bluuebunny/crossref_metadata_2025_split huggingface dataset:
Diversification of glial lineages: A novel method to clone brain cells in vitro on nitrocellulose substratum
Authors: Carnow Thomas B. , Barbarese Elisa , Carson John H. 
Published: January 2005

AbstractWe have developed a novel in vitro method to analyze the diversification of glial cells during development. The primary advantage of the approach is that glial lineages are formed in discrete clones on a nitrocellulose substratum where the relationship of the progeny is strictly defined. This method facilitates the comparison of a large complement of astrocyte and oligodendrocyte lineages under controlled conditions. Clones were formed by plating a brain dissociate on nitrocellulose at very low density (5,000–40,000 cells/154 mm2). However, growth depended on diffusible factors produced by brain cells growing under the nitrocellulose support at high density (feeder layer). The cloning efficiency of cells from mouse forebrain (P0) was 1–3%. This means we can detect 100,000 to 300,000 clonal progenitors in the dissociate (107 cells per forebrain) using the clonal culture technique. Cell phenotypes were determined by immunocytochemical staining with anti‐glial fibrillary acidic protein (GFAP) to label astrocytes and anti‐galactocerebroside (GC) and anti‐myelin basic protein (MBP) to label oligodendrocytes. There was a remarkable diversity of glia represented in different lineages. The number of astrocyte clones was greater than the number of oligodendrocyte clones but combined their total was 90%. Clone sizes were distributed over a wide range, which indicated that growth rates varied. Clones appeared compact or dispersed but astrocyte clones exhibited three different morphologies—fibroblast‐like, stellate, and elongated. Oligodendrocytes had different morphologies distinct from astrocytes. Although there were different glial lineages the cells in most clones were homogeneous, indicating the progeny had the same fate. However, a small number of the clones, approximately 2%, were heterogeneous and contained both astrocytes and oligodendrocytes. The application of this technique to glial lineages demonstrates that intrinsic factors have a role in determining cell fate since different clones formed under the same external conditions. Finally, these results are consistent with the existence of multiple glial progenitors or the continued presence of multipotential progenitors at the time of birth.
DBcAMP effect on the expression of GFAP and of its encoding mRNA in astroglial primary cultures
Authors: Le Prince G. , Fages C. , Rolland B. , Nunez J. , Tardy M. 
Published: January 2005

AbstractShort term and chronic dBcAMP effects on the expression of glial fibrillary acidic protein (GFAP) in astroglial primary cultures are investigated. Short (48 h) and long (more than 7 days) treatments with the cAMP derivative induce both cell shape changes and an increase in GFAP immunolabelling. Such effects are only associated with an increase in GFAP and in GFAP‐mRNA levels in the long term treatment. These results suggest that the short term effect of dBcAMP induces post‐translational modifications of the protein whereas the long term effect is associated with an increase in GFAP mRNA transcription and/or stability.
FGFs stimulate IGF binding protein synthesis without affecting IGF synthesis in rat astroblasts in primary culture
Authors: Loret Camille , Janet Thierry , Labourdette Gérard , Schneid Hélène , Binoux Michel 
Published: January 2005

AbstractThe production of insulin‐like growth factor (IGF)‐I and ‐II and their binding proteins (BPs) has been studied in new‐born rat astroblasts at confluency in primary culture.Under the influence of fibroblast growth factors (FGFs) (acidic and basic), the morphology of the astroblasts was altered, 125I‐deoxyuridine incorporation was increased, and glutamine synthetase activity was stimulated. IGF production and IGF mRNA expression remained unchanged. Production of the 32 kDa BP (IGFBP‐2), the sole or predominant form under base‐line conditions, was enhanced and the 43–39 kDa form (IGFBP‐3) appeared or were increased. Epidermal growth factor (EGF) also stimulated production of these BPs, whereas thrombin and db‐cAMP had no effect.Our data suggest that a relationship exists between FGF‐induced maturation of astroblasts and the forms of BP they produce. The data also indicate that some factors may act specifically on BP synthesis, without affecting IGF synthesis, and in this way play a role in regulating the bioavailability of the IGFs.
Platelet‐derived growth factor is mitogenic for O‐2Aadult progenitor cells
Authors: Wolswijk Guus , Riddle Peter N. , Noble Mark 
Published: January 2005

