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PubMed1984

Astrocyte cell lineage. III. The morphology of differentiating mouse astrocytes in colony culture.

Disaggregated cells of newborn DBA/1J mouse neopallium were grown in colony cultures, and colonies of cells at various stages of differentiation along the astrocyte cell lineage were examined after 3 days, 1, 2 and 4 weeks by electron microscopy and by NBD-phallacidin which demonstrates the distribution of microfilaments. The earliest astrocyte precursor cells or glioblasts are closely apposed epithelial cells that rarely have junctions. Their scanty cytoplasm contains many free ribosomes but few microfilaments. The cells in the next stages of astrocyte lineage or proastroblasts are flat and are separated from each other to a variable degree. They have intercellular junctions associated with microfilaments and contain singly dispersed intermediate filaments. The proastroblasts gradually differentiate into astroblasts which have a similar morphology except that in addition to the singly distributed intermediate filaments they also contain intermediate filaments arranged into bundles of various sizes. The mature fibrous astrocytes have well-defined processes and distinct perikarya. They form from astroblasts in culture and also contain numerous bundles of intermediate filaments. The dibutyryl-cyclic AMP (dBcAMP)-induced astrocytes in culture in contrast are large stellate cells similar to reactive astrocytes found around sites of injury in the brain. On the basis of these and previous immunocytochemical studies of the formation and distribution of intermediate filaments in the cytoplasm of differentiating astrocytes, criteria are proposed for identification of different cells along the astrocyte lineage.

Animals↗

PubMed1994

NMR spectroscopic study of cell cultures of astrocytes and neurons exposed to hypoxia: compartmentation of astrocyte metabolism.

Primary cultures of murine cerebral cortical astrocytes or cerebellar granule neurons were exposed to 7 h of hypoxia (3 h in some cases). The culture medium was analyzed at the end of the hypoxic or normoxic period by 1H NMR spectroscopy and intracellular components were analyzed as perchloric acid extracts by 31P and 1H NMR spectroscopy. Lactate production in astrocytes increased only marginally, whereas high energy phosphate concentrations were reduced, during 7 h of hypoxia and after 17 h of reoxygenation. After 3 h of hypoxia full recovery was possible during reoxygenation. Citrate and glutamine secretion was reduced or unchanged, respectively, during 7 h of hypoxia. Succinate secretion was only observed during normoxia, whereas pyruvate was secreted during hypoxia. Cerebellar granule neurons were more efficient in increasing glycolysis and were, therefore, more resistant to the effects of hypoxia than astrocytes. In the neurons lactate production was doubled and no effects on levels of high energy phosphates were seen after 7 h of hypoxia. Astrocytes were reoxygenated for 17 h after hypoxia or normoxia in a medium containing [2-13C]acetate in order to access if astrocytes were still capable of supplying neurons with essential precursors. The media were subsequently analyzed by 13C NMR spectroscopy. After shorter periods of hypoxia (3 h) full recovery was possible. Citrate and glutamine production remained however decreased during reoxygenation after 7 h of hypoxia. 13C incorporation into glutamine was greatly reduced but that into citrate was unchanged. These results suggest that under the present conditions, neurons are more efficient than astrocytes in switching the energy metabolism from aerobic to anaerobic glycolysis and that astrocytes may suffer long term damage to mitochondria from longer periods of hypoxia. Furthermore, evidence is presented for the existence of several TCA cycles within astrocytes based on labeling ratios. During normoxia the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively.

Adenosine Triphosphate↗

PubMed1991

First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes.

