Morphological Changes In Astrocytes Research Articles

T-PA–specific modulation of a human blood-brain barrier model involves plasmin-mediated activation of the Rho kinase pathway in astrocytes

AbstractTissue-type plasminogen activator (t-PA) can modulate permeability of the neurovascular unit and exacerbate injury in ischemic stroke. We examined the effects of t-PA using in vitro models of the blood-brain barrier. t-PA caused a concentration-dependent increase in permeability. This effect was dependent on plasmin formation and potentiated in the presence of plasminogen. An inactive t-PA variant inhibited the t-PA–mediated increase in permeability, whereas blockade of low-density lipoprotein receptors or exposed lysine residues resulted in similar inhibition, implying a role for both a t-PA receptor, most likely a low-density lipoprotein receptor, and a plasminogen receptor. This effect was selective to t-PA and its close derivative tenecteplase. The truncated t-PA variant reteplase had a minor effect on permeability, whereas urokinase and desmoteplase were ineffective. t-PA also induced marked shape changes in both brain endothelial cells and astrocytes. Changes in astrocyte morphology coincided with increased F-actin staining intensity, larger focal adhesion size, and elevated levels of phosphorylated myosin. Inhibition of Rho kinase blocked these changes and reduced t-PA/plasminogen–mediated increase in permeability. Hence plasmin, generated on the cell surface selectively by t-PA, modulates the astrocytic cytoskeleton, leading to an increase in blood-brain barrier permeability. Blockade of the Rho/Rho kinase pathway may have beneficial consequences during thrombolytic therapy.

Calcium-binding proteins and GFAP immunoreactivity alterations in murine hippocampus after 1 month of exposure to 835 MHz radiofrequency at SAR values of 1.6 and 4.0 W/kg

Widespread use of wireless mobile communication has raised concerns of adverse effect to the brain owing to the proximity during use due to the electromagnetic field emitted by mobile phones. Changes in calcium ion concentrations via binding proteins can disturb calcium homeostasis; however, the correlation between calcium-binding protein (CaBP) immunoreactivity (IR) and glial cells has not been determined with different SAR values. Different SAR values [1.6 (E1.6 group) and 4.0 (E4 group) W/kg] were applied to determine the distribution of calbindin D28-k (CB), calretinin (CR), and glial fibrillary acidic protein (GFAP) IR in murine hippocampus. Compared with sham control group, decreased CB and CR IRs, loss of CB and CR immunoreactive cells and increased GFAP IR exhibiting hypertrophic cytoplasmic processes were noted in both experimental groups. E4 group showed a prominent decrement in CB and CR IR than the E1.6 group due to down-regulation of CaBP proteins and neuronal loss. GFAP IR was more prominent in the E4 group than the E1.6 group. Decrement in the CaBPs can affect the calcium-buffering capacity leading to cell death, while increased GFAP IR and changes in astrocyte morphology, may mediate brain injury due to radiofrequency exposure.

An in vitro model for studying the effects of continuous ethanol exposure on N-methyl- d-aspartate receptor function

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl- d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post–ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I NMDA) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I NMDA from CEE-treated cells were significantly larger than I NMDA from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I NMDA were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial–neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial–neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.

The Rho kinase inhibitor Fasudil up‐regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes

Glutamate transporters play a major role in maintaining brain homeostasis and the astrocytic transporters, EAAT1 and EAAT2, are functionally dominant. Astrocytic excitatory amino acid transporters (EAATs) play important roles in various neuropathologies wherein astrocytes undergo cytoskeletal changes. Astrocytic plasticity is well documented, but the interface between EAAT function, actin and the astrocytic cytoskeleton is poorly understood. Because Rho kinase (ROCK) is a key determinant of actin polymerization, we investigated the effects of ROCK inhibitors on EAAT activity and astrocytic morphology. The functional activity of glutamate transport was determined in murine cultured astrocytes after exposure to the ROCK inhibitors Fasudil (HA-1077) and Y27632 using biochemical, molecular and morphological approaches. Cytochemical analyses assessed changes in astrocytic morphology, F-/G-actin, and localizations of EAAT1/2. Fasudil and Y27632 increased [(3)H]-D-aspartate (D-Asp) uptake into astrocytes, and the action of Fasudil was time-dependent and concentration-related. The rapid stellation of astrocytes (glial fibrillary acidic protein immunocytochemistry) induced by Fasudil was accompanied by reduced phalloidin staining of F-actin and increased V(max) for [(3)H]-D-Asp uptake. Immunoblotting after biotinylation demonstrated that Fasudil increased the expression of EAAT1 and EAAT2 on the cell surface. Immunocytochemistry indicated that Fasudil induced prominent labelling of astrocytic processes by EAAT1/2. These data show for the first time that ROCK plays a major role in determining the cell surface expression of EAAT1/2, providing new evidence for an association between transporter function and astrocytic phenotype. ROCK inhibitors, via the actin cytoskeleton, effect a consequent elevation of glutamate transporter function - this activity profile may contribute to their beneficial actions in neuropathologies.