AbstractWe report that platelet‐derived growth factor (PDGF) is a potent mitogen for oligodendrocyte type‐2 astrocyte (O‐2A) progenitor cells derived from the optic nerves of adult rats. Moreover, O‐2Aadult progenitors cultured in PDGF express the range of properties we have described previously for O‐2Aadult progenitors cultured in the presence of type‐1 astrocytes. Similarly, previous studies have demonstrated that PDGF is able to mimic the influence of type‐1 astrocytes on O‐2Aperinatal progenitors. Specifically, O‐2Aadult progenitors and O‐2Aperinatal progenitors exposed to PDGF express differences in average cell cycle time (59 ± 5 h for O‐2Aadult progenitors versus 20 ± 6 h for O‐2Aperinatal progenitors), average rate of migration (4.1 ± 0.6 μm h−1 versus 24.6 ± 5.4 μm h−1), morphology (unipolar versus bipolar), and antigenic phenotype (04+ vimentin− versus 04− vimentin+). Thus, our present results indicate that a single signalling molecule secreted by type‐1 astrocytes produces markedly different cellular behaviours in two related O‐2A progenitor populations.
Schwann cell plasminogen activator is regulated by neurons
Authors: Clark Mary Blair , Zeheb Ron , White Tracy Keith , Bunge Richard P. 
Published: January 2005

AbstractThe synthesis and release of plasminogen activators (PAs) in co‐cultures of embryonic rat dorsal root ganglion nerve cells (NCs) and Schwann cells (SCs) were examined by metabolic labeling, immunoprecipitation, immunodepletion, SDS‐PAGE, zymography, and a two‐step esterolytic assay. Metabolic labeling of SC cultures followed by immunoprecipitation of the conditioned medium (CM) demonstrated that cultured SCs synthesized and released tissue type PA (tPA). Failure of amilioride to inhibit PA activity in SCCM indicated that urokinase PA (uPA) was unlikely to contribute significantly to PA activity in SCCM. Experimental manipulation of the NCs and SCs suggested that NCs regulated SC derived PA. Total PA activity increased in SCCM 10–14‐fold by 6 days after removal of NCs. Multiple molecular weight forms of PAs were detected by SDS‐PAGE followed by zymography. A PA ∼95 kDa was absent in co‐cultures of SCs + NCs but prominent by 4 days postdenervation; PA ∼50–70 kDa increased through 8 days postdenervation and PA ∼25 kDa, present in SC + NC cultures, was absent 8 days after removal of NCs. Upon reintroduction of NCs to denervated cultures (SCs), the pattern of PAs detected in culture medium was transitional between innervated and denervated cultures. Immunodepletion experiments using conditioned medium from denervated SC cultures indicated that various molecular weight forms of PA detected in SCCM by zymography were immunologically related to tPA. These studies demonstrate that SCs synthesized and released tPA in a tissue culture model of peripheral nerve and that one mechanism for regulation of PA released by SCs was by association with NCs. This regulation occurred in cultures of both myelinating and nonmyelinating Schwann cells and thus was not dependent on the state of myelination.
Quantitative immunohistochemical and biochemical correlates of connexin43 localization in rat brain
Authors: Nagy J. I. , Yamamoto T. , Sawchuk M. A. , Nance D. M. , Hertzberg E. L. 
Published: January 2005

AbstractWe have shown by immunohistochemical methods that the gap junction protein connexin43 is heterogeneously distributed in rat brain (Yamamoto et al: J Comp Neurol 302:853, 1990). Here we have compared quantitatively the relative amount of connexin43 detected on Western blots of seven central nervous system (CNS) regions with the density of connexin43‐immunoperoxidase reactivity in these regions. As has been observed on Western blots of several cell types, homogenates of these CNS regions contained two forms of connexin43, its dephospho form with an apparent mobility of ∼41 kDa and its ∼3 kDa phosphorylated form. While the relative quantities of connexin43 varied considerably among the brain regions, the ratio of the 43/41 kDa forms, 0.71, was relatively uniform (correlation coefficient, r = 0.92). Sections of brain processed for connexin43‐immunolocalization by the peroxidase–antiperoxidase (PAP) method showed that chromogen deposition was linear with incubation time in reaction medium. Optical density of tissue connexin43‐immunoreactivity in each of the seven areas plotted against the density of connexin43 bands on Western blots gave a correlation coefficient of r = 0.90. Connexin43‐immunoreactivity had a similar appearance in sections processed by PAP or immunofluorescense procedures and consisted of isolated or aggregates of puncta. For the brain regions examined we conclude: (1) that although total levels of connexin43 vary widely, the levels of its phosphorylated and non‐phosphorylated forms are held in fairly constant proportion; (2) that there is a high correlation between the relative amounts of connexin43 detected biochemically and the density of connexin43‐immunostaining in tissue sections; and (3) that immunohistochemical staining for connexin43 reflects the relative amounts of connexin43 and possibly the numbers of gap junctions present in individual brain areas.
Prenatal exposure to ethanol alters plasma membrane glycoproteins of astrocytes during development in primary culture as revealed by concanavalin a binding and 5′‐nucleotidase activity
Authors: Renau‐Piqueras Jaime , Guerri Consuelo , Burgal Maria , De Paz Paulino , Saez Rosana , Mayordomo Fernando 
Published: January 2005

