5HT on the SCO secretory activity sion rate in an experimental living animal. To perform this has been studied in some species mainly by means of study, a partial sequence of the rat SCO-spondin mRNA was morphological analyses after chemical serotonergic dener- amplified using primers derived from a 233-bp fragment vation or pharmacological depletion of serotonin neurons. H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 159

which one of the serotonin receptor subtypes is expressed by the SCO. The present report provides immunological evidence that the bovine SCO expresses 5HT2A receptors (and also 5HT2C receptors; A.J.J. and J.M.P-F., unpublished results) and that 5HT down-regulates SCO-spondin tran- scription and decreases the amount of its translation products, but without apparently interfering with their release. The inhibitory role of 5HT was further confirmed by pharmacological depletion of 5HT in rats, which exhibited a SCO-spondin mRNA level significantly higher than that of untreated rats. The pharmacological and molecular characterization of the 5HT receptors operating in the SCO needs to be investigated.

4.1. The bovine SCO expresses 5HT2A receptors. Serotonin can reach the SCO through a direct neural input or via the CSF

Although the SCO is an ancient brain gland and it develops early in ontogeny [28], a direct serotonergic innervation is a postnatal event of mammalian species [33]. This rises the question of the nature of the mechanism controlling the secretory activity of the SCO of the nonmammalian species and of mammalian embryos. It has been suggested that in these species, the control of the SCO could be mediated by compounds present in the CSF, serotonin in particular [14,33,35]. Among mammals, there are marked differences in the serotonergic innervation pattern of SCO [14]. Thus, most of the hypendymal and ependymal secretory cells of the bovine SCO are devoid of a serotonergic neural input (Ref. [14] and the present investigation). Indeed, 5HT fibers are scarce in the two distal thirds of the SCO,

Fig. 5. Immunocytochemistry of fresh and cultured bovine SCO using antisera against RF-glycoproteins (AFRU). (A) Sagittal section of a SCO collected and fixed shortly after death. Immunostaining using AFRU. Intracellular material located in the ependymal (E) and hypendymal (H) cells, and released material aggregated on the surface of the organ (arrows) is strongly reactive. (B–D) SCO explant cultured during 15 days in the presence of 2 AM 5HT, and further cultured for 1 h in the presence of AFRU added to the culture medium. Adjacent serial sections were processed for bcomplete immunostainingQ using AFRU (B) as primary antiserum, and for bincomplete immunostainingQ using anti-rabbit IgG as primary antibody (C). In B, both the intracellular (E) and released (arrow) secretory material is visualized. In C, only the released material that had reacted with AFRU added to the culture medium, is visualized (arrow). (D) Detailed magnification of C, showing the released material labeled in vivo (arrows). n, ; asterisk, supranuclear cytoplasm of SCO Fig. 6. Effect of 5HT on SCO-spondin mRNA expression in rat SCO in ependymocytes. A: Â250, B–C: Â200, D: Â1250. vivo. Total RNA (1 Ag) obtained from the SCO of five-rat pools (n=3 pools for each condition) were subjected to RT-PCR analysis to amplify the expected 127-bp PCR product using the rat amplimer pair shown in Fig. An intriguing observation was that the same treatment (5HT 1A. Lanes and treatments: 1, GAPDH (PBS); 2, SCO-spondin (PBS); 3, pharmacological depletion) rendered opposite results in the SCO-spondin (PCPA); 4, GAPDH (PCPA); 5, DNA size marker (500pb; animals studied [16,37]. No quantitative data were available Gibco BRL). A representative experiment is shown (out of three; the up to now, and there was no possibility to distinguish statistical analysis provided no evidence for a nonspecific effect of the PCPA treatment on GAPDH transcription). Coefficients SCO-spondin/ whether the reported effects of 5HT operated on tran- GAPDH (PCPA) and SCO-spondin/GAPDH (PBS) were 2.90F0.14 and scription, translation, or release of the SCO secretory 1.55F1.06 (meansFS.D.), respectively; the SCO-spondin mRNA expres- glycoproteins. Furthermore, there was no information about sion level was 1.87-fold higher in 5HT-depleted animals. 