Amyloid β Peptide (25–35) Inhibits Na+-Dependent Glutamate Uptake in Rat Hippocampal Astrocyte Cultures

Abstract: Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid β peptide (Aβ), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Aβ including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Aβ increases the vulnerability of neurons to glutamate-induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Aβ. The aim of the current study was to determine if Aβs inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Aβ can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Aβ (25–35) significantly inhibited l -glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular l -glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Aβ toxicity involving astrocytes.     

Computational modeling of paroxysmal depolarization shifts in neurons induced by the glutamate release from astrocytes

Recent experimental studies have shown that astrocytes respond to external stimuli with a transient increase of the intracellular calcium concentration or can exhibit self-sustained spontaneous activity. Both evoked and spontaneous astrocytic calcium oscillations are accompanied by exocytosis of glutamate caged in astrocytes leading to paroxysmal depolarization shifts (PDS) in neighboring neurons. Here, we present a simple mathematical model of the interaction between astrocytes and neurons that is able to numerically reproduce the experimental results concerning the initiation of the PDS. The timing of glutamate release from the astrocyte is studied by means of a combined modeling of a vesicle cycle and the dynamics of SNARE-proteins. The neuronal slow inward currents (SICs), induced by the astrocytic glutamate and leading to PDS, are modeled via the activation of presynaptic glutamate receptors. The dependence of the bidirectional communication between neurons and astrocytes on the concentration of glutamate transporters is analyzed, as well. Our numerical results are in line with experimental findings showing that astrocyte can induce synchronous PDSs in neighboring neurons, resulting in a transient synchronous spiking activity.     

Role of Branched-Chain Aminotransferase Isoenzymes and Gabapentin in Neurotransmitter Metabolism

Abstract: Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with α-ketoglutarate to form the branched-chain α-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-¹⁴C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to α-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an α-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from α-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.     

Synthesis and release of GABA in cerebral cortical neurons co-cultured with astrocytes from cerebral cortex or cerebellum

Cerebral cortical neurons were co-cultured for up to 7 days with astrocytes after plating on top of a confluent layer of astrocytes cultured from either cerebral cortex or cerebellum (sandwich co-cultures). Neurons co-cultured with either cortical or cerebellar astrocytes showed a high stimulus coupled release of gamma-aminobutyric acid (GABA), which is the neurotransmitter of these neurons. When the astrocyte selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol was added during the release experiments, an increase in the stimulus coupled GABA release was seen, indicating that the astrocytes take up a large fraction of GABA released from the neurons. The activity of the GABA synthesizing enzyme glutamate decarboxylase, which is a specific marker of GABAergic neurons, was markedly increased in sandwich co-cultures of cortical neurons and cerebellar astrocytes compared to neurons cultured in the absence of astrocytes whereas in co-cultures with cortical astrocytes this increase was less pronounced. Pure astrocyte cultures did not show any detectable glutamate decarboxylase activity. The astrocyte specific marker enzyme glutamine synthetase (GS) was present at high activity in a glucocorticoid-inducible form in pure astrocytes as well as in co-cultures regardless of the regional origin of the astrocytes. When neurons were cultured on top of the astrocytes, the specific activity of GS was lower compared to astrocytes cultured alone, a result compatible with the notion that neurons are devoid of this enzyme. The results show that cortical neurons develop and differentiate when seeded on top of both homotypic and heterotypic astrocytes. Moreover, it could be demonstrated that the two cell types in the culture system communicate with each other with regard to GABA homeostasis during transmitter release.     

Astrocytes aged in vitro show a decreased neuroprotective capacity

Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100beta increased at 90 days when cells were positive for the senescence beta-galactosidase marker. In long-term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn-superoxide dismutase (SOD-1) and haeme oxygenase-1 (HO-1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90-DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H2O2 exposure. Enhanced glutamate uptake was probably because of up-regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age-related neurodegenerative processes.     