Effects of mild hypothermia on the expression of glial fibrillary acidic protein and ultrastructural changes in astrocytes in the hippocampal CA1 subfield following global cerebral ischemia and reperfusion

Objective To observe the expression of glial fibrillary acidic protein ( GFAP), and the pathological and ultrastructnral changes of astrocytes in the CA1 subfield of the hippocampus following global cerebral ischemia and reperfusion, and to explore the neuroprotective mechanism of mild hypothermia. Methods Global cerebral ischemia was established in rats by a modified version of Pulsinelli's method. Ninety-six rats were divided into three groups including a sham-operated group, a normothermic ischemic reperfusion (IR) group and a hypothermic ischemic reperfusion (HIR) group. Each group had four subgroups which were sacrificed for 6, 12 or 24 hours, or 4 days after reperfusion (for each subgroup n = 8 ). Hematoxylin-eosin (HE) staining was used to observe morphological changes in neurons in the CA1 subfield of the hippocampus. TUNEL methods were used to detect apoptosis among those neurons. Immunohistochemical staining was used to detect the expression of GFAP in the CA1 subfield and the mechanism of astrocyte pathology. GFAP TUNEL double-labeled immunohistochemistry was used with both the shamoperated and experimental groups. Electron microscopy was also used to evaluate morphological changes in astrocytes 24 hours and 4 days after ischemia and reperfusion. Results Compared with the sham-operated group, expression of GFAP immunoreactive positive cells increased gradually in the CA1 subfield of the IR group rats. Compared with the IR group, expression of GFAP immunoreactive positive cells was significantly lower in the HIR group at all time points. Microscopic observation at the 4th day showed that some astrocytes in the CA1 subfield had died through oncosis. Conclusions Mild hypothermia can significantly decrease the expression of GFAP immunoreactive positive cells and the number of apoptotic neurons in the CA1 subfield of the hippocampus, minimize cell oedema and provide protection for neurons. Oncosis kills astrocytes following global cerebral ischemia and reperfusion. Key words: Global cerebral ischemia and reperfusion; Mild hypothermia; Astrocytes; Glial fibrillary acidic protein; Oncosis

Chemical preconditioning-induced reactive astrocytosis contributes to the reduction of post-ischemic edema through aquaporin-4 downregulation

Aquaporins are a family of membrane proteins that promote the transmembrane diffusion of water. Aquaporin-4 (AQP4) is a predominant water channel protein in the brain and is concentrated in the end-feet of astrocytes. A critical question is what role astrocytic AQP4 plays in pathological conditions. Another matter to be elucidated is the relationship between morphological changes in astrocytes and AQP4 expression in such cases. We investigated the correlation between AQP4 expression and post-ischemic brain edema formation with astrocytic molecular markers after 3-nitropropionic acid (3NP) preconditioning. 3NP is a mitochondrial toxin, which can induce tolerance to ischemia at subtoxic levels. Rats were treated with 3NP at the tolerance-inducible and the non-tolerance-inducible stage (TS or NTS) before focal ischemia. The control group was injected with physiological saline. After ischemia, the hemispheric enlargement (HE) was volumetrically measured. Immunohistochemical and immunofluorescence analyses of AQP4, glial fibrillary acidic protein (GFAP), and glutamine synthetase (GS) were also conducted after the 3NP treatment and a vehicle was applied. HE was found to be significantly smaller in the TS group than in the vehicle group or the NTS group. The immunofluorescence analyses demonstrated that the AQP4 immunoreactivity in the cortex and striatum was significantly reduced in the TS group but not in the NTS group. In contrast, both GFAP expression and GS expression in the TS group were enhanced, with reactive astrocytosis. AQP4 downregulation in reactive astrocytosis may be one of the factors contributing to the role of 3NP preconditioning in attenuating post-ischemic edema.