160 H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 whereas in the cephalic third—close to the pineal gland— 4.2. SCO-spondin gene expression is down-regulated by they are numerous (see Ref. [40]). In contrast, the 5HT serotonergic innervation of rat SCO is prominent, forming a conspicuous basal plexus (Ref. [20]; present report). The following findings indicate that 5HT-responsiveness Besides, in this species, the ciliated ependyma lining the may be different depending on the species. Using histo- lateral walls of the third ventricle displays a dense plexus chemical techniques to stain the cat SCO, Sakumoto et al. of serotonergic supraependymal fibers; these fibers could [37] described a decrease of the stainable material after be the source of 5HT that is normally present in the CSF administration of PCPA (a potent inhibitor of 5HT syn- [7]. The use of specific antibodies has allowed to detect thesis), whereas according to Le´ger et al. [16], the same 5HT2A (present report) and 5HT2C (A.J.J. and J.M.P-F., treatment increased the amount of stainable secretory unpublished results) receptors in the bovine and mouse material in the rat SCO. Because no specific methods to SCO. The 5HT2A receptor immunoreactivity was also demonstrate the secretory material of the SCO were used, detected in those neurons of the cerebral cortex that are the morphological analyses carried out by these authors do known to express 5HT2A receptors, supporting the not allow to draw a reliable conclusion. Ultrastructural specificity of this antibody for the 5HT2A receptor. In evidence has been obtained, indicating a significant volume the bovine SCO, the serotonergic fibers penetrating the increase of the rough in the SCO gland basally are scarce; however, there is a strong cells after chemical serotonergic denervation in rats [19], 5HT2A-receptor immunoreactivity at the apical cell pole and under organ culture condition, which implies a complete bathed by CSF (present report), thus supporting the denervation of the explanted SCO [39]. These results have possibility that in this mammalian species the 5HT input been interpreted as an indication of increased rate of to the SCO would be through the ventricular CSF. A synthesis of secretory material after denervation. On the serotonergic control of the SCO via the CSF is further other hand, the rat SCO grafted under the kidney capsule is supported by the fact that 5HT, when added to the culture not reinnervated and its ultrastructure and immunoreactivity medium, inhibited the biosynthesis of RF glycoproteins— to antibodies against RF glycoproteins did not show SCO-spondin included—in the organ cultured bovine indications of an increased secretory activity [32]. These SCO (present report). findings led the authors to challenge the putative inhibitory The 5HT2A/2C receptors are coupled to multiple role of 5HT on the SCO secretory activity postulated by effector pathways, the best characterized being phospho- Mbllgard et al. [19]. lipase C-phosphatidylinositol hydrolysis and phospholi- Recent publications have focused on immunoreactivity pase A2-arachidonic acid release [3].5HT2A/2C receptor variations derived from age/interspecies differences and/or activation may not only change the status of inositol experimental conditions, regarding the 5HT input to the phosphates and arachidonic acid, but also affect Ca2+, SCO in lizards [1] and semidesert rodents [15]. Both ClÀ,Na+,K+, cAMP, and/or PKCa; depending on the publications reported a decreased amount of immunoreac- cellular context and physiological state [10]. However, a tive secretory material under conditions of low/absent 5HT signal transduction pathway coupled to 5HT2A/2C recep- input. 