Glucocorticoids Inhibit Glucose Transport and Glutamate Uptake in Hippocampal Astrocytes: Implications for Glucocorticoid Neurotoxicity

Glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, can damage the hippocampus and impair its capacity to survive coincident neurological insults. This GC endangerment of the hippocampus is energetic in nature, as it can be prevented when neurons are supplemented with additional energy substrates. This energetic endangerment might arise from the ability of GCs to inhibit glucose transport into both hippocampal neurons and astrocytes. The present study explores the GC inhibition in astrocytes. (1) GCs inhibited glucose transport approximately 15-30% in both primary and secondary hippocampal astrocyte cultures. (2) The parameters of inhibition agreed with the mechanisms of GC inhibition of glucose transport in peripheral tissues: A minimum of 4 h of GC exposure were required, and the effect was steroid specific (i.e., it was not triggered by estrogen, progesterone, or testosterone) and tissue specific (i.e., it was not triggered by GCs in cerebellar or cortical cultures). (3) Similar GC treatment caused a decrease in astrocyte survival during hypoglycemia and a decrease in the affinity of glutamate uptake. This latter observation suggests that GCs might impair the ability of astrocytes to aid neurons during times of neurologic crisis (i.e., by impairing their ability to remove damaging glutamate from the synapse).     

17β-Estradiol prevents cell death and mitochondrial dysfunction by an estrogen receptor-dependent mechanism in astrocytes after oxygen-glucose deprivation/reperfusion

17β-Estradiol (E2) has been shown to protect against ischemic brain injury, yet its targets and the mechanisms are unclear. E2 may exert multiple regulatory actions on astrocytes that may greatly contribute to its ability to protect the brain. Mitochondria are recognized as playing central roles in the development of injury during ischemia. Increasing evidence indicates that mitochondrial mechanisms are critically involved in E2-mediated protection. In this study, the effects of E2 and the role of mitochondria were evaluated in primary cultures of astrocytes subjected to an ischemia-like condition of oxygen-glucose deprivation (OGD)/reperfusion. We showed that E2 treatment significantly protects against OGD/reperfusion-induced cell death as determined by cell viability, apoptosis, and lactate dehydrogenase leakage. The protective effects of E2 on astrocytic survival were blocked by an estrogen receptor (ER) antagonist (ICI-182,780) and were mimicked by an ER agonist selective for ERα (PPT), but not by an ER agonist selective for ERβ (DPN). OGD/reperfusion provoked mitochondrial dysfunction as manifested by an increase in cellular reactive oxygen species production, loss of mitochondrial membrane potential, and depletion of ATP. E2 pretreatment significantly inhibited OGD/reperfusion-induced mitochondrial dysfunction, and this effect was also blocked by ICI-182,780. Therefore, we conclude that E2 provides direct protection to astrocytes from ischemic injury by an ER-dependent mechanism, highlighting an important role for ERα. Estrogen protects against mitochondrial dysfunction at the early phase of ischemic injury. However, overall implications for protection against brain ischemia and its complex sequelae await further exploration.     

Astrocytic Gap Junctional Communication Decreases Neuronal Vulnerability to Oxidative Stress-Induced Disruption of Ca2+ Homeostasis and Cell Death

Abstract: We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18-α-glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7-dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO₄ and 4-hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte-astrocyte coupling but no astrocyte-neuron or neuron-neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA-treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO₄/GA. Elevations of neuronal intracellular calcium levels ([Ca²⁺]_i) induced by FeSO₄ were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca²⁺ and the L-type Ca²⁺ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO₄ and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate- and FeSO₄-induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.     

Apolipoprotein E protects astrocytes from hypoxia and glutamate-induced apoptosis

Apolipoprotein E (apoE) is predominantly synthesized by astrocytes in the brain. In this study, we investigated the role of apoE in astrocyte apoptosis. We demonstrated that apoE protects astrocytes from hypoxia-induced apoptosis in a dose-dependent manner. Glutamate release from astrocytic cultures is significantly lower from WT mice than from apoE knockout mice. Furthermore, the protective effect of apoE is mimicked by an NMDA receptor antagonist, MK-801. Finally, the apoE activator T0901317 significantly reduced the effect of glutamate-induced apoptosis of astrocytes. These results suggest that apoE protects astrocytes from hypoxia-induced apoptosis associated to NMDA receptor activation. Approaches that elevate apoE secretion in astrocytes might provide a novel strategy in the protection of neuronal ischemic injury.     