Primary cultures of cerebral cortical neurons or astrocytes or the two cell types together (co-cultures) were incubated with [1-13C]glucose for 20 or 48 h. Subsequently, perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy in order to detect 13C-labeled amino acids (glutamine, glutamate, gamma-aminobutyrate (GABA)) and other metabolites (lactate, tricarboxylic acid cycle (TCA) constituents). NMR spectra of PCA extracts of neurons or co-cultures exhibited distinct peaks for glutamate and GABA whereas the PCA extracts of astrocytes and co-cultures showed peaks corresponding to glutamine and glutamate. This pattern is consistent with the neuronal location of the GABA synthesizing enzyme glutamate decarboxylase and the astrocytic localization of the glutamine synthesizing enzyme, glutamine synthetase. NMR spectra of the incubation media showed clearly that 13C-labeled citrate, alanine and glutamine were synthesized and released from astrocytes since only media from the astrocyte cultures or co-cultures or neurons and astrocytes contained these metabolites in detectable amounts. It may be concluded that astrocytes play an important role supplying neurons with precursors for biosynthesis of glutamate and GABA such as glutamine and TCA cycle constituents. Since among the latter only citrate could be found in significant amounts it may be hypothesized that this may be the quantitatively most important TCA constituent to be released from astrocytes and subsequently utilized by neurons.

Animals↗

PubMed2004

Astrocyte activation and dysfunction and neuron death by HIV-1 Tat expression in astrocytes.

Human immunodeficiency virus type 1 (HIV-1) Tat protein plays an important role in HIV-associated neuropathogenesis. Astrocytosis and neuron death are two hallmarks of HIV-1 infection of the central nervous system (CNS). However, whether there is a direct link between Tat expression, astrocytosis and subsequent neuron death is not known. In this study, we expressed Tat in astrocytes and examined Tat effects on astrocyte function and subsequent neuronal survival. The results showed that Tat expression resulted in a significant increase in glial fibrillary acidic protein (GFAP) expression, a cellular marker of astrocyte activation or astrocytosis. The GFAP promoter-driven reporter gene assay showed that Tat transactivated GFAP expression at the transcriptional level. Furthermore, Tat expression markedly impaired glutamate uptake by astrocytes. Importantly, cell culture supernatants from Tat-expressing astrocytes induced dramatic neuron death. Taken together, these data provide evidence for the first time to directly link Tat expression in astrocytes to astrocytosis, astrocyte dysfunction, and subsequent neuron death. In addition, these data suggest that astrocyte dysfunction contributes, at least in part, to Tat neurotoxicity and subsequently HIV-associated neuropathogenesis.

Animals↗

PubMedDate not supplied

Gene expression patterns in in vivo normal adult astrocytes compared with cultured neonatal and normal adult astrocytes.

This paper presents data on the basal gene expression patterns, determined by microarray analysis, for cultured neonatal and normal adult striatal astrocytes, and for comparison, for astrocytes isolated directly from adult rat striatum (in vivo adult astrocytes). Of the 1176 genes on the Clontech array, 1101 were expressed in one of the three types of astrocyte samples. Nineteen of the genes were expressed only in the astrocytes taken directly from adult rats (in vivo adult). The cultured neonatal astrocytes expressed many genes at a two-fold or greater level than their expression in cultured adult astrocytes, including genes in the adhesion, cytoskeleton, and extracellular matrix (ECM) family, signal transduction genes, and genes related to apoptosis, DNA-binding, and cell cycle regulation. Overall the results support the concept that cultured neonatal astrocytes are more "activated" than cultured adult cells, although the adult cells expressed higher levels of many metabolic enzyme and protease/protease inhibitor genes.

Aging↗

PubMed2001

Interleukin-1beta induces expression of neuropeptide Y in primary astrocyte cultures in a cytokine-specific manner: induction in human but not rat astrocytes.

