Cortical astroglial conditioned medium induces in vitro radial-like forms

Cerebral cortex and striatal cell dissociates obtained from rat fetuses (E17) were subcultured and enriched in astroglial cells before being grown in regional (cerebral cortex, striatum) astroglial conditioned media (CM) or defined basal medium. Incidence of radial-like astroglia (vimentin + or glial fibrillary acid protein, GFAP + ) and length of processes in cortical cell subcultures showed a greater increase when exposed to cerebral cortex CM than to striatal CM or basal medium. Stellate (GFAP + ) forms prevailed in subcultures grown in basal medium while striatal cells exposed to CM of either origin remained undifferentiated. Additionally, cultures were treated with various concentrations of cAMP (0.25 and 0.5 mM) and calcitonin gene related peptide (CGRP) (0.1, 0.5, and 1.0 μ). Under these conditions CM-exposed cultures (with predominant “radial-like” forms) did not increase stellate glial numbers, while fetal calf serum (FCS)-exposed cultures (morphologically undifferentiated) underwent significant degrees of stellate transformation. When CM-exposed cultures were shifted to FCS supplemented basal medium for 24–48 hr and then to basal medium alone prior to treatment, cAMP and CGRP were effective in transforming flat astroglia into stellate morphology. Results are indicative of the existence of astroglial diffusible factors affecting the in vitro expression of astroglial morphotypes from the cerebral cortex. Previous exposure to CM interferes with cytoskeletal astrocytic changes induced by cAMP and CGRP. It is speculated that astroglial factors could act in vivo to maintain the expression of radial-like cells during early developmental stages of the cerebral cortex, but it would not be effective in E 17 striatum. It is also suggested that transformation of radial glia into astrocytes may require previous dedifferentiation of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Immunocytochemical localization of NaCh6 in cultured spinal cord astrocytes

The expression of the alpha-subunit of voltage-gated sodium channel 6 (NaCh6) was examined in cultures of astrocytes from E18 rat spinal cord by using an antibody specific for NaCh6. Stellate cells with processes and flat, pancake-like astrocytes are the two morphological types predominantly present in these cultures. The antibody to NaCh6 labeled clusters at the cell body and along the length of the processes in stellate, process-bearing cells. Weak staining was observed in the flat, pancake-like astrocytes. Together with previous studies (Black et al., Mol Brain Res 23:235-245, 1994, Glia 14:133-144, 1995) that show that stellate cells express NaChs II and III (but not NaCh I) and flat cells express NaCh II, these results support the conclusions that there are different patterns of sodium channel expression between flat and stellate astrocytes and that multiple channel isoforms are expressed within the same cell. This study also suggests that NaCh6 may contribute to the electrical properties found in stellate astrocytes.     

Cerebellar astroglial cells in primary culture: expression of different morphological appearances and different ability to take up [3H]D-aspartate and [3H]GABA

In non-neuronal cultures of cells dissociated from postnatal rat cerebellum astrocytes, identified by the presence of the marker protein glial fibrillary acidic protein (GFAP), displayed two distinct morphological forms. One class was stellate in shape with radially distributed fine processes, while the other class was more varied in shape being polygonal or elongate. [3H]thymidine incorporation experiments revealed that cells of both morphologies were able to incorporate this nucleoside, suggesting the capacity for both cell types to undergo cell division. An autoradiographic study of the uptake of [3H]D-aspartate and [3H]GABA revealed that whilst the two classes of astrocytes took up the aspartate to apparently the same extent, only the stellate cells were found to be heavily labeled following incubation with [3H]GABA. A study of the cultures over a 12-day period showed that there was a disappearance of the stellate astrocytes. The time of disappearance was found to be dependent upon the initial plating density; the stellate morphology was apparent longer in lower density cultures. Time lapse studies suggested that one of the reasons for the disappearance of the stellate cells might be that in fact they underwent a change in shape following certain cell-cell interactions, but cell death also has to be considered as a further possibility. The relationships between the two classes of astroglial cells in these cultures is not yet clear. The possibilities are that they represent two different types of astrocytes, or just one type at different stages of differentiation, or maybe a combination of the two possibilities.     