Dependence of neurones on astrocytes in a coculture system renders neurones sensitive to transforming growth factor beta1-induced glutamate toxicity

Transforming growth factor beta1 (TGF-beta1) has been implicated in formation of astrocyte scars, which prevents axonal regeneration. A coculture system of astrocytes and cerebellar cells was used to investigate possible neurotoxic effects of TGF-beta1. Although not directly neurotoxic, TGF-beta1 was toxic to cerebellar cells in the presence of astrocytes. This toxicity is based on an effect of the cytokine on astrocytes, as conditioned medium from astrocyte cultures treated with TGF-beta1 was more toxic by a similar mechanism. This neurotoxicity was mediated by glutamate present in the culture medium as demonstrated by inhibition by MK-801. Astrocytic ability to metabolise glutamate was compromised by TGF-beta1, as this cytokine increased glutamate concentration. The astrocytes in the coculture system responded to the presence of neurones by secreting neuroprotective interleukin-6, which was partly protective against the TGF-beta1-induced toxicity. In the coculture system, neurones responded to the presence of astrocytes by a reduction in resistance to glutamate toxicity. On addition of TGF-beta1, which compromised astrocytic clearance of glutamate, this reduction in resistance to glutamate toxicity led to a reduction in neuronal survival. These results suggest that when neurones are cocultured with astrocytes they become dependent on astrocytes for survival. This dependence makes neurones susceptible to damage when astrocytes are activated by substances such as TGF-beta1.     

Computational simulation: astrocyte-induced depolarization of neighboring neurons mediates synchronous UP states in a neural network

Although recent reports have suggested that synchronous neuronal UP states are mediated by astrocytic activity, the mechanism responsible for this remains unknown. Astrocytic glutamate release synchronously depolarizes adjacent neurons, while synaptic transmissions are blocked. The purpose of this study was to confirm that astrocytic depolarization, propagated through synaptic connections, can lead to synchronous neuronal UP states. We applied astrocytic currents to local neurons in a neural network consisting of model cortical neurons. Our results show that astrocytic depolarization may generate synchronous UP states for hundreds of milliseconds in neurons even if they do not directly receive glutamate release from the activated astrocyte.     

The role of actin depolymerizing factor in advanced glycation endproducts-induced impairment in mouse brain microvascular endothelial cells

Orexin-A, which is an endogenous neuropeptide, is reported to have a protective role in ischemic stroke. High-concentration glutamic acid (Glu) induced by hypoxia injury in ischemic stroke can be inhibited by glial glutamate transporter GLT-1 which is only expressed in astroglia cells. A previous study reported that Orexin-A may regulate GLT-1 expression. However, the role of orexin-A in the regulation of GLT-1 in ischemic stroke still remains unclear. In this study, we aimed to investigate the effect and the underlying mechanism of orexin-A on Glu uptake in astrocytes in vitro and this effect on protecting the neurons from anoxia/hypoglycemic injury. The expression of GLT-1 significantly increased in the astrocytes with orexin-A treatment under anoxia/hypoglycemic conditions, promoting the uptake of Glu and inhibiting the apoptosis of co-cultured cells of astrocytes and neurons. However, these effects were significantly weakened by treatment with orexin-A receptor 1 (OX1R) antagonist. Orexin-A significantly up-regulated the expressions of PKCα and ERK1/2 under anoxia/hypoglycemic conditions in astrocytes, whereas the OX1R antagonist markedly reversed the effect. Furthermore, PKCα or ERK1/2 inhibitor significantly constrained the GLT-1 expression in astrocytes and facilitated the apoptosis of co-cultured cells, and GLT-1 overexpression could reverse those effects of PKCα or ERK1/2 inhibitor. Taken together, orexin-A promoted the GLT-1 expression via OX1R/PKCα/ERK1/2 pathway in astrocytes and protected co-cultured cells against anoxia/hypoglycemic injury.     