AbstractWe have investigated the effect of prenatal exposure to ethanol on the extent of binding and surface distribution of the lectin concanavalin A (con A) on rat cortical astrocytes during the periods of proliferation and differentiation in primary culture. The enzymatic activity of the plasma membrane glycoprotein 5′‐nucleotidase was also assessed. The cells were obtained from control fetuses (no exposure to ethanol) and from fetuses prenatally exposed to ethanol.The main findings were: (1) both proliferating and differentiating control astrocytes showed two distinct types of surface con A receptors that could correspond to high‐ and low‐affinity binding sites; (2) the extent of con A binding was greater in mature than in proliferating control cells; (3) the distribution of con A on cell surface components changed with differentiation; (4) the activity of 5′‐nucleotidase showed a substantial increment during the period of differentiation; and (5) prenatal exposure to ethanol clearly decreased the ability of astrocytes to bind con A, altered the surface distribution of the receptors for this lectin, and decreased the activity of 5′‐nucleotidase. These effects were more marked in proliferating cells.In conclusion, it is shown that the extent of con A labeling and the activity of 5′‐nucleotidase in astrocytes are dependent on the stage of cell differentiation and that prenatal exposure to ethanol alters the plasma membrane structure of these cells during development.
Spontaneous changes in intracellular calcium concentration in type I astrocytes from rat cerebral cortex in primary culture
Authors: Fatatis Alessandro , Russell James T. 
Published: January 2005

AbstractMeasurement of fura‐2 fluorescence in type I astrocytes from rat cerebral cortex showed that the intracellular calcium ion concentration undergoes very large spontaneous changes. These spike‐like changes ranged from resting levels of calcium of 50–250 nM to as high as 1–2 μM. The spikes were found to be irregular in frequency and amplitude and were frequently synchronous in confluent cultures. The synchronous events appeared as propagating waves that spread over many cells. The spontaneous spikes persisted when the extracellular calcium concentration was reduced to below micromolar levels suggesting that the source for the increases in [Ca2+]i was intracellular. Treatment of the astrocytes with tetrodotoxin did not abolish the spontaneous changes, nor did blockade of voltage‐dependent calcium channels with nimodipine and D‐600. Ryanodine, a blocker of the sarcoplasmic reticulum calcium‐induced calcium release channel, was also without effect. These changes in [Ca2+]i were different in character from both agonist‐induced oscillations and depolarization‐induced increases in intracellular calcium concentration. Depolarization using 25–100 mM [K+]o resulted in a prompt rise in intracellular calcium concentration, which returned to near resting levels, and this response was sensitive to removal of extracellular calcium and voltage‐gated calcium channel antagonists. L‐glutamate (0.5–100 μM) caused large increases in [Ca2+]i that were associated with discrete periodic oscillations in some cells. The cellular trigger for the spontaneous spikes is currently not understood. We conclude that spontaneous changes in [Ca2+]i in astrocytes are distinct from agonist‐induced and membrane potential depolarization‐induced changes.
Uptake of [3H]serotonin and [3H]glutamate by primary astrocyte cultures. I. Effects of different sera and time in culture
Authors: Kimelberg H. K. , Goderie S. K. , Conley P. A. , Higman S. , Goldschmidt R. , Amundson R. H. 
Published: January 2005

AbstractNa+‐dependent, fluoxetine‐sensitive high‐affinity uptake of serotonin and Na+‐dependent uptake of glutamate were studied in primary astrocyte cultures from 1‐day‐old rat neocortex. This uptake was independent of time in culture from 1 to 6 weeks. High‐affinity serotonin uptake was decreased when cells were grown in horse serum as compared to fetal bovine serum and was almost absent when cells were grown in chemically defined medium. In contrast, glutamate uptake was unaffected by the composition of the medium in which the cultures were grown. The serum effect on serotonin uptake was not due to the greater level of serotonin in the fetal bovine serum and was only reversed by a change of serum over a time period of days. © 1992 Wiley‐Liss, Inc.
PDGF increases the expression of fos and jun in newly formed oligodendrocytes that have become resistant to the mitogenic effect of PDGF
Authors: Hart I. K. , Richardson W. D. , Raff M. C. 
Published: January 2005

