Serge Herbuté

Ependymal and choroidal cells in culture: Characterization and functional differentiation

During the past 10 years, our teams developed long‐term primary cultures of ependymal cells derived from ventricular walls of telencephalon and hypothalamus or choroidal cells (modified ependymal cells) derived from plexuses dissected out of fetal or newborn mouse or rat brains. Cultures were established in serum‐supplemented or chemically defined media after seeding on serum‐, fibronectin‐, or collagen‐laminin‐coated plastic dishes or semipermeable inserts. To identify and characterize cell types growing in our cultures, we used morphological features provided by phase contrast, scanning, and transmission electron microscopy. We used antibodies against intermediate filament proteins (vimentin, glial fibrillary acidic protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin, and fibronectin in single or double immunostaining, and monoclonal antibodies against epitopes of ependymal or endothelial cells, to recognize ventricular wall cell types with immunological criteria. Ciliated or nonciliated ependymal cells in telencephalic cultures, tanycytes and ciliated and nonciliated ependymal cells in hypothalamic cultures always exceeded 75% of the cultured cells under the conditions used. These cells were characterized by their cell shape and epithelial organization, by their apical differentiations observed by scanning and transmission electron microscopy, and by specific markers (e.g., glial fibrillary acidic protein, ciliary rootlet proteins, DARPP 32) detected by immunofluorescence. All these cultured ependymal cell types remarkably resembled in vivo ependymocytes in terms of molecular markers and ultrastructural features. Choroidal cells were also maintained for several weeks in culture, and abundantly expressed markers were detected in both choroidal tissue and culture (Na+‐K+‐dependent ATPase, DARPP 32, G proteins, ANP receptors). In this review, the culture models we developed (defined in terms of biological material, media, substrates, duration, and subculturing) are also compared with those developed by other investigators during the last 10 years.

Multiple-Unit Activity in the Pineal Gland of the Japanese Quail: Spontaneous Firing and Responses to Photic Stimulations

The present study was devoted to the recording ofspontaneous multiple-unit discharges in the pineal gland of the quail to investigate the influence of light stimulation on the multiunit pineal activit

Persistence of tight junctions and changes in apical structures and protein expression in choroid plexus epithelium of rats after short-term head-down tilt.

Major alterations of choroidal cell polarity and protein expression were previously shown to be induced in rats by long-term adaptation to space flight (14 days aboard a space shuttle) or anti-orthostatic suspension (14 and 28 days) performed by tilting rats head-down (i.e. using a ground-based model known to simulate several effects of weightlessness). In rabbits, it was hypothesized that the blood-CSF barrier was opened in choroid plexus, after a short head-down suspension. To understand the early responses to fluid shifts induced by head-down tilts and evaluate the tightness of the choroidal junctions, we have investigated the effects of acute adaptations to anti-orthostatic restraints, using hindlimb-suspended Sprague-Dawley and Wistar rats. Ultrastructural and immunocytochemical studies were performed on choroid plexuses from lateral, third and fourth ventricles, after 30, 90 and 180 minutes of head-down tilt. Alterations were not perceptible at the level of choroidal tight junctions, as shown by freeze-fracture, claudin-1 and ZO-1 immunolocalizations and conventional electron microscopy, after intravenous injection of cytochrome C. The apical surface of choroidal cells was clearly more affected. Microvilli were longer and thinner and ezrin was over-expressed during all the periods of time considered, showing an early cytoskeletal response. Several proteins involved in the choroidal production of cerebrospinal fluid (sodium-potassium ATPase, carbonic anhydrase II, aquaporin 1) appeared first increased (30 minutes after the tilt), and then, returned to the control level or were lowered (after a 3-hour head-down suspension). Although head-down tilts do not seem to damage the blood-cerebrospinal fluid barrier in choroid plexus, it seemed that the expression of several apical proteins is affected very early.