2+ tors not involving [Ca ]i or cAMP changes is difficult to The present results show a significant increase in the envisage. A recent report has shown that 5HT does not expression of the SCO-spondin mRNA (encoding for the 2+ trigger changes in [Ca ]i or cAMP in bovine SCO cells major glycoprotein secreted by the SCO) and also in the collected freshly or obtained from explants cultured for amount of RF glycoproteins present in the SCO, under months [40]. The authors suggested that 5HT2, 5HT4, experimental conditions in which 5HT input to the SCO is 5HT6, and/or 5HT7 receptors would be missing in the reduced or missing, indicating a down-regulation at least at SCO. However, in the present investigation, 5HT2A the transcriptional level. This inhibitory control mediated by receptors have been detected by immunocytochemistry 5HT may derive from two input pathways, the serotonergic and immunoblotting in the fresh bovine SCO. We have fibers innervating the SCO and/or the 5HT present in the no explanation to offer for this discrepancy with CSF (see above). Schfniger et al.’s [40] findings in fresh SCO cells; in the case of SCO cells obtained from cultured SCO, the 4.3. 5HT negative control of SCO-spondin gene expression 2+ lack of effect of 5HT on [Ca ]i could be explained by a seems to operate on transcription, but not on translation receptor down-regulation under prolonged culture con- and/or release ditions (months). This latter possibility led to the experimental design used in the present investigation, in A decrease in the level of SCO-spondin mRNA and RF which the SCO was organ cultured for a shorter period glycoproteins (SCO-spondin included) in SCO explants (15 days) in the presence or absence of 5HT. Under these cultured in the presence of 5HT was detected. These conditions, the expected 5HT2A receptor polypeptide was findings support the possibility that 5HT activates a second consistently immunodetected in protein extracts of the messenger cascade leading to transcriptional repression. SCO explants. Indeed, the high correlation between the extent to what H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162 161 transcriptional and translational rates are decreased is CYT, Chile, and FIS PI030756 and Red Cien to J.M.P.-F., remarkable, and indicates that the reduced RF glycoproteins from Instituto de Salud Carlos III and Servicio Andaluz de (SCO-spondin included) level derives from SCO-spondin Salud, Spain. H.G.R was a recipient of a fellowship from transcriptional down-regulation. CONICYT, Chile. It must be kept in mind that SCO-spondin secretion may be controlled by mechanisms other than serotonergic input, because this protein appears to be involved in different References functions [17,33,35], and that in fact several peptidergic and neurotransmitter systems innervate the SCO secretory cells [1] S. Ahboucha, M. Didier-Bazes, A. Meiniel, M. Fe`vre-Montagne, H. and thus may regulate SCO activity [14,27]. However, the Gamrani, Secretory activity and serotonin innervation of subcommis- present results, together with the observation that the SCO sural organ, NeuroReport 11 (2000) 1821–1825. in some mammalian species is richly innervated by [2] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. serotonergic fibers and poorly innervated by several other Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. neural systems [14,27], indicate that 5HT would play a 25 (1997) 3389–3402. prominent role in the regulation of the SCO secretory [3] K.A. Berg, S. Maayani, W.P. Clarke, Interactions between effectors activity. Given that the bovine SCO expresses at least two linked to serotonin receptors, Ann. N. Y. Acad. Sci. 861 (1998) 111–120. 