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

The expression of sodium channel α-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 cultured 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. © 1995 Wiley-Liss, Inc.     

The relationship of expression of statin, the nuclear protein of nonproliferating cells, to the differentiation and cell cycle of astroglia in cultures and in situ

Cells in the quiescent, nonproliferative state express a protein, statin, in their nuclei. When the cells reenter the cell cycle, statin disappears and another protein, cyclin, appears. We have examined mouse astroglia at various stages of differentiation in cultures and astroglia in adult mouse brains for the presence of statin. In cultures initiated from the neopallium of newborn mice, the glial fibrillary acidic protein (GFAP)+ stellate astrocytes were statin-negative (statin-) but cyclin-positive (cyclin+). In the same cultures, large flat cells (senescent cells) were statin+ but cyclin-. In frozen sections of the brains of adult mice and in brain smears, GFAP+ astrocytes were statin-. Neither stellate astrocytes grown in cultures for 30 or more days nor astrocytes in adult mouse brain were labeled when pulsed with bromodeoxyuridine (BudR). When astroglia were treated with dibutyrl cyclic adenosine monophosphate (dBcAMP), large stellate cells that closely resemble reactive astrocytes in situ formed. These cells were all statin+ from 11-62 days in vitro; however, reactive astrocytes in mouse neopallium, 4-50 days after a stab wound, were statin-. In colony cultures, senescent cells became statin+, whereas stellate astrocytes and their precursor cells remained statin-. These observations indicate that normal astrocytes both in cultures and in situ retain the potential to divide and probably progress through the cell cycle at a very slow rate.     

Gangliosides of cultured astroglia

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.     

Microglial cells in culture express a prominent glutathione system for the defense against reactive oxygen species

To obtain information on the glutathione metabolism of microglial cells, the content of glutathione and activities of enzymes involved in the defense against peroxides were determined for microglia-rich cultures from rat brain. These cultures contain approximately 90% microglia cells as determined by immunocytochemical staining for glial markers, by the phagocytosis activity of the cells and by the production of superoxide after stimulation of the cells with phorbolester. For these cultures, a glutathione content of 41.2 +/- 11.2 nmol/mg protein and a specific activity of glutathione reductase of 15.2 +/- 3.2 nmol/(min x mg protein) were determined. These values are significantly higher than those found for astroglial or neuronal cultures. In addition, with 68.7 +/- 23.5 nmol/(min x mg protein), the specific activity of glutathione peroxidase in microglial cultures was 78% higher than in cultured neurons. The specific catalase activity of microglial cultures was less than 40% that of astroglial or neuronal cultures. Microglial cultures contain only marginal amounts of oxidized glutathione. However, on application of oxidative stress by incubation of microglial cultures with hydrogen peroxide or with the superoxide-producing hypoxanthine/xanthine oxidase system, cellular glutathione was rapidly oxidized. These results demonstrate that microglial cells have a prominent glutathione system, which is likely to reflect the necessity for self-protection against reactive oxygen species when produced by these or surrounding brain cells.     

Preparation and characterization of type 1 astrocytes cultured from adult rat cortex, cerebellum, and striatum.

Astrocytes have been prepared from adult rat cortex, cerebellum, and striatum, using a modification of the McCarthy-DeVellis (J Cell Bio 85:890, 1980) method. The cultures consist of 99% type 1 polygonal astrocytes, which divide more slowly than cells from newborn animals. One day after preparing the cultures, 90% of the cells are glial fibrillary acidic protein (GFAP)-positive and 80% are vimentin-positive by immunohistochemical staining, suggesting that they are present de novo and not derived from precursor cells. The astrocytes from adult brain respond to an elevation of intracellular cyclic AMP, following treatment with forskolin, by becoming more stellate in shape and putting out fine ramified processes. They contain the same amount of GFAP per mg protein, measured by immunoblot, as cells from newborn animals. These cultures thus offer the possibility of comparing the biochemical properties of astrocytes derived from adult animals with those from newborn animals, or with cultures of reactive astrocytes isolated from lesioned brain.     

