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Id genes encoding inhibitors of transcription are expressed during in vitro astrocyte differentiation and in cell lines derived from astrocytic tumors.

Id proteins belong to a class of nuclear transcription factors known as helix-loop-helix proteins. It has been reported that Id genes function as negative regulators of differentiation, and Id gene expression is down-regulated during cell differentiation. We examined the regulation of Id genes during astrocyte differentiation in a murine nervous system precursor cell line, NSEHip2-28, which is able to differentiate along the astroglial lineage, as well as in human astroglial tumor cell lines. Upon induction of NSEHip2-28 differentiation, at a time when glial fibrillary acidic protein expression became detectable, the expression of all four Id family members initially increased dramatically, and subsequently decreased. Furthermore, varying levels of Id gene expression were found in astroglial tumor cell lines displaying variable degrees of lineage-specific differentiation. These results suggest that the expression of Id family members may play an important role in the control of astrocyte differentiation.

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Glutamate up-regulates alpha 1 and alpha 2 subunits of the sodium pump in astrocytes of mixed telencephalic cultures but not in pure astrocyte cultures.

Prior work employing an in vitro model of the cerebral cortex has shown that sodium pump activity is a critical determinant for neuronal survival of glutamate stimulation. We have hypothesized that up-regulation of total brain sodium pump activity will protect against potential excitotoxins. Increased sodium pump activity could theoretically occur by changes in the reaction rate (short-term) and/or by increased levels of sodium pump protein (long-term) and is potentially complex since the three catalytic (a) subunit isoforms of the sodium pump are distributed in a highly variable, cell-specific pattern in the brain. Short-term regulation (seconds to minutes) has been well studied: brain sodium pump exhibits a large dynamic range. In contrast, the possibility of long-term modulation of sodium pump activity has not been extensively explored. We used isoform specific antibodies and [3H]ouabain binding to determine whether prolonged stimulation of sodium pump activity in rodent telencephalic cultures increased total sodium pump enzyme. Exposure of mixed neuronal-glial cultures to high levels of glutamate (10 mM) for 18 h, which is highly toxic to neurons, was associated with an approximately 80% increase in alpha 1 and alpha 2 subunit expression by glia. Induction of alpha 2 subunit immunoreactivity was also associated with comparable changes in [3H]ouabain binding, suggesting that the up-regulation corresponded to functional alpha 2 protein. Shorter (30 min) glutamate treatments, which also killed neurons, did not produce similar changes in sodium pump expression. In contrast to mixed cultures, pure astrocyte cultures had undetectable alpha 2 and alpha 3 and moderate levels of alpha 1 protein, as confirmed by low levels of [3H]ouabain binding. Glutamate treatment using this protocol was associated with a decrease in alpha 1 sodium pump expression. We conclude that long-term regulation of the sodium pump can be demonstrated in glia which have developed in the presence of neurons. Both alpha 1 and alpha 2 isoforms of the sodium pump are involved in this response to glutamate.

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Cultivation of immature astrocytes of mouse cerebellum in a serum-free, hormonally defined medium. Appearance of the mature astrocyte phenotype after addition of serum.

A hormonally defined culture medium is described which supports survival and proliferation of astroglia from primary cultures of early postnatal mouse cerebellum. This medium consists of bovine serum albumin, insulin, transferrin, selenium, hyaluronic acid, protease inhibitor aprotinin, and epidermal growth factor. Trypsin-dissociated single cerebellar cell suspensions are plated in this medium on poly-l-coated glass coverslips and maintained for two weeks before subcultivation. After subcultivation into defined medium more than 99% of all cells are vimentin-positive and fibronectin- and almost completely glial fibrillary acid (GFA) protein-negative, indicating that these cells are less mature astrocytes. After replacement of defined medium by culture medium containing 10% horse serum, expression of GFA protein is detectable in addition to vimentin by indirect immunofluorescence.

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Astrocyte subtypes in the rat olfactory bulb: morphological heterogeneity and differential laminar distribution.