Dysfunction of Retinal Cell and Optic Nerve by Continuous Cerebroventricular Infusion of Glucosamine

Abstract － We have investigated the effect of glucosamine on the retinal cells after continuous infusion into cerebroventricle by using osmotic minipump to avoid peripheral effect. Continuous intracerebroventricular (i.c.v) infusion of glucosamine with the rate of 0.1 μmol/10 μl/hr for 7 days resulted in morphological changes of the optic nerve in electron microscopic level as well as morphological changes of the retina in light microscopic level. Retinal sections were immunostained for the detection of morphological changes of astro-cytes. GFAP immunoreactivity appeared not only in the Muller cells but also many of the radial processes of Muller cells. The optic nerve showed deformed axon and slight lamellar separation of myelin sheath after continuous infusion of glucosamine in observing with electron microscope. Interestingly, vacuoles were observed in deformed axons and retinal layers were folded and detached. These results suggested that glucosamine plays a role in induction of morphological dysfunction in retina and optic nerves.Keywords: Glucosamine, Retinal cell, O-glycosylation, GFAP, Vimentin, Osmotic minipump

Essential role of mitogen-activated protein kinase pathways in protease activated receptor 2-mediated nitric-oxide production from rat primary astrocytes

Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-κB (NF-κB) DNA binding and transcriptional activity as well as IκBα phosphorylation. Inhibitors of NF-κB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-κB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-κB pathways.

The organotin compounds trimethyltin (TMT) and triethyltin (TET) but not tributyltin (TBT) induce activation of microglia co-cultivated with astrocytes

The organotin compounds trimethyltin (TMT), triethyltin (TET) and tributyltin (TBT) show different organotoxicities in vivo. While TMT and TET induce a strong neurotoxicity accompanied by microglial and astroglial activation, TBT rather effects the immune system. Previously, we have shown in an in vitro co-culture model that microglial cells can be activated by TMT in the presence of astrocytes. In this study, we wanted to investigate (a) if the neurotoxic organotin compound TET can also activate microglial cells in vitro similar to TMT and (b) if differences between the neurotoxicants TMT and TET on the one side and TBT on the other exist concerning microglial activation. Therefor, purified microglial and astroglial cell cultures from neonatal rat brains were treated either alone or in co-cultures for 24 h with different concentrations of TMT, TET or TBT and the basal cytotoxicity and nitric oxide formation was determined. Furthermore, morphological changes of astrocytes were examined. Our results show that microglial activation can be increased in subcytolethal concentrations, but only in the presence of astrocytes and not in microglial cell cultures alone. This increase was induced by the neurotoxicants TMT and TET but not by TBT. Taken together, the differing microglia activating effect of the organotin compounds may contribute to the differing neurotoxic potential of this group of chemicals in vivo. In addition, our results emphasize the need for co-culture systems when studying interactions between different cell types for toxicity assessment.

Morphological changes in hippocampal astrocytes induced by environmental enrichment in mice

Environmental enrichment is known to induce plastic changes in the brain, including morphological changes in hippocampal neurons, with increases in synaptic and spine densities. In recent years, the evidence for a role of astrocytes in regulating synaptic transmission and plasticity has increased, and it is likely that morphological and functional changes in astrocytes play an important role in brain plasticity. Our study was designed to evaluate changes in astrocytes induced by environmental enrichment in the CA1 region of the hippocampus, focusing on astrocytic density and on morphological changes in astrocytic processes. After 8 weeks of environmental enrichment starting at weaning, male CF-1 mice presented no significant changes in astrocyte number or in the density of glial fibrillary acidic protein (GFAP) immunoreactivity in the stratum radiatum. However, they did present changes in astrocytic morphology in the same region, as expressed by a significant increase in the ramification of astrocytic processes measured by the Sholl concentric circles method, as well as by an increase in the number and length of primary processes extending in a parallel orientation to CA1 nerve fibers. This led astrocytes to acquire a more stellate morphology, a fact which could be related to the increase in hippocampal synaptic density observed in previous studies. These findings corroborate the idea that structural changes in astrocytic networks are an integral part of plasticity processes occurring in the brain.

