Molecular Brain 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 serotonin 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 third ventricle that releases glycoproteins into the cerebrospinal fluid, 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 central nervous system 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 cerebral aqueduct, 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 raphe nuclei 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 brains (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.