Despite increased recognition of the importance and heterogeneity of astrocyte functions throughout the central nervous system (CNS) relatively little attention has been paid to morphological diversity among astrocytes. Recent studies have indicated that subsets of astrocytes are involved in glial-axonal interactions critical to both development and reinnervation of the rat olfactory bulb. Here, we have characterized the morphologies and distribution of astrocytes within anatomically and functionally distinct layers of the adult main olfactory bulb (MOB). Using a known immunohistochemical marker for astrocytes, glial fibrillary acidic protein (GFAP), and the classic gold sublimate method, we identified six astrocyte subtypes based on their morphology and distribution: (1) unipolar, (2) irregular, (3) wedge-shape, (4) circular, (5) semicircular, and (6) elongate. Unipolar, irregular and wedge-shape astrocytes have not been previously described in the CNS. The unipolar and irregular types are located exclusively in the olfactory nerve layer. Wedge-shape astrocytes are unique to, and are the major subtype in, the glomerular layer. These three morphologically unique astrocyte subtypes may correspond to olfactory nerve layer (ONL) and glomerular layer (GL) astrocytes, which express molecules that regulate axonal growth or synaptogenesis during development and/or regeneration of the olfactory nerve. In the glomerular layer, astrocytes are highly organized with respect to the glomeruli. Individual astrocytes are loyal to a single glomerulus. In the external plexiform layer, astrocytes are spaced relatively uniformly. In the granule cell layer, astrocytes appear to compartmentalize granule cell aggregates, recently shown to be coupled by tight junctions. The distribution and patterns of astrocyte processes and the density of GFAP immunoreactivity are distinctive for each of the layers of the olfactory bulb. The spacing of astrocytes and the organization of their processes may be important to compartmentalization of neuronal functions. High levels of GFAP immunoreactivity correlated with layers of high neuronal plasticity. The morphological diversity and differential distribution of astrocytes in the olfactory bulb reported here support growing evidence for functional diversity of astrocytes and important interactions among specific astrocyte and neuron subtypes. It is reasonable to hypothesize, therefore, that as for neurons, morphologically distinctive astrocyte subtypes may correspond to functionally specific classes.

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Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.

4-Aminopyridine↗

The use of astrocytes in culture as model systems for evaluating neurotoxic-induced-injury.

The prevailing thought that astrocytes function predominantly as passive metabolic or even physical support for neurons has faded over the last 20 years. Today these stellar shaped cells are credited with an expanded role, playing key functions in CNS development, homeostasis, and pathology. In probing their expanded roles, primary astrocyte culture systems have proven to be an indispensable tool. Astrocytes have been implicated in both a defensive and facilitatory capacity for many toxic injuries. Evidence for a protective role of astrocytes in modulating CNS toxicity is afforded by observations that the toxicity of glutamate to cortical neurons is diminished upon astrocytic enrichment of the cell culture (Rosenberg and Aizenman, 1989). In cultures of rat cerebral cortex in which astrocyte proliferation is stringently suppressed, glutamate neurotoxicity occurs at low glutamate concentrations similar to those which are normally found in the extracellular space in the hippocampus. In the presence of excess astrocytes, concentrations of glutamate one-hundred fold higher are required to produce equivalent neurotoxicity (Rosenberg and Aizenman, 1989). Astrocytes can facilitate the action of neurotoxins via a modulating process which takes place within the astrocyte or by a direct cytotoxic effect. Whereas primary astrocyte cultures remain unaffected by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; Marini et al., 1989), they function prominently in the selective destruction of dopaminergic neurons of the nigrostriatal pathway in humans, other primates and rodents (Davis et al. 1979; Langston et al., 1983; Burns et al., 1983; Langston et al., 1984; Heikkila et al., 1984; Jarvis and Wagner, 1985). Thus, while MPTP by itself is not toxic to cerebellar cells in co-culture with cerebellar astrocytes, MPTP is toxic to the granule cells (Marini et al, 1989). This is thought to be due to an astrocyte-mediated conversion of MPTP to its highly polar and toxic metabolite, 1-methyl-4-phenylpyridinium ion (MPP+; Chiba et al. 1984). There is compelling evidence that astrocytes respond directly or indirectly to a number of other neurotoxins. Direct cytotoxic effects on astrocytes constitute the major morphologic feature in hyperammonemia (Norenberg, 1981), a condition implicated as an etiologic factor in several CNS disorders. In addition, a predisposition of astrocytes for methylmercury uptake (Aschner et al., 1990 a,b) offers a possible explanation for the observed neurotoxicity of this heavy metal, since a direct toxic effect on astrocytes would result in failure of astrocyte homeostatic functions, indirectly resulting in neuronal impairment, injury and death.