TNF alpha increases activity of gamma-glutamyl transpeptidase in cultured rat astroglial cells

To investigate the presence of gamma-glutamyl transpeptidase (gammaGT) in brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells were studied. Astroglial cultures contained a specific gammaGT activity of 2.3 +/- 0.9 nmol/min/mg protein. A similar specific gammaGT activity was measured for oligodendroglial cultures, whereas microglial cells and neurons contained less than 30% of the specific gammaGT activity of astroglial cultures. The activity of gammaGT in astroglial cultures was elevated strongly by the presence of tumor necrosis factor-alpha (TNFalpha) in a time- and concentration-dependent manner. Maximal activity of gammaGT was observed after incubation of astroglial cultures for 3 days with 30 ng/mL TNFalpha. Under these conditions the specific gammaGT activity was increased by threefold compared to controls. Presence of the gammaGT-inhibitor acivicin completely inhibited gammaGT activity both in TNFalpha-treated and in control cells. In addition, the increase in astroglial gammaGT activity after application of TNFalpha was prevented completely by the presence of the protein synthesis inhibitor cycloheximide. gammaGT is involved in extracellular processing of glutathione (GSH) that is exported by astroglial cells. After TNFalpha-treatment the concentration of GSH in the medium of astroglial cells was reduced significantly compared to control cells. In conclusion, the data presented demonstrate that TNFalpha stimulates gammaGT synthesis in astroglial cells and thereby improves the capacity to process GSH exported by these cells.     

Role of Thyroid Hormone in the Morphological Differentiation and Maturation of Astrocytes: Temporal Correlation with Synthesis and Organization of Actin

Morphological changes and the molecular mechanisms associated with the maturation of astrocytes were studied under normal and thyroid hormone-deficient conditions using long-term (30 days) primary cultures derived from the neonatal rat brain. Immunocytochemical staining of cells with a monoclonal antibody specific to glial fibrillary acidic protein demonstrated for the first time that, similar to their maturation in vivo, astrocytes maintained in normal serum-containing medium can undergo complete maturation involving two distinct stages of morphological differentiation (from radial glia to flat polygonal cells with epithelioid morphology and then to mature process-bearing cells with stellate morphology). Deficiency of thyroid hormone delays the first step and totally blocks the second stage of differentiation in the maturation process. Comparative staining of normal and thyroid hormone-deficient astrocytes with filamentous actin-specific fluorescein isothiocyanate-phalloidin and quantitation of the various forms of intracellular actin using an improved DNase I assay demonstrated that maturation of astroglial cells is associated with characteristic alterations in the level of cytoskeletal and non-cytoskeletal filamentous (F) actin. In particular, the maintenance of the epithelioid form of the hypothyroid astrocytes is associated with a progressive increase in the level of cytoskeletal F-actin and a concomitant decline in the level of non-cytoskeletal F-actin. Quantitation of actin mRNA by Northern blot analysis and studies on the rate of actin synthesis at various stages of differentiation showed that the initial transformation into the epithelioid form is associated with an increase in the rate of synthesis of actin and the expression of its mRNA, while the final transformation into the mature process-bearing form is correlated with a decline in these parameters. The results indicate that thyroid hormone plays an obligatory role in promoting the differentiation and maturation of astrocytes, and that during this process the hormone regulates the expression of actin and its intracellular organization in a way conducive to morphological differentiation.     

Subpopulations of rat cerebellar astrocytes in primary culture: morphology, cell surface antigens and [3H]GABA transport

