RESUMO
The subcommissural organ (SCO) is a brain circumventricular organ formed by ependymal and hypendymal secretory cells. It secretes glycoproteins into the cerebrospinal fluid of the third ventricle where they condense into a thread-like structure known as Reissner's fiber (RF). The present study was designed to investigate whether or not the bovine SCO continues to synthesize and release glycoproteins after a long-term culture. Cultured explants of SCO survive for several months. The content of the secretory granules present in the cultured ependymocytes displayed immunoreactive and lectin-binding properties similar to those of the core glycosylated glycoproteins found in the bovine SCO. The explants actively incorporated (35)S-cysteine. In the cultured ependymocytes, the pattern of distribution of the radioactive label and that of the immunoreactive secretory material was similar, thus indicating that this material has been synthesized during culture. At the ultrastructural level, the cultured tissue exhibited a high degree of differentiation comparable to that of the bovine SCO in situ. A striking finding was the observation of similar results when cerebrospinal fluid was used as a culture medium. The addition of antibodies against RF-glycoproteins into the culture medium allowed visualization, by means of different immunocytochemistry protocols, deposits of extracellular immunoreactive secretory material on the free surface of the cultured ependymocytes, indicating that release of secretory glycoproteins into the culture medium does occur. Primary culture of dispersed SCO ependymocytes, obtained either from fresh or organ cultured bovine SCO, showed that these cells release RF-glycoproteins that aggregate in the vicinity of each cell. The present investigation has shown that: (1) two types of secretory ependymocytes become evident in the cultured SCO; (2) under culture conditions, the SCO cells increase their secretory activity; (3) explants of bovine SCO synthesize RF-glycoproteins and release them to the culture medium; (4) after release these proteins aggregate but do not form a RF; (5) a pulse of anti-RF antibodies into the culture medium blocks the secretion of RF-glycoproteins for several days.
Assuntos
Órgão Subcomissural/crescimento & desenvolvimento , Órgão Subcomissural/metabolismo , Animais , Bovinos , Células Cultivadas , Líquido Cefalorraquidiano , Meios de Cultura , Meios de Cultura Livres de Soro , Epêndima/citologia , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Técnicas de Cultura de Órgãos/métodos , Órgão Subcomissural/ultraestrutura , Fatores de TempoRESUMO
The cell bodies of hypothalamic secretory neurons are localized in areas protected by the blood-brain barrier (BBB), whereas their axon terminals are localized in the median eminence, which lacks a BBB. This implies a complex barrier system, allowing neurons of the central nervous system to secrete into the blood stream without making the BBB leaky. In the present study, three experimental protocols were applied to clarify certain relevant aspects of the barriers operating in the medial basal hypothalamus of the rat. We established that the milieu of the arcuate nucleus is exposed to both the ventricular and the subarachnoidal cerebrospinal fluid (CSF). The median eminence milieu, the perivascular space of the portal vessels, and the subarachnoid space appear to be in open communication; also, beta2-tanycytes establish an efficient barrier between the median eminence milieu and the ventricular CSF. Similarly, beta1-tanycytes establish a lateral barrier, separating the intercellular space of the median eminence from that of the arcuate nucleus. We also found that the glucose transporter I (GLUT I), a BBB marker, is localized throughout the whole plasma membrane of beta1-tanycytes, but is missing from beta2-tanycytes. Expression of GLUT I by tanycytes progressively develops during the first postnatal weeks; while the degree of damage of the arcuate nucleus by administration of monosodium glutamate, at different postnatal intervals, parallels that of the GLUT I immunoreactivity of beta1-tanycytes. An explanation is offered for the selective destruction of the arcuate neurons by the parenteral administration of monosodium glutamate to infant rats.