Previous studies have demonstrated that astrocyte cultures express neuropeptide Y (NPY) in a regulated manner, namely, phorbol ester leads to an increase in proNPY-mRNA and NPY production. In this respect, the behavior of astrocytes derived from the human fetal or rat neonatal brain is similar (Regul. Pept. 75 (1998) 293). Since astrocytes can be exposed to high levels of IL-1beta, we addressed the question: Does IL-1beta regulate NPY expression by the astrocytes? Primary astrocytes derived from the human fetal or rat neonatal cortex were cultured in serum-free medium. IL-1beta, but not IL-6 or TNF-alpha, led to an increase in NPY production dose-dependently. IL-1beta action manifested in the human but not in the rat astrocytes and it was completely abolished by IL-1 receptor antagonist. The responsiveness to IL-1beta did not diminish upon sub-culture of the astrocytes (five passages). In addition, IL-1beta led to an increase in the abundance of proNPY-mRNA, which was preceded by a rapid and transient increase in cFos-mRNA and a rapid and sustained increase in JunB-mRNA. In contrast to cFos/JunB, IL-1beta did not alter the abundance of cJun-mRNA. In summary, we demonstrate that IL-1beta induction of NPY expression in astrocytes is species- and cytokine-specific and that IL-1 receptor is involved. Moreover, induction of NPY expression is preceded by a rapid increase in the expression of two transcription factors (cFos, JunB) that have been previously (Oncogene 9 (1994) 2369; J. Neurochem. 70 (1998) 1887) implicated in transcriptional regulation of the human NPY gene.

Animals↗

PubMed2002

Astrocyte mGlu(2/3)-mediated cAMP potentiation is calcium sensitive: studies in murine neuronal and astrocyte cultures.

Signal transduction mechanisms of group II metabotropic glutamate receptors (mGlu(2/3)) remains a matter of some controversy, therefore we sought to gain new insights into its regulation by studying cAMP production in cultured neurons and astrocytes, and by examining inter-relationships of mGlu(2/3)-induced signalling with cellular calcium and various signalling cascades. mGlu(2/3) agonists 2R,4R-4-aminopyrrolidine-2,4-dicarboxylic acid (2R,4R-APDC) and (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) inhibited 10 microM forskolin-stimulated production of cAMP in murine cortical neurons, striatal neurons and forebrain astrocytes in the absence of extracellular Ca(2+). These agonists potentiated cAMP production in the presence of 1.8 mM Ca(2+) in astrocytes only. This potentiation was dependent on the extracellular Ca(2+) concentration (0.001-10 mM) and inhibited by the mGlu(2/3) antagonist LY341495 (1 microM), adenosine deaminase (1 U/ml) and the adenosine A(2A) receptor antagonist ZM241385 (1 microM). Pre-incubation with the phospholipase C (PLC) inhibitor U73122 (10 microM), L-type Ca(2+)-channel blockers nifedipine (1 microM) and nimodipine (1 microM), the calmodulin kinase II (CaMKII) inhibitor KN-62 (10 microM) or pertussis toxin (100 ng/ml) inhibited this potentiation. In the absence of 1.8 mM Ca(2+), thapsigargin (1 microM) facilitated the potentiation of cAMP production. Measurement of the Ca(2+)-binding dye Fluo-3/AM showed that, compared to Ca(2+)-free conditions, thapsigargin and 1.8 mM Ca(2+) elevated [Ca(2+)](i) in astrocytes; the latter effect being prevented by L-type Ca(2+)-channel blockers. Potentiation of cAMP production was also demonstrated when astrocytes were stimulated with the beta-adrenoceptor agonist isoprenaline (10 microM) in the presence of 1.8 mM Ca(2+), but not with the adenosine agonist NECA (10 microM) or the group I mGlu receptor agonist DHPG (100 microM). BaCl(2) (1.8 mM) in place of Ca(2+) did not facilitate forskolin-stimulated mGlu(2/3)-potentiation of cAMP. In short, this study in astrocytes demonstrates that under physiological Ca(2+) and adenylate cyclase stimulation an elevation of cAMP production is achieved that is mediated by PLC/IP(3)- and CaMKII-dependent pathways and results in the release of endogenous adenosine which acts at G(s) protein-coupled A(2A) receptors. These findings provide new insights into mGlu(2/3) signalling in astrocytes versus neurons, and which could determine the functional phenotypy of astrocytes under physiological and pathological conditions.

Animals↗

PubMedDate not supplied

Astrocyte and oligodendrocyte connexins of the glial syncytium in relation to astrocyte anatomical domains and spatial buffering.