Glial fibrillary acidic protein (GFAP)-positive astrocytes in preconfluent cultures derived from postnatal rat cerebellum have been previously shown to display two distinct morphologies, one stellate and the other irregularly epithelioid. The immunofluorescence studies described here showed that these cells also possess unique surface characteristics. In cultures derived from 8-day-old animals stellate cells bound the monoclonal antibody A2B5 whereas the epithelioid cells bound another monoclonal antibody against rat neural antigen-2 (RAN2). Some stellate cells derived from 2-day-old animals also bound tetanus toxin. The A2B5 labelling of the stellate cells made it possible to follow their fate in vitro. In confirmation of previous time-lapse studies, they underwent a shape transformation as confluence was approached, ultimately attaining a form resembling that of the epithelioid cells. Autoradiographic transport studies using two tritiated gamma-aminobutyric acid (GABA) analogues cis-1,3-aminocyclohexane carboxylic acid (ACHC) and beta-alanine revealed further differences between the two types of astrocytes. Whereas [3H]ACHC was taken up solely by the stellate cells [3H]beta-alanine was transported by both cell types. In other experiments in which various inhibitors of [3H]GABA transport were used ACHC virtually eliminated uptake into the stellate astrocyte, but had little effect on the epithelioid ones. The 'neuron-like' [3H]GABA transport process in the stellate astrocytes was confirmed in experiments comparing the effect of another compound which has been proposed as an astrocyte-selective GABA transport inhibitor, 4,5,6,7-tetrahydroisoxazolo-(4,5-C)pyridin-3-ol (THPO). No discrimination was found in its effect on the uptake of [3H]GABA into either neurons or stellate astrocytes. Further autoradiographic studies following the uptake of [3H]GABA by postnatal cerebellar slices showed that astrocytes in all layers of the cerebellar cortex and white matter transported [3H]GABA in contrast to the situation in culture where the amino acid is taken up predominantly by the stellate astrocytes. The possibility is discussed that the stellate astrocytes represent a population of cerebellar fibrous astrocytes whereas the identity of the epithelioid astrocytes is less certain.     

Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture

We have used 4 cell-type-specific markers to identify individual glial and neuronal cells in dissociated cell cultures of neonatal rat sciatic nerve, dorsal root ganglia (DRG), optic nerve, cerebellum, corpus callosum, cerebral cortex and leptomeninges. Schwann cells were identified with antibodies against rat neural antigen-1 (Ran-1), neurons with tetanus toxin, astrocytes with antibody against the glial fibrillary acidic protein (GFAP) and oligodendrocytes with antibody against galactocerebroside. All of these ligands react with cell surface molecules except for anti-GFAP antibody which binds to intracellular glial filaments. Using two-fluorochrome immunofluorescence we have studied the distribution of various glycoproteins and glycolipids on these 4 major neural cell types in short-term cultures. We have found that (1) although Ran-1 is expressed on glial and neuronal tumours, it was not found on normal astrocytes, oligodendrocytes or neurons; (2) Thy-1 was present on fibroblasts and some neurons but not on the majority of leptomeningeal cells or on oligodendrocytes or astrocytes in short-term cultures (however, it was expressed on some astrocytes in longer term cultures); (3) the 'large external transformation sensitive' (LETS) protein could be detected on fibroblasts and leptomeningeal cells but not on neurons or glial cells; (4) GM1 was present on all neurons, most oligodendrocytes and approx. 50% of other cell types; sulfatide and GM3 were only detectable on oligodendrocytes, while globoside was only found on some neurons. In addition, we were able to identify putative microglial cells by the presence of cell surface receptors for IgG and by their phagocytic activity; they did not express and of the cell-type-specific defining markers.     

Astrocyte spreading in response to thrombin and lysophosphatidic acid is dependent on the Rho GTPase

Astrocytes are typically star shaped cells playing diverse roles in the function of the nervous system. In astrocyte cultures established from the cerebral hemispheres of newborn rats, the cells have generally a polygonal fibroblast-like morphology, but acquire a stellate shape upon serum removal. When the serine protease thrombin or the bioactive lipid lysophosphatidic acid is added, the stellate cells revert to the flat morphology. Here we show that the effect of these agents is mediated via activation of the small GTP-binding protein Rho. Neither thrombin nor lysophosphatidic acid induced spreading of astrocytes microinjected with C3 transferase, an exoenzyme which ADP-ribosylates and thereby inactivates Rho. In contrast, the response of cells injected with a dominant negative form of Rac was unaffected. In addition, the injection of active Rho into stellate astrocytes mimicked the effect of thrombin and lysophosphatidic acid and an injection of C3 into flat cells grown in serum induced stellation. The conversion from a stellate to a spread morphology upon activation of Rho resulted in the formation of stress fibers and focal adhesions which most probably are key events in establishing and stabilizing the altered cytoarchitecture. These results suggest that Rho plays a crucial role in determining the shape of astrocytes and thereby may modulate their interaction with neurons in vivo. GLIA 21:244–252, 1997. © 1997 Wiley-Liss, Inc.     

