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Results for “Astrocyte morphology”

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PubMed2000

The p44/42 mitogen-activated protein kinase cascade is involved in the induction and maintenance of astrocyte stellation mediated by protein kinase C.

The mitogen-activated protein kinase (MAPK) is known to be involved in the differentiation of various types of cells. To understand the role of p44/42 MAPK (ERK1/2) in astrocyte differentiation, we investigated the effects of U0126 and PD98059, specific inhibitors of the MAPK-activating enzyme MEK, on astrocyte morphology in culture. Cultured rat cortical astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but differentiated into process-bearing stellate cells in response to the membrane-permeable cyclic AMP analog dibutyryl cyclic AMP (dBcAMP) or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). dBcAMP-induced astrocyte stellation was not affected by MEK inhibitors, while PMA-induced astrocyte stellation was significantly blocked by U0126 (0.1-10 microM) and PD98059 (10-30 microM). Western blot analysis with an antibody specific for phosphorylated ERK1/2 revealed that PMA, but not dBcAMP, induced phosphorylation of ERK1/2 in a time- and concentration-dependent manner. The PMA-induced astrocyte stellation and ERK1/2 phosphorylation were blocked by specific PKC inhibitors, GF-109203X (0.01-1 microM) and calphostin C (1 microM). In addition, when U0126 or PD98059 was added after treatment with PMA, stellate astrocytes returned to polygonal. These results suggest that the MEK/ERK cascade is involved in the induction and maintenance of astrocyte stellation mediated by PKC, but not by cyclic AMP signaling.

Animals↗
PubMed1992

Ultrastructural characterization of transitional cells in oligodendrogliomas.

In oligodendroglial tumors the expression of glial fibrillary acidic protein (GFAP) is found in cells with an astrocytic morphology representing preexistent or neoplastic astrocytes. In addition, a proportion of the GFAP-positive cells has the morphology of miniature gemistocytes (minigemistocytes) or oligodendrocytes (gliofibrillary oligodendrocytes or GFOC). Both minigemistocytes and GFOC are considered as cells transitional between astrocytic and oligodendroglial lineage. Though minigemistocytes can readily be distinguished in routinely stained histological sections, GFAP immunostaining is obligatory for the identification of the GFOC. In the present study, the GFOC is characterized at the ultrastructural level using an immunogold-silver stain on semithin (1 micron) slides for identification of GFAP immunoreactivity and subsequent processing of the adjacent slide for immunoelectron microscopy. In analogy with the minigemistocytes, the glial filaments in the GFOC are arranged in parallel bundles. The finding of cells with ultrastructural features intermediate between those of GFOC and minigemistocytes suggests a close relationship and a possible interconvertibility between the two transitional cell types in oligodendrogliomas.

Adult↗
PubMed2005

A truncated tropomyosin-related kinase B receptor, T1, regulates glial cell morphology via Rho GDP dissociation inhibitor 1.

Through tropomyosin-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

Amino Acid Motifs↗
PubMed2004

Beta-hydroxy-butyrate alters the extracellular content of S100B in astrocyte cultures.

Astrocytes have a variety of roles in maintaining neural tissue physiology, including energetic support, uptake and metabolism of glutamate and secretion of neurotrophic factors. Glutamate toxicity has been implicated in neurodegenerative disorders associated with conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. Glucose is the main energetic substrate for brain cells but, in some circumstances, the ketone bodies are used as a supplementary source and have been suggested to be neuroprotective agents against seizure disorders. Here, we investigate some possible biochemical changes in astrocyte cultures induced by beta-hydroxy-butyrate, the predominant blood ketone body. Its effect upon S100B secretion, astrocyte morphology and glutamate uptake was particularly investigated. S100B, a calcium-binding protein expressed and secreted by astrocytes, has neurotrophic activity and a possible role in epileptogenesis. Cell morphology was investigated by phase-contrast microscopy and immunocytochemistry for actin, GFAP and S100B. Our data show that beta-hydroxy-butyrate induces dramatic changes in astrocyte morphology and, independent of this, causes changes in the extracellular content of S100B. We observed an increment in S100B 1 h after beta-hydroxy-butyrate addition and a decrease 24 h later. No changes were observed in glutamate uptake. These astrocytic modifications may be associated with reduced neuronal excitability observed in the ketogenic condition.