Astroctyes express a set of three connexins (Cx26, Cx30, and Cx43) that are contained in astrocyte-to-astrocyte (A/A) gap junctions; oligodendrocytes express a different set of three connexins (Cx29, Cx32, and Cx47) that are contained in the oligodendrocyte side of necessarily heterotypic astrocyte-to-oligodendrocyte (A/O) gap junctions, and there is little ultrastructural evidence for gap junction formation between individual oligodendrocytes. In addition, primarily Cx29 and Cx32 are contained deeper in myelin sheaths, where they form autologous gap junctions at sites of uncompacted myelin. The presence of six connexins in macroglial cell populations has revealed unprecedented complexity of potential connexin coupling partners, and with restricted deployment of gap junctional intercellular communication (GJIC) within the "pan-glial" syncytium. New implications for the organization and regulation of spatial buffering mediated by glial GJIC are derived from recent observations of the existence of separate astrocyte anatomical domains, with only narrow regions of overlap between astrocyte processes at domain borders. Thus, widespread spatial buffering in the CNS may occur not successively through a multitude of processes arising from different astrocytes, but rather in a more orderly fashion from one astrocyte domain to another via intercellular coupling that occurs only at restricted regions of overlap between astrocyte domains, augmented by autocellular coupling that occurs within each domain.

Animals↗

PubMed1995

Decrease of NCAM expression and astrocyte-neurone interaction in long-term cultured astrocytes.

In order to assess the characteristics of the older astrocyte, we obtained long-term cultured rat astrocytes (20 months) and examined the features of protein expression in relation to neuronal interaction. In short-term cultured astrocytes, NCAM expressed strongly in contrast to weak expression of laminin by both immunocytochemical and ELISA assay. On the contrary, in long-term cultured astrocytes, a marked decrease of NCAM expression was observed along with increased laminin expression compared with short-term cultured astrocytes. The long-term cultured astrocytes remained positive to anti-GFAP antibody and showed a much lower ability to interact with neurones than the short-term cultured astrocytes. NCAM may be one of the responsible molecules related to the astrocyte-neurone interaction in the developing and ageing nervous system.

Animals↗

PubMed1986

Microtubule-associated protein 2 (MAP2) is present in astrocytes of the optic nerve but absent from astrocytes of the optic tract.

Using the peroxidase-antiperoxidase staining technique at the light- and electron-microscope levels and two monoclonal antibodies against microtubule-associated protein 2 (MAP2), we found that astrocytes located at the periphery of the rat optic nerve were strongly stained, while those in the central region were very weakly stained. MAP2 immunoreactivity was present in astrocytes of the optic chiasm, but was absent from astrocytes in the optic tract. Inside astrocytes, MAP2 immunoreactivity was excluded from bundles of glial filaments. Treatment of animals with beta,beta'-iminodipropionitrile (IDPN), which caused axonal atrophy, enhanced the staining intensity of all optic nerve astrocytes. Axons and oligodendrocytes remained unstained. Using PAGE and Western immunoblots, we found that extracts from bovine optic nerve contained MAP2. Astrocytes in any other region of the nervous system were negative for MAP2 immunoreactivity, except of the pituicytes and the astrocytes of the fimbria of hippocampus. The optic nerve, neurohypophysis, and hippocampal fimbria are white matter tracts that travel unsupported and free of surrounding nervous tissue. These findings suggest that MAP2 is expressed in astrocytes that are under excessive mechanical stress and further indicate that MAP2 may function as a cytoskeletal rigidifying agent in certain cells.

Animals↗

PubMed1996

Growth stimulation and chemotropic attraction of rat retinal ganglion cell axons in vitro by co-cultured optic nerves, astrocytes and astrocyte conditioned medium.