5HT receptor subtypes (5HT2A, present report; and 5HT2C, A.J.J. and J.M.P-F., unpublished results), it cannot be [4] M.M. Bradford, A rapid and sensitive method for the quantitation of micrograms quantities of protein utilizing the principle of protein–dye excluded that more than one 5HT receptor subtype may binding, Anal. Biochem. 72 (1976) 248–254. account for the inhibitory effect of 5HT on the SCO activity. [5] T. Caprile, S. Hein, S. Rodrı´guez, H. Montecinos, E. Rodrı´guez, In this context, an interesting observation was that 5HT Reissner fiber binds and transports away monoamines present in the down-regulates SCO-spondin gene transcription to a similar cerebrospinal fluid, Mol. Brain Res. 110 (2003) 177–192. extent in both in vitro bovine SCO and in vivo rat SCO. On [6] G. Cattoretti, S. Pileri, C. Parravicini, M.H. Becker, S. Poggi, C. Bifulco, G. Key, L. D’Amato, E. Sabattini, E. Feudale, Antigen the other hand, the similar rate of SCO-spondin transcrip- unmasking on formalin-fixed, paraffin-embedded tissue sections, J. tional down-regulation displayed by the bovine and rat SCO Pathol. 171 (1993) 83–98. is surprising considering their marked differences in the [7] V. Chan-Palay, Serotonin in the supra- and subependymal amount of serotonergic fibers innervating these cells. Hence, plexuses and in the leptomeninges; their roles in local alterations of the possibility that 5HT reaching the SCO from the CSF, cerebrospinal fluid and vasomotor activity, Brain Res. 102 (1976) 103–130. rather than from a direct innervation, may account for most [8] R. Didier, I. Creveaux, R. Meiniel, A. Herbet, B. Dastugue, A. of its effect on SCO activity in different species, must be Meiniel, SCO-spondin and RF-Gly I: two designations for the same considered. glycoprotein secreted by the subcommissural organ, J. Neurosci. Res. To better understand the regulatory role of 5HT in the 61 (2000) 500–507. SCO secretory pathway, we also explored the pharmaco- [9] G. Estivill-Torrus, M. Cifuentes, J.M. Grondona, E. Miranda, F.J. Bermu´dez-Silva, P. Ferna´ndez-Llebrez, J. Pe´rez, Quantification of the logical effect of this neurotransmitter on the release of SCO secretory glycoproteins of the subcommissural organ by a sensitive secretory material. Explants of bovine SCO cultured for sandwich ELISA with a polyclonal antibody and a set of monoclonal several weeks and months secrete RF glycoproteins into the antibodies against the bovine Reissner’s fiber, Cell Tissue Res. 294 culture medium [9,39]. By using the experimental approach (1998) 407–413. introduced by Schoebitz et al. [39]—to visualize aggregated [10] C.C. Gerhardt, H. Heerikhuizen, Functional characteristics of heterologously expressed 5-HT receptors, Eur. J. Pharmacol. 334 extracellular secretory material released by the SCO—the (1997) 1–23. present investigation has shown that bovine SCO explants [11] S. Gobron, H. Monnerie, R. Meiniel, I. Creveaux, W. Lehmann, D. cultured in the presence of 5HT continue to secrete RF Lamalle, B. Dastugue, A. Meiniel, SCO-spondin: a new member of glycoproteins to the culture medium, suggesting that this the thrombospondin family secreted by the subcommissural organ is a neurotransmitter does not interfere with the release mech- candidate in the modulation of neuronal aggregation, J. Cell. Sci. 109 (1996) 1053–1061. anism of these proteins. [12] S. Gobron, I. Creveaux, R. Meiniel, R. Didier, A. Herbet, M. Bamdad, In brief, the present results provide direct evidence F. El Bitar, B. Dastugue, A. Meiniel, Subcommissural organ/Reissner’s