Gap-junction blocker carbenoxolone differentially enhances NMDA-induced cell death in hippocampal neurons and astrocytes in co-culture

The beneficial or detrimental role of gap junction communication in the pathophysiology of brain injury is still controversial. We used co-cultures of hippocampal astrocytes and neurons, where we identified homocellular astrocyte-astrocyte and heterocellular astrocyte-neuron coupling by fluorescence recovery after photobleaching, which was decreased by the gap junction blocker carbenoxolone (CBX). In these cultures, we determined the cell type-specific effects of CBX on the excitotoxic damage caused by N-methyl-D-aspartate (NMDA). We determined in both astrocytes and neurons the influence of CBX, alone or together with NMDA challenge, on cytotoxicity using propidium iodide labeling. CBX alone was not cytotoxic, but CBX treatment differentially accelerated the NMDA-induced cell death in both astrocytes and neurons. In addition, we measured mitochondrial potential using rhodamine 123, membrane potential using the oxonol dye bis(1,3-diethylthiobarbituric acid)trimethine oxonol, cytosolic Ca(2+) level using fura-2, and formation of reactive oxygen species (ROS) using dihydroethidium. CBX alone induced neither an intracellular Ca(2+) rise nor a membrane depolarization. However, CBX elicited a mitochondrial depolarization in both astrocytes and neurons and increased the ROS formation in neurons. In contrast, NMDA caused a membrane depolarization in neurons, coinciding with intracellular Ca(2+) rise, but neither mitochondrial depolarization nor ROS production seem to be involved in NMDA-mediated cytotoxicity. Pre-treatment with CBX accelerated the NMDA-induced membrane depolarization and prevented the repolarization of neurons after the NMDA challenge. We hypothesize that these effects are possibly mediated via blockage of gap junctions, and might be involved in the mechanism of CBX-induced acceleration of excitotoxic cell death, whereas the CBX-induced mitochondrial depolarization and ROS formation are not responsible for the increase in cytotoxicity. We conclude that both in astrocytes and neurons gap junctions provide protection against NMDA-induced cytotoxicity.     

Effect of Overexpression of Protective Genes on Mitochondrial Function of Stressed Astrocytes

Antiapoptotic members of the Bcl-2 family have been shown to reduce ischemic brain injury in vivo and in vitro. Understanding early changes in respiration are important in understanding the cells response to stress and the mechanisms of protection afforded by overexpression of protective genes. This mini-review summarizes current knowledge regarding early responses of astrocytes to ischemia-like stress and the effects of overexpression of protective Bcl-2 family genes on astrocyte mitochondrial function. Overexpression of Bcl-x(L) improves mitochondrial respiratory function, normalizes mitochondrial membrane potential, and reduces production of free radicals early after the imposition of a stress in primary cultured murine astrocytes.     

Astrocyte Mitochondrial Mechanisms of Ischemic Brain Injury and Neuroprotection

Research on ischemic brain injury has established a central role of mitochondria in neuron death. Astrocytes are also damaged by ischemia, although the participation of mitochondria in their injury is ill defined. As astrocytes are responsible for neuronal metabolic and trophic support, astrocyte dysfunction will compromise postischemic neuronal survival. Ischemic alterations to astrocyte energy metabolism and the uptake and metabolism of the excitatory amino acid transmitter glutamate may be particularly important. Despite the significance of ischemic astrocyte injury, little is known of the mechanisms responsible for astrocyte death and dysfunction. This review focuses on differences between astrocyte and neuronal metabolism and mitochondrial function, and on neuronal-glial interactions. The potential for astrocyte mitochondria to serve as targets of neuroprotective interventions is also discussed.     

Metabolic and Biosynthetic Alterations in Cultured Astrocytes Exposed to Hypoxia/Reoxygenation

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to ± 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.     