The effects of explants of optic nerves of different ontogenetic ages (P0 P14, adult), and of cultured astrocytes of various ages on the neurite regeneration of rat retinal ganglion cells (RGC) were assessed in vitro, using a three-dimensional culture system which allows the co-cultivation of various explants. Both co-cultured P0-P12 optic nerves and astrocyte cultures from P2 cerebral cortex stimulated the regeneration of neurites from the retinal explants after 3 days in culture. By contrast, P14 and older explants of the optic nerve, astrocytes from P17 optic nerve and astrocytes that had previously been grown in culture for more than 6 weeks had no effect on RGC neurite outgrowth. Moreover, both the P0-P12 optic nerve explants and the astrocytes from P2 cerebral cortex also seemed to have a chemotropic effect on the regenerating neurites, because the latter were longer on the side facing the co-explantat. The absence of a cellular bridge between retinal and optic nerve explants suggests that the effects are mediated by astroglia-derived diffusible neurite growth promoting factors. Accordingly, astrocyte-conditioned medium from P2 astrocytes also stimulated the outgrowth of neurites from the retinal explants. These findings show that immature astrocytes of a limited ontogenetic period release as yet unknown diffusible neurite growth-promoting factors which stimulate the regeneration of neurites from retinal explants.

Animals↗

PubMed2003

Thyroid hormone modulates the extracellular matrix organization and expression in cerebellar astrocyte: effects on astrocyte adhesion.

The effects of thyroid hormone (T(3)) on extracellular matrix (ECM) expression and organization in cerebellar astrocytes were studied. Control astrocytes exhibit laminin immunostaining distributed in a punctate configuration and fibronectin concentrated in focal points at the cell surface. These cells attach to the substratum by membrane points, as shown by scanning microscopy, possibly by focal points stained to fibronectin. In contrast, after T(3) treatment, laminin assumes a fibrillary pattern and fibronectin becomes organized in filaments homogeneously distributed on the cell surface; the cells acquire a very flat and spread morphology. T(3) treatment also modulates astrocyte adhesion. In addition, increased expression of both laminin and fibronectin was detected by Western blot. These alterations in fibronectin and/or laminin production and organization may be involved in the flat and spread morphology and in altered adhesion. We observed that fibroblast growth factor-2 (FGF(2)) added to cultures had similar effects to those described to T(3). Neutralizing antibodies against FGF(2) reversed T(3) effects on fibronectin and laminin distribution. We also observed that cerebellar neurons co-cultured on T(3)-treated astrocytes had an increase in the number of cells and presented longer neurites. Thus, we propose a novel mechanism of the effect of thyroid hormone on cerebellar development mediated by astrocytes: T(3) may induce astrocyte secretion of growth factors, mainly FGF(2), that autocrinally stimulate astrocyte proliferation, reorganization in ECM proteins, and alterations in cell spreading and adhesion. These effects may indirectly influence neuronal development.

Animals↗

PubMed2004

Focal astrocyte loss is followed by microvascular damage, with subsequent repair of the blood-brain barrier in the apparent absence of direct astrocytic contact.

Blood-brain barrier (BBB) breakdown is a feature of cerebral ischaemia, multiple sclerosis, and other neurodegenerative diseases, yet the relationship between astrocytes and the BBB integrity remains unclear. We present a simple in vivo model in which primary astrocyte loss is followed by microvascular damage, using the metabolic toxin 3-chloropropanediol (S-alpha-chlorohydrin). This model is uncomplicated by trauma, ischaemia, or primary immune involvement, permitting the study of the role of astrocytes in vascular endothelium integrity, maintenance of the BBB, and neuronal function. Male Fisher F344 rats given 3-chloropropanediol show astrocytic damage and death at 4-24 h in symmetrical brainstem and midbrain nuclear lesions, while neurons show morphological changes at 24-48 h. Fluorescent 10 kDa dextran tracers show the BBB leaking from 24 h, progressing to petechial haemorrhage after 48-72 h, with apparent repair after 6 days. BBB breakdown, but not the earlier astrocytic death, is accompanied by a delayed increase in blood flow in the inferior colliculus. An ED1 inflammatory response develops well after astrocyte loss, suggesting that inflammation may not be a factor in starting BBB breakdown. This model demonstrates that the BBB can self-repair despite the apparent absence of direct astrocytic-endothelial contact. The temporal separation of pathological events allows pharmacological intervention, and the mild reversible ataxia permits long-term survival studies of repair mechanisms.