indicating that 5HT down-regulates SCO-spondin biosyn- fiber complex: characterization of SCO-spondin, a glycoprotein with thesis while it would not affect its release, and suggest that potent activity on neurite outgrowth, Glia 32 (2000) 177–191. such a regulatory input may reach the SCO via the CSF. The [13] J. Hess, G. Sterba, Studies concerning the function of the complex subcommissural organ–liquor fibre: the binding ability of the liquor pharmacological and molecular characterization of the 5HT fibre to pyrocatechin derivatives and its functional aspects, Brain Res. receptors operating in the SCO needs to be investigated. 58 (1973) 303. [14] A.J. Jime´nez, P. Ferna´ndez-Llebrez, J.M. Pe´rez-Fı´gares, Neural input and neural control of the subcommissural organ, Microsc. Res. Tech. Acknowledgements 52 (2001) 520–533. [15] A. Laalaoui, S. Ahboucha, M. Didier-Bazes, M. Fe`vre-Montagne, A. Meiniel, H. Gamrani, Postnatal secretion of the subcommissural organ This work was supported by grants 2990011 and of the Meriones shawi: control of serotonin innervation, Dev. Brain 4000001 to H.G.R. and 1000435 to E.M.R., from FONDE- Res. 126 (2001) 75–80. 162 H.G. Richter et al. / Molecular Brain Research 129 (2004) 151–162

[16] L. Le´ger, A. Degueurce, J.J. Lundberg, J.F. Pujol, K. Mbllgard, Origin [32] E.M. Rodrı´guez, S. Rodrı´guez, K. Schoebitz, C.R. Yulis, P. Hoffmann, and influence of the serotoninergic innervation of the subcommissural V. Manns, A. Oksche, Light- and electron-microscopic investigation organ in the rat, Neuroscience 10 (1983) 411–423. of the rat subcommissural organ grafted under the kidney capsule, [17] A. Meiniel, R. Meiniel, R. Didier, I. Creveaux, S. Gobron, H. with particular reference to immunocytochemistry and lectin histo- Monnerie, B. Dastugue, The subcommissural organ and Reissner’s chemistry, Cell Tissue Res. 258 (1989) 499–514. fiber complex: an enigma in the central nervous system? Prog. [33] E.M. Rodrı´guez, A. Oksche, S. Hein, R. Yulis, Cell biology of the Histochem. Cytochem. 30 (1996) 1–66. subcommissural organ, Int. Rev. Cytol. 135 (1992) 39–121. [18] A. Meiniel, SCO-spondin, a glycoprotein of the subcommissural [34] E.M. Rodrı´guez, P. Jara, H. Richter, H. Montecinos, B. Fla´ndez, R. organ/Reissner’s fiber complex: evidence of a potent activity in Wiegand, A. Oksche, Evidence for the release of CSF-soluble neuronal development in primary cell cultures, Microsc. Res. Tech. 52 secretory material from the subcommissural organ, with particular (2001) 484–495. reference to the situation in human, in: A. Oksche, E.M. Rodrı´guez, P. [19] K. Mbllgard, J.J. Lundberg, L. Wiklund, L. Lochenmajer, H.G. Ferna´ndez-Llebrez (Eds.), The Subcommissural Organ. An Ependy- Baumgarten, Morphological consequences of serotonin neurotoxin mal Brain Gland, Springer, Berlin, 1993, pp. 121–131. administration: neurontarget cell interaction in the rat subcommissural [35] E.M. Rodrı´guez, S. Rodrı´guez, S. Hein, The subcommissural organ, organ, Ann. N. Y. Acad. Sci. 305 (1978) 262–288. Microsc. Res. Tech. 41 (1998) 98–123. [20] K. Mbllgard, L. Wiklund, Serotoninergic synapses on ependymal and [36] S. Rodrı´guez, K. Vio, C. Wagner, M. Barrı´a, E.H. Navarrete, V.D. hypendymal cells of the rat subcommissural organ, J. Neurocytol. 