AbstractBipotential glial (O‐2A) progenitor cells give rise to oligodendrocytes on a predictable schedule in the developing rat optic nerve. The loss of mitotic responsiveness to platelet‐derived growth factor (PDGF) that is seen when O‐2A progenitor cells initially differentiate into oligodendrocytes seems to reflect blocks or deficiencies downstream to some of the early intracellular signalling events induced by PDGF. Here we show that PDGF increases the expression of the Fos and Jun nuclear proteins in newly formed oligodendrocytes in vitro, suggesting that at least one intracellular signalling pathway to the nucleus is activated by PDGF in these cells even though they do not synthesize DNA in response to PDGF. © 1992 Wiley‐Liss, Inc.
5′‐Nucleotidase immunoreactivity of perineuronal microglia responding to rat facial nerve axotomy
Authors: Schoen S. W. , Graeber M. B. , Kreutzberg G. W. 
Published: January 2005

AbstractThe ecto‐enzyme 5′‐nucleotidase was localized immunocytochemically in the axotomized rat facial nucleus. As revealed by the monoclonal antibody 5N4–2,5′‐nucleotidase immunoreactivity markedly increased on perineuronal microglia during the first week following axotomy, and gradually disappeared from these cells by the end of the third post‐operative week. Interestingly, parenchymal microglia were not or only weakly stained. These findings indicate that 5′‐nucleotidase 5N4‐2‐immunoreactivity may serve as a marker for perineuronal microglia, a population of satellite glial cells that appear to be actively engaged in lesion‐induced synaptic changes during regeneration. © 1992 Wiley‐Liss, Inc.
Development and differentiation of glial precursor cells in the rat cerebellum
Authors: Levine Joel M. , Stincone Francis , Lee Ying‐Shuan 
Published: January 2005

AbstractThe development and differentiation of bipotential glial precursor cells has been studied extensively in tissue culture, but little is known about the distribution and fate of these cells within intact animals. To analyze the development of glial progenitor cells in the developing rat cerebellum, we utilized immunofluorescent, immunocytochemical, and autoradiographic techniques. Glial progenitor cells were identified with antibodies against the NG2 chondroitin‐sulfate proteoglycan, a cell‐surface antigen of 02A progenitor cells in vitro, and the distribution of this marker antigen was compared to that of marker antigens that identify immature astrocytes, mature astrocytes, oligodendrocyte precursors, and mature oligodendrocytes.Cells expressing the NG2 antigen appeared in the cerebellum during the last 3‐4 days of embryonic life. Over the first 10 days of postnatal life, the NG2‐labeled cells incorporated 3H‐thymidine into their nuclei and their total number increased. At all ages examined, the NG2‐labeled cells did not contain either vimentin‐like or glial fibrillary acidic protein (GFAP)‐like immunoreactivity, suggesting that they do not develop along an astrocytic pathway. NG2‐labeled cells of embryonic animals expressed GD3 ganglioside antigens, a property of oligodendrocyte precursors, whereas NG2‐positive cells of postnatal animals did not express GD3 immunoreactivity. Nevertheless, the NG2‐labeled cells of the nascent white matter expressed oligodendrocyte‐specific marker antigens. Cells lyingoutside of the white matter continued to express the NG2 antigen. In adult animals, the NG2‐labeled cells incorporated 3H‐thymidine. Glial cells isolated from adult animals and grown in tissue culture express the NG2 antigen and display the phenotypic plasticity characteristic of 02A progenitor cells. These findings demonstrate that a population of glial progenitor cells is extensive within both young and adult animals.
Cell type‐specific regulation of major histocompatibility complex (MHC) Class I gene expression in astrocytes, oligodendrocytes, and neurons
Authors: Massa Paul T. , Ozato Keiko , McFarlin Dale E. 
Published: January 2005