Assuntos
Barreira Hematoencefálica/fisiologia , Hipotálamo Médio/irrigação sanguínea , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/fisiologia , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Núcleo Arqueado do Hipotálamo/patologia , Feminino , Transportador de Glucose Tipo 1 , Peroxidase do Rábano Silvestre/farmacocinética , Hipotálamo Médio/citologia , Hipotálamo Médio/crescimento & desenvolvimento , Hipotálamo Médio/metabolismo , Técnicas In Vitro , Masculino , Eminência Mediana/irrigação sanguínea , Eminência Mediana/citologia , Eminência Mediana/efeitos dos fármacos , Eminência Mediana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Ratos , Ratos Endogâmicos , Glutamato de Sódio/farmacologia , Distribuição TecidualRESUMO
The subcommissural organ (SCO) is a brain gland that secretes glycoproteins into the cerebrospinal fluid (CSF). It is an ancient and conserved secretory structure of the brain, developing very early in ontogeny. However, the function of the SCO is unknown. The secretory cells of the SCO are arranged into a single or double, irregularly shaped layer located at the interface of the CSF and nervous tissue. This has prevented its selective surgical destruction. The present investigation was designed to destroy the secretory cells of 30-day-old explants of bovine SCO by use of an immunological approach. A membrane preparation enriched with plasma membrane of the secretory cells of the bovine SCO was obtained. This preparation was further processed to separate the structural proteins. A similar procedure was applied to obtain a fraction of integral proteins of the plasma membrane of a nonsecretory ciliated ependyma. Antisera were prepared against both preparations of integral proteins. The antiserum against the fraction obtained from the SCO cells immunostained the plasma membrane of the bovine SCO cells and in immunoblot it reacted with several proteins of the membrane preparation from SCO cells. When added to the culture medium this antibody bound to the apical plasma membrane of the secretory ependyma of the bovine SCO kept in culture; it caused the lysis of these cells when used together with complement. None of these properties were displayed by the antiserum raised against the integral proteins of the plasma membrane of the ciliated ependyma. This antiserum, however, immunostained the bovine ciliated ependyma neighboring the SCO. These results indicate that immunological surgery of the SCO in living animals may be possible to achieve.
Assuntos
Órgão Subcomissural/citologia , Órgão Subcomissural/imunologia , Animais , Especificidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Western Blotting , Bovinos , Membrana Celular/química , Células Cultivadas , Plexo Corióideo/química , Plexo Corióideo/imunologia , Eletroforese em Gel de Poliacrilamida , Epêndima/citologia , Imuno-Histoquímica , Proteínas de Membrana/análise , Microscopia Eletrônica , Sistemas Neurossecretores/citologia , Coelhos , Ratos , Órgão Subcomissural/ultraestruturaRESUMO
The subcomissural organ (SCO) is an ancient and conserved brain gland secreting glycoproteins into the cerebrospinal fluid which condense to form Reissner's fiber (RF). The SCO of an elasmobranch species, the dogfish Scyliorhinus canicula, was investigated applying morphological and biochemical methods. The SCO of 34 dogfishes were processed for the following techniques: (1) conventional transmission electron microscopy; (2) light and electron microscopy lectin histochemistry (Concanavalin A, Con A; wheat germ agglutinin, WGA; Limax flavus agglutinin, LFA); (3) light and electron microscopy immunocytochemistry using antisera raised against the glycoproteins of the bovine RF (anti-bovine RF), and the secretory material of the dogfish SCO (anti-dogfish SCO). The former reacts with the SCO of virtually all vertebrate species [19] (conserved epitopes); the latter reacts only with the SCO of elasmobranchs [Cell Tissue Res., 276 (1994) 515-522] (class-specific epitopes). At the light microscopic level both antisera immunoreacted selectively with the SCO and RF; no other structure of the central nervous system was reactive. Within the SCO the binding sites for WGA (affinity = glucosamine, sialic acid) and LFA (affinity = sialic acid) displayed the same density and intracellular distribution. At the ultrastructural level two types of granules were distinguished. Type I granules (200-400 nm) were numerous, reacted with both antisera, bound WGA but not Con A. Type II granules (0.8-1.8 microns) reacted with the anti-bovine RF serum but not with the anti-dogfish SCO serum, bound Con A and WGA. The content of dilated cisternae of the rough endoplasmic reticulum reacted with both antisera and bound Con A; it did not bind WGA. The SCOs of 4500 dogfishes were extracted in ammonium bicarbonate. This extract was used for SDS-PAGE and blotting. Blots were processed for immunolabeling using anti-bovine RF and anti-dogfish SCO sera, and for lectin binding (Con A, WGA and LFA). The anti-bovine RF revealed four compounds with apparent molecular weights of 750, 380, 145 and 35 kDa. The two former also reacted with the anti-dogfish SCO serum and bound Con A. Only the 380 kDa compound bound WGA and LFA. The findings indicate that both the conserved and the class-specific epitopes are part of the same compounds (780, 380 kDa), which would be stored in type I granules. The lectin binding properties of these compounds point to the 780 kDa compound as a precursor form and the 380 kDa polypeptide as a processed form.