8 Ramı´rez, J.M. Pe´rez-Fı´gares, E.M. Rodrı´guez, Changes in the (1979) 445–467. cerebrospinal-fluid monoamines in rats with an immunoneutralization [21] H. Monnerie, O. Boespflug-Tanguy, B. Dastugue, A. Meiniel, of the subcommissural organ-Reissner’s fiber by maternal delivery of Reissner’s fiber supports the survival of chick-cortical neurons in antibodies, Exp. Brain Res. 128 (1999) 278–290. primary mixed cultures, Cell Tissue Res. 282 (1995) 81–91. [37] T. Sakumoto, H. Sakai, D. Salvert, H. Sakai, H. Kimura, T. Maeda, M. [22] H. Monnerie, B. Dastugue, A. Meiniel, In vitro differentiation of chick Jouvet, Possible role of serotonin in the secretory activity of the spinal cord neurons in the presence of Reissner’s fibre, an ependymal subcommissural organ of the cat, Neurosci. Res. 1 (1984) 191–197. brain secretion, Dev. Brain Res. 102 (1997) 167–176. [38] K. Schoebitz, O. Garrido, M. Heinrichs, L. Speer, E.M. Rodrı´guez, [23] H. Monnerie, B. Dastugue, A. Meiniel, Effect of synthetic peptides Ontogenetical development of the chick and duck subcommissural derived from SCO-spondin conserved domains on chick cortical and organ, Histochemistry 84 (1986) 31–40. spinal-cord neurons in cell cultures, Cell Tissue Res. 293 (1998) [39] K. Schoebitz, C. Gonza´lez, B. Peruzzo, C.R. Yulis, E.M. Rodrı´guez, 407–418. Organ culture of the bovine subcommissural organ. Evidence for [24] W.W. Naumann, Immunohistochemische untersuchungen zur onto- synthesis and release of the secretory material, Microsc. Res. Tech. 52 genese des subcommissuralorgans, Acta Histochem., Suppl. 33 (1986) (2001) 496–509. 265–272. [40] S. Schfniger, M.D.A Kopp, C. Schomerus, E. Maronde, F. Dehghani, [25] F. Nualart, S. Hein, E.M. Rodrı´guez, A. Oksche, Identification and A. Meiniel, E.M. Rodrı´guez, H.-W. Korf, F. Nqrnberger, Effects of partial characterization of the secretory glycoproteins of the bovine neuroactive substances on the activity of subcommissural organ cells subcommissural organ–Reissner’s fiber complex. Evidence for the in dispersed cell and explant cultures, Cell Tissue Res. 307 (2002) existence of two precursor forms, Mol. Brain Res. 11 (1991) 227–238. 101–114. [26] F. Nualart, S. Hein, C.R. Yulis, A.M. Za´rraga, A. Araya, E.M. [41] L.A. Sternberger, P.H. Hardy Jr., J.J. Cuculis, H.G. Meyer, The Rodrı´guez, Partial sequencing of Reissner’s fiber glycoprotein I (RF- unlabeled antibody enzyme method of immunohistochemistry. Prep- Gly I), Cell Tissue Res. 292 (1998) 239–250. aration and properties of soluble antigen–antibody complex (horse- [27] F. Nqrnberger, S. Schfniger, Presence and functional significance of radish-peroxidase-anti peroxidase) and its use in identification of neuropeptide and neurotransmitter receptors in subcommissural organ spirochetes, J. Histochem. Cytochem. 18 (1970) 315–333. cells, Microsc. Res. Tech. 52 (2001) 534–540. [42] Y. Terada, K. Tomita, H. Nonoguchi, T. Yang, F. Marumo, Different [28] R. Olsson, Reissner’s fiber mechanisms: some common denominators, localization and regulation of two types of receptor in: A. Oksche, E.M. Rodrı´guez, P. Ferna´ndez-Llebrez (Eds.), The messenger RNA in microdissected rat nephron segments using reverse Subcommissural Organ. An Ependymal Brain Gland, Springer, Berlin, transcription polymerase chain reaction, J. Clin. Invest. 92 (1993) 1993, pp. 33–39. 2339–2345. [29] H.G. Richter, R.I. Mun˜oz, C.S. Milla´n, M.F. Guin˜azu´, C.R. Yulis, [43] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of E.M. Rodrı´guez, The floor plate cells from bovines express the proteins from polyacrylamide gels to nitrocellulose sheets: procedure mRNA encoding for SCO-spondin and its translation products, Mol. and some applications, Proc. Natl. Acad. Sci. U. S. A. 76 (1979) Brain Res. 93 (2001) 137–147. 4350–4354. [30] S. Rodrı´guez, T. Caprile, Functional aspects of the subcommissural [44] G.R. Walker, K.D. Feather, P.D. Davis, K.K. Hines, SuperSignalTM organ–Reissner’s fiber complex with emphasis in the clearance of CL-HRP: a new enhanced chemiluminescent substrate for the brain monoamines, Microsc. Res. Tech. 52 (2001) 564–572. development of the horseradish peroxide label in Western blotting [31] E.M. Rodrı´guez, A. Oksche, S. Hein, S. Rodrı´guez, R. Yulis, applications, J. NIH Res. 7 (1995) 76. Comparative immunocytochemical study of the subcommissural organ, Cell Tissue Res. 237 (1984) 427–441.