Animals↗

PubMed2006

Sphingosine-1-phosphate is released by cerebellar astrocytes in response to bFGF and induces astrocyte proliferation through Gi-protein-coupled receptors.

The mitogenic role of sphingosine-1-phosphate (S1P) and its involvement in basic fibroblast growth factor (bFGF)-induced proliferation were examined in primary cultures of cerebellar astrocytes. Exposure to bFGF resulted in a rapid increase of extracellular S1P formation, bFGF inducing astrocytes to release S1P, but not sphingosine kinase, in the extracellular milieu. The SK inhibitor N,N-dimethylsphingosine inhibited S1P release as well as bFGF-induced growth stimulation. S1P application in quiescent astrocytes caused a dose-dependent increase in DNA synthesis. This gliotrophic effect was induced by a brief exposure to low nanomolar S1P, mimicked by the S1P receptor agonist dihydro-S1P, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S1P also induced activation of extracellular signal-regulated kinase that was inhibited again by PTX. Moreover, the S1P lyase inhibitor 4-deoxypyridoxine induced the cellular accumulation of S1P but did not affect DNA synthesis. These results support the view that S1P exerted a mitogenic effect on cerebellar astrocytes extracellularly, most likely through cell surface S1P receptors. In agreement, mRNAs for S1P1, S1P2, and S1P3 receptors are expressed in cerebellar astrocytes (Anelli et al., 2005. J Neurochem 92:1204-1215). Ceramide, a negative regulator of astrocyte proliferation and down-regulated by bFGF (Riboni et al., 2002. Cerebellum 1:129-135), efficiently inhibited S1P-induced proliferation. The S1P action appears to be part of an autocrine/paracrine cascade stimulated by bFGF and, together with ceramide down-regulation, essential for astrocytes to respond to bFGF. The results suggest that S1P and bFGF/S1P may play an important role in physiopathological glial proliferation, such as brain development, reactive gliosis and brain tumor formation.

Animals↗

PubMed1983

Primary astrocyte cultures--a key to astrocyte function.

Morphological studies have established the ubiquitous nature of astrocytes in the CNS. Their processes surround capillaries and synapses, form the subpial and subependymal layers, and seemingly invest every neuronal surface not covered by other neuronal surfaces or oligodendroglial membranes. Although such interrelationships have long suggested that astrocytes may play many critical roles, there still remains relatively little experimental information on the functions and properties of these cells. About a decade ago it became evident that primary cultures from neonatal rodent brains can consist predominantly of normal astrocytes. Based on these findings there is now an increasing number of studies in which such primary cultures are being used to help unravel the continuing enigma of the properties and functions of astrocytes. Aspects of this work are reviewed in this article. Such work has already shown that astrocytes in primary culture exhibit the basic electrophysiological characteristics which had been the only functional property well established for these cells in situ. Further studies of the electrophysiological properties of these cells, which can be correlated with ion transport studies, are beginning to show that astrocytes may have more complex electrophysiological properties than had previously been supposed, as well as a number of important electrically silent ion fluxes. In addition, astrocytes in primary culture show uptake of and receptors for a number of transmitters, properties which have wide-ranging implications. Studies in culture also support work in vivo that astroglia may have an important role in neuronal development.

Animals↗

PubMed1984

Astrocyte cell lineage. IV. Changes in the organization of microfilaments and adhesion patterns during astrocyte differentiation in culture.