3-Hydroxybutyric Acid↗
PubMed2005

Morphological alterations and cell death provoked by the branched-chain alpha-amino acids accumulating in maple syrup urine disease in astrocytes from rat cerebral cortex.

1. Maple syrup urine disease (MSUD) is an inherited metabolic disorder predominantly characterized by neurological dysfunction and cerebral atrophy whose patophysiology is poorly known. 2. We investigated here whether the branched-chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine (Val), which are the biochemical hallmark of this disorder, could alter astrocyte morphology and cytoskeleton reorganization by exposing cultured astrocytes from cerebral cortex of neonatal rats to various concentrations of the amino acids. A change of cell morphology from the usual polygonal to the appearance of fusiform or process-bearing cells was caused by the BCAA. Cell death was also observed when astrocytes were incubated in the presence of BCAA for longer periods. 3. Val-treated astrocytes presented the most dramatic morphological alterations. Immunocytochemistry with anti-actin and anti-GFAP antibodies revealed that all BCAA induced reorganization of actin and GFAP cytoskeleton. In addition, lysophosphatidic acid, an activator of RhoA GTPase pathway, was able to totally prevent the morphological alterations and cytoskeletal reorganization induced by Val, indicating that the RhoA signaling pathway was involved in these effects. 4. Furthermore, creatine attenuated the morphological alterations provoked by the BCAA, the protection being more pronounced for Val, suggesting that impairment of energy homeostasis is partially involved in BCAA cytotoxic action. The data indicate that the BCAA accumulating in MSUD are toxic to astrocyte cells, a fact that may be related to the pathogenesis of the neurological dysfunction of MSUD patients.

Amino Acids, Branched-Chain↗
PubMed1987

Morphological changes in cultured astrocytes following exposure to calcium ionophores.

Induced differentiation of cultured astrocytes to a process-bearing morphology has been described with a variety of agents known to increase intracellular cyclic AMP. The ionophores A23187 and ionomycin were found to induce a reversible change in astrocyte morphology to a process-bearing form. Cell death also occurs following exposure to either A23187 or ionomycin. This change in morphology which occurs following exposure to ionomycin in calcium- and serum-containing Dulbecco's Modified Eagle's medium also occurs in the absence of extracellular calcium. These findings suggest a role for other second messengers than cyclic AMP in controlling astrocyte morphology.

Animals↗
PubMed1997

Pharmacological characterization of protease-activated receptor (PAR-1) in rat astrocytes.

The proteolytic action of thrombin on its receptor (protease-activated receptor-1 or PAR-1) results in a conformational change in which the new N-terminal sequence auto-activates the receptor. Peptide analogs of this N-terminal sequence (TRAPs) are able to mimic the effect of thrombin and an extensive search has led to the definition of the structural requirement for the agonist and antagonist activity on thrombin receptors in several peripheral systems. Thrombin plays an important role in central and peripheral nervous system development and PAR-1 is present in neurons and astrocytes. We have now characterized thrombin receptors pharmacologically in cultured rat astrocytes by using [3H]thymidine incorporation and reversal of stellation induced by Bt2cAMP as end-points. Thrombin increased [3H]thymidine incorporation into DNA with an EC50 of 1 nM and induced a complete reversion of cell stellation. The effects of thrombin on [3H]thymidine incorporation were mimicked by TRAP-14 (EC50 = 3 microM) and a peptide containing non-natural amino acids Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (A6Y; EC50 = 0.8 microM). Similarly, these two peptides reversed Bt2cAMP-induced stellation. The effect of thrombin, TRAP-14 and A6Y on [3H]thymidine incorporation into DNA was significantly prevented by L9R, a 9-amino-acid peptide (Leu-Val-Arg-D-Cys-Gly-Lys-His-Ser-Arg; IC50 = 180 microM against thrombin and TRAP-14 and 800 microM against A6Y) previously described as an antagonist in human platelet aggregation. L9R antagonized also thrombin effects on astrocyte morphology. These results demonstrate that rat astrocytes express PAR-1 receptors which are pharmacologically similar to those previously characterized in human platelets.