Lipocalin-2 Is an Autocrine Mediator of Reactive Astrocytosis

Astrocytes, the most abundant glial cell type in the brain, provide metabolic and trophic support to neurons and modulate synaptic activity. In response to a brain injury, astrocytes proliferate and become hypertrophic with an increased expression of intermediate filament proteins. This process is collectively referred to as reactive astrocytosis. Lipocalin 2 (lcn2) is a member of the lipocalin family that binds to small hydrophobic molecules. We propose that lcn2 is an autocrine mediator of reactive astrocytosis based on the multiple roles of lcn2 in the regulation of cell death, morphology, and migration of astrocytes. lcn2 expression and secretion increased after inflammatory stimulation in cultured astrocytes. Forced expression of lcn2 or treatment with LCN2 protein increased the sensitivity of astrocytes to cytotoxic stimuli. Iron and BIM (Bcl-2-interacting mediator of cell death) proteins were involved in the cytotoxic sensitization process. LCN2 protein induced upregulation of glial fibrillary acidic protein (GFAP), cell migration, and morphological changes similar to characteristic phenotypic changes termed reactive astrocytosis. The lcn2-induced phenotypic changes of astrocytes occurred through a Rho-ROCK (Rho kinase)-GFAP pathway, which was positively regulated by nitric oxide and cGMP. In zebrafishes, forced expression of rat lcn2 gene increased the number and thickness of cellular processes in GFAP-expressing radial glia cells, suggesting that lcn2 expression in glia cells plays an important role in vivo. Our results suggest that lcn2 acts in an autocrine manner to induce cell death sensitization and morphological changes in astrocytes under inflammatory conditions and that these phenotypic changes may be the basis of reactive astrocytosis in vivo.

Reactive Astrocytes, Vesicle Traffic and Regulated Exocytosis

Event Abstract Back to Event Reactive Astrocytes, Vesicle Traffic and Regulated Exocytosis Robert Zorec1* 1 University of Ljubljana, Laboratory of Neuroendocrinology-Molecular Cell Physiology, Slovenia Astrocytes are important communication cells with neurons and integrators of the brain function. In pathological conditions they become reactive and upregulate also the expression of cytoskeletal intermediate filaments, but the function of this event is poorly understood. Communication between astrocytes and neurons is importantly supported by exocytotic release of gliotransmitters (such as glutamate, neuroactive peptides and ATP) from membrane-bound vesicles and it may be affected by physiological and morphological changes in astrocytes. Prior and after fusing with the plasma membrane, membrane-bound vesicles are transported through the cytoplasm. In order to study the role of the cytoskeleton in their trafficking we labelled different types of vesicles and monitored their traffic in different experimental conditions. Prefusion transport of peptidergic vesicles was studied by fluorescently labelled ANP (atrial natriuretic peptide; ANP); and postfusion transport of glutamatergic and peptidergic vesicles was studied by labelling vesicles with antibodies against specific membrane or lumen vesicle proteins. To monitor the mobility of these vesicles we used confocal microscopy. Studies of the prefusion mobility of fluorescently labeled peptidergic ANP vesicles in the cytoplasm of single rat and mouse astrocytes in culture and studies of postfusion transport of glutamatergic and peptidergic vesicles revealed the dependence of the vesicle traffic on the cytoskeleton. We reported that delivery of vesicles to the plasma membrane for exocytosis and their retrieval involves an interaction with the cytoskeleton, in particular microtubules and actin filaments, which is similar to neurons and excitable secretory cells. Moreover, the experiments in mouse astrocytes deficient in the intermediate filament proteins, glial fibrillary acidic protein and vimentin, showed that intermediate filaments may play a global role in exocytotic vesicle traffic and potentially affect the capacity of astrocytes to communicate with neighbouring cells via regulated exocytosis. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Symposium10 – Neuron-glial communications in the normal and pathological brain Citation: Zorec R (2009). Reactive Astrocytes, Vesicle Traffic and Regulated Exocytosis. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.040 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Nov 2009; Published Online: 19 Nov 2009. * Correspondence: Robert Zorec, University of Ljubljana, Laboratory of Neuroendocrinology-Molecular Cell Physiology, Ljubljana, Slovenia, robert.zorec@mf.uni-lj.si Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Robert Zorec Google Robert Zorec Google Scholar Robert Zorec PubMed Robert Zorec Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Neurometabolic Disorders: Urea-Cycle Disorder, Outcomes, Development and Treatment