Suprachiasmatic nucleus lesions abolish photoperiod-induced changes in the testis function and GnRH immunoreactivity in the mink, a short-day breeder

Testicular activity (testis volume and plasma testosterone) and immunoreactive GnRH hypothalamic system were examined after suprachiasmatic nucleus (SCN) lesion in the mink, a short-day breeding mammal, whose sexual activity is inhibited by day lengths exceeding 10 h. In animals maintained under a natural photoperiod, SCN destruction performed during the period of maximum sexual activity (February) was shown to have no effect on onset of the testicular inactive period which begins at the end of winter and continues through spring. On the other hand, while gonadal activity began again at the end of autumn in intact animals, minks that had undergone SCN destruction remained sexually inactive until the end of the experiment period (February). The SCN could thus be crucial to the onset of sexual activity triggered by the reduction of day length, whereas onset of sexual inactivity is a spontaneous phenomenon. This was confirmed in a second experiment demonstrating that a short photoperiod (4 L:20 D), highly gonadostimulatory in intact animals, had no effect on testicular activity after SCN destruction. An immunocytochemical study of the hypothalamic GnRH system (staining intensity and number of labeled perikarya and immunoreactive endings in the external layer of the median eminence) also showed consistent by very low rates of immunoreactivity and number of labeled perikarya and endings in operated animals.

Effects of an 11-day spaceflight on the choroid plexus of developing rats

Cellular distributions of ezrin, a cytoskeletal protein involved in apical cell differentiation in choroid plexus, and carbonic anhydrase II, which is partly involved in the cerebrospinal fluid production, were studied by immunocytochemistry, at the level of choroidal epithelial cells from the lateral, third and fourth ventricles in normal or experimental fetuses, in parallel with the ultrastructure of apical microvilli, observed by transmission electron microscopy. We compared choroid plexuses from developing normal rats (gestational day 15 to birth) with choroid plexuses from 20-day-old rat fetuses, developed for 11 days in space, aboard a space shuttle (NASA STS-66 mission, NIH-R1 experiments), from gestational day 9 to day 20. The main changes observed in fetuses developed in space were demonstrated by immunocytochemistry and concerned the distribution of ezrin and carbonic anhydrase II. Thus, in fetuses developing in space, ezrin was strongly detected in the choroidal cytoplasm and weakly associated to the membrane in the apical domain of the choroid plexus from the fourth ventricle. Such alterations suggested that choroid plexus from rat fetal brain displays a delayed maturation under a micro-gravitational environment. In contrast, intense immunoreactions to anti-carbonic anhydrase II antibodies showed that this enzyme is very abundant in rats developed in space, compared to ground control fetuses.

Heart and plasma atrial natriuretic peptide (ANP) in response to long-term endurance training in rats

Long-term endurance training effects on heart and plasma ANP were investigated in male Wistar rats. Maximal O2 uptake (VO2max) was significantly higher in trained groups, when they are used as their own control. After 3, 4, and 5 weeks of endurance training, VO2max was respectively increased by 7.7% (p less than 0.05), 13.7% (p less than 0.01), and 18.4% (p less than 0.001). Plasma ANP and glomerular ANP receptor density showed no clear variations in trained rats. However, cardiac ANP content decreased significantly in left and right atrial tissues by 35-36% (p less than 0.05) after 5 weeks of training. ANP immunoreactivity was investigated to show the distribution of ANP within the atria. ANP was found in diffuse and granular forms. The diffuse pattern (immature ANP) disappeared in cardiocytes of trained rats, while the granular form persisted, especially in the left atrial tissue. These data suggest that chronic endurance training might cause a decrease in ANP synthesis with no change in ANP storage. Such results are in agreement with the hypothesis that the left atrium could be especially involved in long-term fluid volume control.

Distribution of arrestin-like protein and β-subunit of GTP-binding proteins in quail choroid plexuses

Monoclonal antibodies (Mabs) directed against retinal arrestin (S-antigen) were used to detect and characterize this protein in choroid plexus (CP) of quails maintained during eight days, either under long-day photoperiods or in constant darkness. Immunocytochemistry and Western blotting confirmed the presence and the distribution of an arrestin-like protein in quail CP. Arrestin-like immunoreactivities in CP were compared with those obtained with Mabs to beta 36-subunit of G proteins (G beta), alpha-subunit of transducin and rhodopsin. Rhodopsin-like and transducin-like proteins could not be detected in choroidal cells, whereas intense positive reactions were observed with anti-G beta and anti-arrestin Mabs. The strongest immunoreactivities were found in choroidal ependymocytes of the lateral and IIIrd ventricles. In CP epithelial cells lining the IVth ventricle, very weak or no immunoreactivity could be detected with Mabs to arrestin, while Mab against G beta subunit always provided a positive reaction. In quails maintained in constant darkness, arrestin- and G beta-immunoreactivities of CP epithelial cells displayed changes in cellular distribution and intensity (decrease or disappearance of the immunoreactions). The strong arrestin-like immunoreaction located in the apical region of ependymocytes suggests the preferential association of the protein with choroidal microvilli and a possible role in cerebrospinal fluid production assumed by CP cells.