Animals↗
PubMed2006

High extracellular K(+) evokes changes in voltage-dependent K(+) and Na (+) currents and volume regulation in astrocytes.

[K(+)](e) increase accompanies many pathological states in the CNS and evokes changes in astrocyte morphology and glial fibrillary acidic protein expression, leading to astrogliosis. Changes in the electrophysiological properties and volume regulation of astrocytes during the early stages of astrocytic activation were studied using the patch-clamp technique in spinal cords from 10-day-old rats after incubation in 50 mM K(+). In complex astrocytes, incubation in high K(+) caused depolarization, an input resistance increase, a decrease in membrane capacitance, and an increase in the current densities (CDs) of voltage-dependent K(+) and Na(+) currents. In passive astrocytes, the reversal potential shifted to more positive values and CDs decreased. No changes were observed in astrocyte precursors. Under hypotonic stress, astrocytes in spinal cords pre-exposed to high K(+) revealed a decreased K(+) accumulation around the cell membrane after a depolarizing prepulse, suggesting altered volume regulation. 3D confocal morphometry and the direct visualization of astrocytes in enhanced green fluorescent protein/glial fibrillary acidic protein mice showed a smaller degree of cell swelling in spinal cords pre-exposed to high K(+) compared to controls. We conclude that exposure to high K(+), an early event leading to astrogliosis, caused not only morphological changes in astrocytes but also changes in their membrane properties and cell volume regulation.

Animals↗
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

Gene expression in astrocytes is affected by subculture.

We have investigated the effects of cell passaging and time in culture on astrocyte morphology, transferrin expression and the expression of two main astrocyte markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS: EC 6.3.1.2). When primary astrocytes were subcultured, giving rise to secondary and tertiary cultures, their morphology changed, regardless of the split ratio used to passage the cells. Correlating with this morphological change, a dramatic increase in the accumulation of GFAP and GS mRNAs was observed after cells had been passaged. This effect was in marked contrast to the moderate increase in the levels of GFAP and GS mRNAs observed over several weeks in primary culture. Hydrocortisone induction of GS gene expression was not affected by cell passage. Transferrin mRNA, which is not normally found in astrocytes in vivo, was expressed at a high level in primary cultures of astrocytes. However, transferring mRNA almost completely disappeared after the second passage. Astrocyte-conditioned media, or co-cultures with oligodendrocytes, modified transferrin gene expression. Taken together, these results show that subculturing of primary rat astrocytes leads to a dramatic change in the genetic expression of several proteins and provides a new approach to modify astrocyte differentiation in vitro.

Animals↗
PubMed1999

HMBA inhibits growth and induces differentiation of astrocytes from neonatal rat brain.

HMBA, a potent differentiation inducer belonging to the class of hybrid polar compounds, inhibited the growth of astrocytes from neonatal rat brain. At a concentration of 5.0 mM, which is necessary for growth inhibition and differentiation of a number of leukemic and solid tumour cell lines, astrocyte proliferation was reduced by 74.5+/-5.5%. On HMBA treatment astrocyte morphology changed from flat polygonal with phase translucent cytoplasm to compact retracted appearance with phase dark cytoplasm. HMBA also increased expression of GFAP, a marker for mature astrocytes. HMBA thus induces both morphological and biochemical differentiation of astrocytes. Like dbcAMP and staurosporine, two other known differentiation inducers of astrocytes, HMBA was also found to induce expression of the immediate early gene c-fos. However, unlike dbcAMP and staurosporine, which also induce fra-1, HMBA repressed cycloheximide-induced fra-1 gene expression.

Acetamides↗
PubMed1998

Adenosine stimulates stellation of cultured rat cortical astrocytes.