The urea-cycle disorders (UCDs) are a group of congenital enzyme and carrier deficiencies predisposing to fatal hyperammonemia. Hyperammonemia causes alterations of neurotransmitter function and cell volume, leading to cerebral edema. Neuropathological findings of UCDs reflect changes in astrocyte morphology. Neurological features accompanying acute hyperammonemia include changes in behavior and consciousness in the short term, and potential for impairments in memory and executive function as long-term effects. Plasma measures of ammonia and glutamine, although useful for clinical monitoring, are poor markers of CNS function. Treatment combines dietary protein restriction and nitrogen-scavenging agents. Despite this regimen, patients remain at risk for repeated hyperammonemic events and further neurological damage. Thus, neuroprotective strategies should be explored as adjunctive therapies.

A new hepatic encephalopathy model to monitor the change of neural amino acids and astrocytes with behaviour disorder

To elucidate the pathogenesis of hepatic encephalopathy (HE), we developed a new HE model with behaviour disorder. Male Wistar rats were divided into four treatment groups: a HE model: acetaminophen (APAP)+3-methylcholanthrene (3-MC) group (APAP+MC group); control group: acetaminophen group; 3-methylcholanthrene group; and a no-treatment group. We monitored the changes of neural amino acids in the synaptic cleft and astrocytes in the brain during behaviour disorder. In the APAP+MC group, alanine amino transferase, blood ammonia and glucose increased from 3 h and total bilirubin increased at 6 h. Prothrombin time was prolonged from 3 h in the APAP+MC group. The APAP+MC group exhibited centrilobular necrosis in the liver after 8 h. In the APAP+MC group, rats jumped vertically and this vertical activity increased significantly from 4 to 7 h. During the behaviour disorder, we found that glutamate and aspartate increased in the synaptic cleft from 4 h after treatment with APAP+3-MC, glutamate increased 23.9-fold at 7 h and aspartate increased 16.1-fold at 4 h, whereas glutamine did not change. At that time, we observed morphological changes of the astrocytes by immunostaining for the glial fibrillary acidic protein. Our new HE model demonstrated that increased excitatory neural amino acids and morphological change in astrocytes were involved in the behaviour disorder that occurs with HE.

PKCε upregulates voltage‐dependent calcium channels in cultured astrocytes

Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.

Enantio-selective effects of clenbuterol in cultured neurons and astrocytes, and in a mouse model of cerebral ischemia

Neuroprotective effects of the lipophilic β 2-adrenoceptor agonist clenbuterol have been established in neuronal cultures and in various rodent models of stroke. In previous studies, however, clenbuterol was always applied as a racemate, while it has not been established whether the enantiomers differ in their neuroprotective activities. Here, we demonstrate that R,S-clenbuterol and S(+)-clenbuterol, but not the R(−)-enantiomer protect cultured neurons against glutamate-mediated excitotoxicity and staurosporine-induced apoptosis. Similar to previous findings with clenbuterol racemate, the neuroprotective effect of S(+) -clenbuterol correlated well with morphological changes of astrocytes which transformed into dense stellate cells with dendritic processes indicating β 2-adrenoceptor-mediated activation. Most importantly, the S(+)-enantiomer but not R(−)-clenbuterol reduced ischemic brain damage similar to the effect of the racemate. The selective β 2-adrenoceptor antagonist butoxamine blocked this neuroprotective effect of S(+)-clenbuterol. In addition, S(+)-clenbuterol significantly reduced blood pressure, enhanced blood glucose levels and increased glucocorticoid levels compared to vehicle-or R(−)-clenbuterol-treated controls. These results clearly demonstrate that S(+)-clenbuterol is the eutomer that mediates neuroprotective effects of the β 2-adrenoceptor agonist but also according changes of physiological parameters.