The presence of glutathione in primary neuronal and astroglial cultures from rat cerebral cortex and brain stem

Summary The concentration of the tripeptide glutathione (GSH) was measured in primary cultures of neurons and astroglial cells from rat cerebral cortex and brain stem. The concentration of GSH was found to be approximately 20 nmol/ mg protein in the neuronal culture from the cerebral cortex and ca. 40 nmol/ mg protein in the neuronal brain stem cultures. A GSH concentration of approximately 20 nmol/mg was observed in the astrocyte cultures from both brain regions. The possibility to increase the GSH concentration was tested by incubating the cultures in the presence of the GSH precursor -glutamylcysteine (-GC). In the cultured astrocytes -GC produced a dose-dependent increase in GSH. A similar increase was observed in the neuronal cultures, but this effect failed to reach statistical significance.     

Vimentin-GFAP transition in primary dissociated cultures of rat embryo spinal cord

Primary dissociated cultures derived from 15-day-old rat embryo spinal cord with or without dorsal root ganglia (DRG) were grown on polylysine, Primaria and laminin substrates. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. After 2 days in culture, the number of cells stained with GFAP antibodies progressively increased within the vimentin-positive monolayer surrounding the neuronal aggregates. These astrocytes had the typical appearance of astrocytes in primary dissociated cultures derived from late fetal or early neonatal murine brain, i.e. large flat or stellate cells with thick processes staining equally well with GFAP and vimentin antibodies. Astrocytes found within the neuronal aggregates in 4-5 day cultures were markedly different, i.e. small stellate cells with slender processes forming a delicate mesh throughout the aggregate. These GFAP-positive cells stained only weakly with vimentin antibodies. Spinal cord neurons formed aggregates on laminin substrates but failed to extend neurites and rapidly degenerated. The large flat cells in the surrounding monolayer gradually invaded the aggregates. These cells stained with both GFAP and vimentin antibodies. DRG neurons developed equally well on Primaria and laminin substrates, extending their neurites on the vimentin-positive flat cells forming the monolayer regardless of their reactivity with GFAP antibodies.     

Argininosuccinate synthetase: Localization in astrocytes and role in the production of glial nitric oxide

An antiserum raised against the peptide representing the partial sequence 196–222 of mouse liver argininosuccinate synthetase (ASS) was used to detect and localize the enzyme in cells of neural primary cultures. No ASS immunoreactivity was detected by Western blotting in homogenates of mouse pure astroglial cultures and rat astroglia-rich cultures. However, when the cultures had been treated with bacterial lipopolysaccharide, interferon-γ, or a combination of both, ASS immunoreactivity was disclosed. Immunocytochemical examination of rat astroglia-rich cultures revealed a colocalization of ASS with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were some GFAP-positive cells showing no specific staining for ASS, and vice versa. Colocalization of ASS with the inducible isoform of nitric oxide synthase in the same cell was shown only occasionally; nitric oxide synthase was predominantly expressed in microglial cells. In rat neuron-rich primary cultures astroglial cells as well as neurons expressed ASS. Cells of mouse pure astroglial cultures were able to synthesize arginine and, consequently, nitric oxide from citrulline, but not from ornithine. The findings demonstrate that ASS is expressed in astroglial cells under conditions that stimulate long-lasting production of nitric oxide; a functional role of this enzyme in the latter process is implicated. GLIA 24:428–436, 1998. © 1998 Wiley-Liss, Inc.     