We investigated the effect of adenosine on astrocyte morphology by using cell cultures prepared from the cerebral cortices of neonatal rats. Cultured rat cortical astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but differentiated into process-bearing stellate cells in response to adenosine (1-1000 microM). Adenosine-induced astrocyte stellation was abolished by treatment with microtubule inhibitors, colchicine and paclitaxel, indicating the involvement of cytoskeletal elements. The effect of adenosine was mimicked by other adenosine receptor agonists, and blocked by adenosine receptor antagonists and guanosine 5'-O-(2-thiodiphosphate), indicating that the effect of adenosine is mediated by G protein-coupled adenosine receptors. Although adenosine receptors are known to be linked to adenylate cyclase or phospholipase C, adenosine did not change intracellular cyclic AMP level nor intracellular Ca2+ concentration in astrocytes. Alternatively, adenosine-induced stellation was abolished by tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide, suggesting that adenosine causes astrocyte stellation through tyrosine dephosphorylation. Adenosine may function as a factor regulating astrocyte differentiation.

Adenosine↗
PubMed2004

Intracellular pH-dependent peroxynitrite-evoked synergistic death of glucose-deprived astrocytes.

Previously, we reported that glucose-deprived astrocytes were highly vulnerable to peroxynitrite (ONOO-). Here we demonstrate that the increased vulnerability caused by glucose deprivation and ONOO- depends on intracellular pH. The ONOO- releasing reagent 3-morpholinosydnonimine (SIN-1) markedly induced the release of lactate dehydrogenase (LDH, the marker of cytotoxicity) in glucose-deprived astrocytes. Morphological studies and caspase activity assay showed that astrocytes treated together with glucose deprivation and ONOO- died mostly in a necrotic mode. Alkalinization of pH from 7.4 to 7.8 increased LDH release, whereas acidification from pH 7.4 to 7.0 decreased it. However, intracellular pH (pHi), not extracellular pH (pHe), appeared to play a critical role in the synergistic death. Thus, without a change in pHe (7.4) cytosolic acidification by a weak acid salt, sodium acetate, and a Na+/H+ antiporter inhibitor, amiloride, reduced LDH release. In contrast, a weak base, NH4Cl, and a Na+/H+ antiporter stimulator, monensin, increased pHi and greatly enhanced LDH release. The augmented death was found to be due, in part, to the preceding decrease in the level of reduced glutathione, the ONOO- scavenger, and collapse of the mitochondrial transmembrane potential at alkaline pH.

Amiloride↗
PubMed1991

Single Channel Recording from Glial Cells on the Untreated Surface of the Frog Optic Nerve.

The patch clamp technique has been used to record single channel currents from the untreated surface of the intact frog optic nerve after the meninges and basal lamina have been mechanically removed. Cells filled via dialysis with Lucifer yellow (LY) from the patch pipette had a typical astrocyte morphology and were dye-coupled to adjacent astrocytes. This is consistent with the electron-microscopic observation that all the cells on the surface of this nerve are astrocytes. Two types of ion channels were studied in detached patches. One, identified as a K+ channel, had a conductance of 88 +/- 4 (S.E.) n=9 pS and an equilibrium potential of -59 +/- 8 mV in physiological K+ solutions. The steady-state open probability was not significantly altered by changing the membrane potential. A second channel had a large conductance of 300 - 1200 pS, a reversal potential of approximately 0 mV in symmetrical and non-symmetrical solutions, and was open only in the voltage range of +/-20 mV. These are the characteristics of a large anionic channel described in other preparations including cultured astrocytes.

Journal Article↗
PubMed2003

Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation.

There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders.

Animals↗
PubMed1992

[Regulation by chloride ion of astroglial cell functions and morphological transformation].