Ammonia↗

Sodium channel expression in optic nerve astrocytes chronically deprived of axonal contact.

Immunocytochemical and electrophysiological methods were used to examine the effect of retinal ablation on the expression of sodium channels within optic nerve astrocytes in situ and in vitro. Enucleation was performed at postnatal day 3 (P3), and electron microscopy of the enucleated optic nerves at P28-P40 revealed complete degeneration of retinal ganglion axons, resulting in optic nerves composed predominantly of astrocytes. In contrast to control (non-enucleated) optic nerve astrocytes, which exhibited distinct sodium channel immunoreactivity following immunostaining with antibody 7493, the astrocytes in enucleated optic nerves did not display sodium channel immunoreactivity in situ. Cultures obtained from enucleated optic nerves consisted principally (greater than 90%) of glial fibrillary acidic protein (GFAP)+/A2B5- ("type-1") astrocytes, as determined by indirect immunofluorescence; GFAP+/A2B5+ ("type-2") astrocytes were not present, nor were GFAP-/A2B5+ (O-2A) progenitor cells. Sodium channel immunoreactivity was not present in GFAP+/A2B5- astrocytes obtained from enucleated optic nerves; in contrast, GFAP+/A2B5- astrocytes from control optic nerves exhibited 7493 immunostaining for the first 4-6 days in culture. Sodium current expression, studied using whole-cell patch-clamp recording, was attenuated in cultured astrocytes derived from enucleated optic nerves. Whereas 39 of 50 type-1 astrocytes cultured from intact optic nerves showed measurable sodium currents at 1-7 days in vitro, sodium currents were present in only 6 of 38 astrocytes cultured from enucleated optic nerves. Mean sodium current densities in astrocytes from the enucleated optic nerves (0.66 +/- 0.3 pA/pF) were significantly smaller than in astrocytes from control optic nerves (7.15 +/- 1.1 pA/pF). The h infinity-curves of sodium currents were similar in A2B5- astrocytes from enucleated and control rat optic nerves. These results suggest that there is neuronal modulation of sodium channel expression in type-1 optic nerve astrocytes, and that, following chronic loss of axonal association in vivo, sodium channel expression is down-regulated in this population of optic nerve astrocytes.

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Isoform-specific expression of sodium channels in astrocytes in vitro: immunocytochemical observations.

The expression of sodium channel alpha-subunit isoforms in astrocytes cultured from P-0 rat spinal cord and P-7 rat optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and isoform-specific amino acid sequences of the rat brain sodium channel. In spinal cord cultures at 5 days in vitro (DIV), both stellate and flat astrocytes were immunostained with antibody SP20, which recognizes a conserved sequence common to sodium channel types I, II/IIA, and III. Antibody SP11-I, which is directed against a subtype-specific sequence in sodium channel I, did not yield detectable staining in spinal cord astrocytes. Antibody SP11-II, which is directed against a subtype-specific sequence in sodium channel II, immunostained both stellate and flat spinal cord astrocytes, although with less intensity than SP20. Antibody SP32-III, which is directed against a subtype-sequence in sodium channel III, immunostained stellate but not flat spinal cord astrocytes. SP20, SP11-II, and SP32-III staining persisted in stellate spinal cord astrocytes through 14-21 DIV, while SP20 and SP11-II immunostaining in flat spinal cord astrocytes was attenuated with time in culture. In optic nerve cultures at 5 DIV, SP20 staining was present in both stellate and flat astrocytes, but at reduced levels compared to spinal cord astrocytes. With increased time in culture SP20 staining was maintained in stellate optic nerve astrocytes but was gradually lost in flat optic nerve astrocytes. Stellate optic nerve astrocytes exhibited low levels of staining with SP11-I, SP11-II, and SP32-III. Flat optic nerve astrocytes lacked or displayed very low SP11-II staining, and SP11-I and SP32-III staining was not detectable. These observations demonstrate that cultures astrocytes are immunoreactive to antibodies generated against conserved and isotype-specific peptide sequences of rat brain sodium channels, and further suggest that there are different patterns of sodium channel expression between flat vs. stellate astrocytes and in astrocytes derived from different regions of the CNS.

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