AbstractMechanisms of major histocompatibility complex (MHC) class I gene regulation in cells of the CNS have been studied in vitro. Astrocytes in primary cultures, but neither oligodendrocytes nor neurons, constitutively expressed cell surface MHC class I molecules. Interferon‐gamma (IFN‐γ) treatment led to induction of MHC class I expression in astrocytes and oligodendrocytes but not in neurons. The conserved up‐stream sequence containing the juxtaposed nuclear factor (NF)‐kB‐like region I and IFN‐response consensus sequence (ICS) constitutively enhanced MHC class I gene promoter activity in astrocytes, but not in oligodendrocytes or in neurons. Nuclear extracts from astrocytes, but not from oligodendrocytes and neurons, had a binding activity specific for the NF‐kB‐like region I sequence, indicating that constitutive expression of MHC class I genes is governed by the upstream region I enhancer and its binding factor. IFN‐γ treatment led to induction of MHC class I promoter activity in astrocytes and oligodendrocytes, but not in neurons. In accordance with this observation, a nuclear factor that binds to the ICS was induced in astrocytes and oligodendrocytes but not in neurons following IFN‐γ treatment. This study illustrates cell type‐specific regulation of MHC class I genes in the CNS that correlates with the expression of DNA binding factors relevant to MHC class I gene transcription. © 1993 Wiley‐Liss, Inc.
Receptor‐mediated phospholipase D activity in primary astroglial cultures
Authors: Gustavsson Lena , Lundqvist Christofer , Hansson Elisabeth 
Published: January 2005

AbstractPhospholipase D, an enzyme involved in signal transduction cascades, catalyses the formation of phosphatidic acid and, when ethanol is present, the formation of phosphatidylethanol. In the present study we demonstrate that stimulation of muscarinic acetylcholine receptors as well as P2‐purinergic receptors induces activation of phospholipase D in primary cultures of astroglial cells. Both the hydrolysis and the transphosphatidylation reactions were stimulated by receptor agonists. Carbachol and ATP induced a rapid increase in the amount of [3H]phosphatidic acid in astroglial cells prelabelled with [3H]oleic acid. When ethanol (150 mM) was present, phosphatidylethanol was formed. Furthermore, the receptor‐mediated increase in the concentration of phosphatidic acid was inhibited by ethanol, indicating that the phosphatidic acid production was indeed mediated by phospholipase D. The formation of phosphatidylethanol was concentration dependent, with a half‐maximal effective concentration of 5 × 10−5 M for carbachol and 10−5 M for ATP. The carbachol‐induced response was almost completely inhibited by atropine, indicating activation of phospholipase D via muscarinic receptors. The purinergic response is most probably mediated via P2‐receptors since ADP was almost as efficient as ATP in inducing phosphatidylethanol formation, whereas AMP was significantly less potent. We conclude that astroglial cells in primary culture display muscarinic and purinergic receptors coupled to phospholipase D. The relationship to cell function needs to be further investigated. © 1993 Wiley‐Liss, Inc.
Increase of intracellular calcium induced by oxytocin in hypothalamic cultured astrocytes
Authors: Di Scala‐Guenot Dominique , Mouginot Didier , Strosser Marie‐Thérèse 
Published: January 2005

AbstractA recent study demonstrated oxytocin (OT) receptors on hypothalamic cultured astrocytes (Di Scala‐Guenot and Strosser, 1992). The attempt in the present paper was to determine a possible intracellular calcium mobilization induced by OT receptor activation in these cells. Using the microspectrofluorimetric technique with fura‐2 as calcium indicator, brief applications of OT on single astrocytes induced a transient and reversible dose‐dependent increase of intracellular calcium concentration ([Ca2+]i) in most of the cells tested. In a few cells, OT application triggered intracellular calcium oscillations. Repetitive applications of OT generally produced a decreasing calcium signal, suggesting a desensitization of the receptor. OT‐induced calcium release was prevented by a prior or simultaneous application of an OT antagonist. The origin of the calcium mobilization was assessed during conditions where no extracellular calcium was available. Neither removal of extracellular calcium nor addition of a calcium channel blocker, cadmium 100 μM, in the bathing solution, did affect the calcium response to OT, demonstrating that release of intracellular calcium is solely involved in the OT‐induced [Ca2+]i increase. The OT‐induced calcium mobilization was abolished after thapsigargin application (100 nM). This indicates that the calcium response to OT application was principally associated with activation of the IP3‐sensitive calcium stores. Taken together these results demonstrate that OT receptors previously detected on hypothalamic cultured astrocytes are functional receptors which transduction pathways involve calcium mobilization from IP3‐sensitive stores. © 1994 Wiley‐Liss, Inc.