The organization of microfilaments using NBD-phallacidin and cell adhesion to substratum by surface reflection interference microscopy was examined during differentiation of astrocytes in colony cultures and correlated with motile behaviour of cells. Disaggregated cells from neopallium of 12-day-old or newborn DBA/1J mouse embryos were used to establish colonies and astrocyte precursor cells at various stages of differentiation along the astrocyte lineage were examined after 3 days, 1, 2 and 4 weeks in culture. The earliest astrocyte precursor cells, the glioblasts, are stationary and form epithelial-type colonies which adhere to the substratum primarily around the edge where large microfilament bundles are found. Bundles of microfilaments are also present around the apical ends of closely packed cells. As the epithelial cells start to separate and transform into flat proastroblasts, adherens-type junctions which have a zig-zag appearance and are associated with microfilaments form between adjacent cells. In the highly motile astroblasts these junctional regions break down into multiple smaller regions where the separated cells remain in contact through fine processes. The astroblasts also have stress fibres, focal contacts with substratum, foci from which microfilament bundles radiate and a complex pattern of fine, circumferentially oriented bundles of microfilaments. This elaborate organization of microfilaments disappears as the motile astroblasts differentiate into stationary fibrous astrocytes that have little polymerized actin and lack focal contacts. These results show that stationary astrocyte precursor cells in vitro go through a highly motile stage having a characteristic distribution of microfilaments and focal contacts before becoming stationary again. We consider that the motile stage could correspond to the stage in vivo when astrocyte precursor cells migrate from the ventricular and subventricular regions to take up position in different parts of the developing brain.

Actins↗

PubMed2005

A new approach to inhibiting astrocytic IP3-induced intracellular calcium increase in an astrocyte-neuron co-culture system.

Astrocytes exhibit dynamic Ca2+ mobilization, such as Ca2+ wave and Ca2+ oscillation, via an inositol 1,4,5-triphosphate-induced Ca2+ release (IICR)-dependent mechanism. The physiological functions of astrocytic Ca2+ mobilization, however, are poorly understood. To investigate this issue, we created a plasmid encoding an enhanced green fluorescent protein-tagged inositol 1,4,5-triphosphate absorbent protein and expressed it in cultured astrocytes. Expression of this protein inhibited both IICR and the Ca2+ wave in cultured astrocytes. By combining this method to the single cell electroporation technique, we were able to inhibit IICR specifically in astrocytes in an astrocyte-neuron co-culture system. Our approach provides a useful tool for direct examination of the physiological role of astrocytic Ca2+ signaling on neuronal function.

Adenosine Triphosphate↗

PubMed2004

The expression of PEA-15 (phosphoprotein enriched in astrocytes of 15 kDa) defines subpopulations of astrocytes and neurons throughout the adult mouse brain.

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is an abundant phosphoprotein in primary cultures of mouse brain astrocytes. Its capability to interact with members of the apoptotic and mitogen activated protein (MAP) kinase cascades endows PEA-15 with anti-apoptotic and anti-proliferative properties. We analyzed the in vivo cellular sources of PEA-15 in the normal adult mouse brain using a novel polyclonal antibody. Immunohistochemical assays revealed numerous PEA-15-immunoreactive cells throughout the brain of wild-type adult mice while no immunoreactive signal was observed in the brain of PEA-15 -/- mice. Cell morphology and double immunofluorescent staining showed that both astrocytes and neurons could be cellular sources of PEA-15. Closer examination revealed that in a given area only part of the astrocytes expressed the protein. The hippocampus was the most striking example of this heterogeneity, a spatial segregation restricting PEA-15 positive astrocytes to the CA1 and CA3 regions. A PEA-15 immunoreactive signal was also observed in a few cells within the subventricular zone and the rostral migratory stream. In vivo analysis of an eventual PEA-15 regulation in astrocytes was performed using a model of astrogliosis occurring along motor neurons degeneration, the transgenic mouse expressing the mutant G93A human superoxyde-dismutase-1, a model of amyotrophic lateral sclerosis. We observed a marked up-regulation of PEA-15 in reactive astrocytes that had developed throughout the ventral horn of the lumbar spinal cord of the transgenic mice. The heterogeneous cellular expression of the protein and its increased expression in pathological situations, combined with the known properties of PEA-15, suggest that PEA-15 expression is associated with a particular metabolic status of cells challenged with potentially apoptotic and/or proliferative signals.

3T3 Cells↗