Activity-dependent neuronal control of gap-junctional communication in astrocytes

A typical feature of astrocytes is their high degree of intercellular communication through gap junction channels. Using different models of astrocyte cultures and astrocyte/neuron cocultures, we have demonstrated that neurons upregulate gap-junctional communication and the expression of connexin 43 (Cx43) in astrocytes. The propagation of intercellular calcium waves triggered in astrocytes by mechanical stimulation was also increased in cocultures. This facilitation depends on the age and number of neurons, indicating that the state of neuronal differentiation and neuron density constitute two crucial factors of this interaction. The effects of neurons on astrocytic communication and Cx43 expression were reversed completely after neurotoxic treatments. Moreover, the neuronal facilitation of glial coupling was suppressed, without change in Cx43 expression, after prolonged pharmacological treatments that prevented spontaneous synaptic activity. Altogether, these results demonstrate that neurons exert multiple and differential controls on astrocytic gap-junctional communication. Since astrocytes have been shown to facilitate synaptic efficacy, our findings suggest that neuronal and astrocytic networks interact actively through mutual setting of their respective modes of communication.     

Loss of the glutamate transporter splice-variant GLT-1b in inferior colliculus and its prevention by ceftriaxone in thiamine deficiency

Downregulation of astrocytic glutamate transporters is a feature of thiamine deficiency (TD), the underlying cause of Wernicke's encephalopathy, and plays a major role in its pathophysiology. Recent investigations suggest that ceftriaxone, a β-lactam antibiotic, stimulates GLT-1 expression and confers neuroprotection against ischemic and motor neuron degeneration. Thus, ceftriaxone treatment may be a protective strategy against excitotoxic conditions. In the present study, we examined the effects of ceftriaxone on the glutamate transporter splice-variant GLT-1b in rats with TD and in cultured astrocytes under TD conditions. Our results indicate that ceftriaxone protects against loss of GLT-1b levels in the inferior colliculus during TD, but with no significant effect in the thalamus and frontal cortex by immunoblotting and immunohistochemistry. Ceftriaxone also normalized the loss of GLT-1b in astrocyte cultures under conditions of TD. These results suggest that ceftriaxone has the ability to increase GLT-1b levels in astrocytes during TD, and may be an important pharmacological strategy for the treatment of excitotoxicity in this disorder.     

Cytosine arabinofuranoside-induced activation of astrocytes increases the susceptibility of neurons to glutamate due to the release of soluble factors

Activation of astrocytes occurs during many forms of CNS injury, but its importance for neuronal survival is poorly understood. When hippocampal cultures of neurons and astrocytes were treated from day 2–4 in vitro (DIV 2–4) with 1 μM cytosine arabinofuranoside (AraC), we observed a stellation of astrocytes, an increase in glial fibrillary acidic protein (GFAP) level as well as a higher susceptibility of the neurons to glutamate compared with cultures treated from DIV 2–4 with vehicle. To find out whether factors released into the culture medium were responsible for the observed differences in glutamate neurotoxicity, conditioned medium of AraC-treated cultures (MCMAraC) was added to vehicle-treated cultures and conditioned medium of vehicle-treated cultures (MCMvh) was added to AraC-treated cultures 2 h before and up to 18 h after the exposure to 1 mM glutamate for 1 h. MCMAraC increased glutamate neurotoxicity in vehicle-treated cultures and MCMvh reduced glutamate neurotoxicity in AraC-treated cultures. Heat-inactivation of MCMvh increased, whereas heat-inactivation of MCMAraC did not affect glutamate toxicity suggesting that heat-inactivation changed the proportion of factors in MCMvh inhibiting and exacerbating the excitotoxic injury. Similar findings were obtained using conditioned medium of pure astrocyte cultures of DIV 12 treated from DIV 2–4 with vehicle or 1 μM AraC suggesting that heat-sensitive factors in MCMvh were mainly derived from astrocytes. Treatment of hippocampal cultures with 1 mM dibutyryl-cAMP for 3 days induced an activation of the astrocytes similar to AraC and increased neuronal susceptibility to glutamate. Our findings provide evidence that activation of astrocytes impairs their ability to protect neurons after excitotoxic injury due to changes in the release of soluble and heat-sensitive factors.     

Pre-conditioning induces the precocious differentiation of neonatal astrocytes to enhance their neuroprotective properties

Hypoxic preconditioning reprogrammes the brain''s response to subsequent H/I (hypoxia–ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O₂). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.