Recently, several lines of evidence have indicated the important roles of glial cells, especially astrocytes, in the regulation of neuronal functions. The neuron-glia interaction is one of the most important issues in neuroscience, including neuropharmacology. I reviewed the present status and perspectives on the physiologic and pathologic functions of astrocytes in relation to the roles of intracellular Cl-. Astrocytes have different types of Cl- transport systems, such as voltage-sensitive and ligand-gated channels; HCO3(-)-Cl- exchange; and Na+, K+, Cl- cotransport systems. Anion exchange and cotransport systems are responsible for intracellular pH regulation and astrocytic volume regulation, respectively. Especially, astrocytic volume regulation is physiologically important for reducing the concentrations of K+ and glutamate in the extracellular space by their uptake systems. Disturbance of astrocytic volume regulation is expressed as astrocytic swelling, which is usually observed in various brain pathologic states including ischemia. Experimentally, glutamate caused a typical swelling of astrocytes in culture by Cl- and Ca(++)-dependent processes. Glutamate-induced swelling is qualitatively different from reversible swelling induced by hypoosmotic medium. Recently, we found that Cl- is intracellular factor for modulating the receptor-adenylate cyclase system in brain slices. Similarly, the receptor- and forskolin-stimulated adenylate cyclase of astrocytes showed a clear Cl- dependence. This was functionally confirmed by astrocytic morphological transformation induced by the cyclic AMP system.

Astrocytes↗
PubMed1998

Lysophosphatidic acid stimulates actomyosin contraction in astrocytes.

Lysophosphatidic acid (LPA) is an extracellular signaling molecule that can enter the central nervous system following injury or diseases that disrupt the blood-brain-barrier. Using a combination of time-lapse microscopy, immunocytochemistry, and biochemical techniques, we demonstrate that LPA stimulates profound changes in astrocyte morphology that are due to effects on the actomyosin cytoskeleton. Flat astrocytes in primary culture display prominent actin stress fibers. Treatment with the myosin light chain kinase inhibitor, ML-9, causes stress fiber dissolution and dramatic morphology changes including rounding of the cell body and the formation of processes. LPA can stabilize actin stress fibers and inhibit the morphology changes in ML-9-treated cells. Furthermore, this activity is dependent upon activation of the GTP-binding protein Rho as evidenced by the ability of C3 exoenzyme, a specific inhibitor of Rho, to block the effect. Phosphorylation of the regulatory light (RLC) chain initiates conformational changes in myosin II that result in the formation of myosin filaments and the recruitment of actin into contractile stress fibers. LPA-induced stabilization of stress fibers is accompanied by increases in phosphorylation of the RLC of myosin. Furthermore, astrocytes grown on flexible silicone undergo rapid contraction in response to LPA treatment. The forces generated by these cells manifest themselves as increased wrinkling in the silicone. The observed contraction and accompanying increases in regulatory light chain phosphorylation suggest that LPA-induced signaling cascades in astrocytes regulate actin/myosin interactions.

ADP Ribose Transferases↗
PubMed2001

Astrocytic clasmatodendrosis in hippocampal organ culture.

Mechanisms by which astrocytes are irreversibly injured from ischemic brain injury remain incompletely defined. More than 90 years ago Alzheimer showed that astrocytes lose their distal processes (i.e., undergo "clasmatodendrosis") when irreversibly injured by a reduction in blood flow, a process shown by Friede and van Houten (1961) to be due to energy failure and acidosis. Such alterations in astrocytic morphology can relate directly to changes in cell function. However, astrocytic clasmatodendrosis has largely been lost to the modern literature, perhaps because of a inability to study it under controlled conditions. In the present study, novel four-dimensional (4D)and digital deblurring imaging of glial fibrillary acidic protein (GFAP) immunostaining changes in hippocampal organ cultures (HOTCs) were used to establish an in vitro model of astrocytic clasmatodendrosis. Also, astrocytes in primary culture were transfected with green fluorescent protein (GFP) to show the occurrence of clasmatodendrosis via a parallel and separate means. In HOTCs, a significant reduction in astrocytic process length occurred 15 min (and remained for 60 min) after exposure to acidic Ringer's and mitochondrial inhibition in the pyramidal cell body layer. Time-lapsed images of primary cultures showed thinning of cell processes within 15 min of exposure to acidic Ringer's and mitochondrial inhibition. Distal processes subsequently broke away but retained their fluorescence for minutes before disintegrating along with their parent cell bodies. This report shows the spatiotemporal occurrence of clasmatodendrosis in astrocytes of HOTCs closely parallels that seen in vivo. Thus, HOTCs, where microenvironmental conditions can be controlled and single, identified cells can be followed in space and time, can be applied to study the interrelations between energy metabolism and pH that result in clasmatodendrosis.

Acidosis↗