Three‐dimensional confocal morphometry reveals structural changes in astrocyte morphology in situ

Neuronal activity and many pathological states in the CNS are accompanied by transient astrocytic swelling, which affects excitability, extrasynaptic transmission, and neuron-glia interactions. By using three-dimensional confocal morphometry (3DCM), we quantified the morphometric parameters of astrocytes in intact tissue. In experiments performed in brain cortex slices from transgenic GFAP/EGFP mice, we applied 3DCM to study the dynamic changes in astrocyte morphology during hypotonic stress. Our morphometric analysis showed that the effect of a 10-min application of hypotonic solution (200 mmol/kg) on the swelling of different cell compartments was dependent on the extent of the swelling of the total astrocyte volume. If the swelling of the whole cell, i.e., soma and processes, was less than approximately 10%, there were no differences between the swelling of the soma and the processes. However, if the swelling of the total cell volume was greater than 10%, the swelling of the processes was greater than the swelling of the soma. Analyzing the effect of hypotonic solution on the morphology of these astrocytes revealed that the total cell volume increased; however, certain cell compartments were distinguished in which the volume increased, whereas in other compartments cell volume decreased or apparently did not change, and the structure of some compartments was altered. Our data show that astrocytes in brain slices undergoing hypotonic stress display cell volume regulation as well as transient changes in morphology.

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.

L‐Glutamate activates RhoA GTPase leading to suppression of astrocyte stellation

The actin cytoskeleton is known to support cellular morphological changes. Rho family small GTPases function as switching molecules to promote the convergence of both extracellular and intracellular signals in regulating cytoskeletal organization. Evidence indicates that L-glutamate suppresses morphological changes of astrocytes over a broad spectrum. To test the possibility that L-glutamate affects cytoskeletal reorganization, we investigated its effect on morphological changes induced by manganese exposure. L-glutamate concentration-dependently prevented and reversed manganese-induced astrocyte stellation and cytoskeletal disruption. The suppressive effect of L-glutamate on manganese-induced stellation was mediated by the activation of the glutamate transporter rather than ionotropic or metabotropic glutamate receptors. Pharmacological and biochemical approaches revealed the involvement of Ras homolog gene family, member A (RhoA) activation in L-glutamate-mediated suppression of manganese-induced stellation. The activation of RhoA by L-glutamate was partly through the up-regulation of guanine nucleotide exchange factor phosphorylation and was abrogated by competitive nonsubstrate inhibitors. Furthermore, the hyperphosphorylation of myosin light chain and cofilin through the activation of RhoA following L-glutamate treatment synergistically stabilized actin stress fibres. These results suggest that manganese-induced stellation is suppressed by a mechanism involving glutamate transporters. Our in vitro findings also strongly indicate that astrocyte morphological plasticity is under the control of RhoA and that manganese and L-glutamate regulate astrocyte morphology by modulating this switching molecule under culture conditions.

Regulation of astrocytic glutamate transporter expression by Akt: evidence for a selective transcriptional effect on the GLT‐1/EAAT2 subtype

In the nervous system, astrocytes express different ratios of the two glial glutamate transporters, glutamate transporter subtype 1 (GLT-1) and glutamate/aspartate transporter (GLAST), but little is known about the signaling pathways that independently regulate their expression. Treatment with dibutyryl-cAMP, epidermal growth factor (EGF) or other growth factors both induces expression of GLT-1 and increases expression of GLAST in astrocyte cultures. The induction of GLT-1 is correlated with morphological and biochemical changes that are consistent with astrocyte maturation. Pharmacological studies suggest that phosphatidylinositol 3-kinase (PI-3K) and the nuclear transcription factor-kappaB (NF-kappaB) may be involved in the induction of GLT-1 expression. In several signaling systems Akt, also known as protein kinase B (PKB), functions downstream of PI-3K. In these present studies we used lentiviral vectors engineered to express dominant-negative (DN), constitutively active (CA), or null variants of Akt to study the possible involvement of Akt in the regulation of GLT-1. Expression of DN-Akt attenuated the EGF-dependent induction of GLT-1. Expression of CA-Akt caused a dose- and time-dependent increase in GLT-1 protein, increased GLT-1 mRNA levels, increased dihydrokainate-sensitive (presumably GLT-1 mediated) transport activity, and caused a change in astrocyte morphology to a more stellate shape, but had no effect on GLAST protein levels. Finally, the expression of CA-Akt increased the expression of a reporter construct containing a putative promoter fragment from the human homolog of GLT-1, called EAAT2. From these studies, we conclude that Akt induces the expression of GLT-1 through increased transcription and that Akt can regulate GLT-1 expression without increasing GLAST expression in astrocytes.