Morphological modulation of cultured rat brain astroglial cells: antagonism by ganglioside GM1

Secondary cultures of neonatal rat astroglial cells, maintained in a serum-free, chemically defined medium were treated with several agents thought to activate cyclic AMP-synthesizing systems. Dibutyryl cyclic AMP (dBcAMP), forskolin and cholera toxin promoted, within 2 h, the near-complete conversion of 1-day-old (D1) astroglial cells from a flat, epithelioid morphology to a stellate (star-shaped) morphology. With all 3 agents, cell susceptibility to morphological change declined with culture age, 5-day-old cultures failing to respond altogether. D1 cultures, after 48 h of treatment, had reverted to the flat morphology. Gangliosides reported to stimulate adenylate cyclase were also tested, using purified GM1 X GM1 failed to stimulate the conversion to stellate morphologies. GM1, however, did affect these astroglial cells by causing a block or reversal of their morphological response to dBcAMP, forskolin or cholera toxin. The GM1 response was specific for the intact ganglioside molecule, asialo GM1 and sialic acid having no effect. Gangliosides GD1a, GD1b and GT1b were also active, being effective at ca. 4-fold lower concentrations. The response to GM1 appeared to involve a direct interaction with the astroglial cell, rather than influencing either substratum or medium components.     

Cloning and expression of inducible nitric oxide synthase from rat astrocytes

In primary cultures of rat astroglial cells exposure to bacterial endotoxin lipopolysaccharide (LPS) causes induction of a Ca²⁺-independent form of the nitric oxide synthase (iNOS) enzyme. We have now cloned the mRNA encoding astroglial iNOS using a combination of cDNA library screening and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides directed against conserved regions of all NOS enzymes. The sequence of astroglial iNOS cDNA is highly similar to the mouse macrophage sequence, having an overall homology of 92% at the DNA level and 93% at the protein level. As in other NOSs, canonical binding sites for redox cofactors are present. The 3′-untranslated region displays 4 consensus AU-pentamers, 2 polyadenylation sites, and terminates in a stretch of 17 adenosine residues. In situ hybridization studies with LPS-treated astrocyte cultures demonstrated the presence of iNOS mRNA in the majority of astroglial cells, identified by antibody staining to the glial fibrillary acidic protein (GFAP). PCR analysis showed that LPS stimulated synthesis of astrocyte iNOS mRNA, which was detected as early as 2 hr after exposure to LPS, peaked at 4 hr, and slowly declined over the next 20 hr. These results confirm that astrocytes can express iNOS and provide tools for the subsequent analysis of iNOS gene expression in rodent brain © 1994 Wiley-Liss, Inc.     

Astrocytes in cat visual cortex studied by GFAP and S-100 immunocytochemistry during postnatal development.

A monoclonal antibody to glial fibrillary acidic protein (GFAP) and a polyclonal antiserum to the S-100 protein were used to study the expression of these astrocytic proteins in the postnatal visual cortex of the cat. Three changes in antigen expression of these astroglial markers could be distinguished over development. First, the density of cells in the white matter, which are heavily labelled with both antibodies from birth until adulthood, diminishes after the third postnatal weeks. By intracellular filling with Lucifer Yellow the reduction of the cell density can be attributed to the disappearance of large astrocytes with a morphology of transforming radial glia, present only in early postnatal development. Second, heavily labelled, large cells present in the grey matter at the seventh postnatal day have disappeared by the fifth postnatal week. On the basis of their morphology these cells can also be classified as radial glial cells. Finally, astroglial cells of the adult-like stellate form appear to be labelled in the cortical layers between the third and seventh postnatal weeks. While the density of these cells and the S-100 immunoreactivity of the cell bodies is adult-like at the fourth postnatal week, there is a gradual increase of the staining intensity with the GFAP antibody up to the seventh postnatal week. This developmental period is paralleled by the appearance of S-100-positive astrocytic processes. The gradual expression of GFAP immunoreactivity and the increased expression of S-100 is interpreted as reflecting the time course of astrocytic maturation. A possible relation of the maturation of astrocytes and cortical development, both of which are prominent in the time period between the third and seventh postnatal week, is discussed.