Astrocytes regulate N-methyl-D-aspartate receptor subunit composition increasing neuronal sensitivity to excitotoxicity

We have examined the dependence of rat cerebellar granule neurons (CGNs) for protection against glutamate toxicity. Under co-culture conditions, rat CGNs require astrocytes to protect against glutamate. The CGNs become more sensitive to glutamate toxicity in co-culture than when grown in cultures with only low numbers of astrocytes. If the protection of the astrocytes was withdrawn or blocked, this sensitivity led to neuronal death. Differing changes in NMDA receptor subunit subtype composition were noted depending on the conditions in which the CGNs were grown. Suppression of individual NMDA subunit subtypes by oligonucleotide knockdown resulted in inhibition of toxicity. This result implies that astrocytes regulate the expression of NMDA receptor subunit subtypes which influence neuronal sensitivity to glutamate toxicity.     

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.     

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.     

Astrogliosis Is a Possible Player in Preventing Delayed Neuronal Death

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing (GFAP⁺) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, GFAP⁺ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including S100β, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.     

Prostaglandin E(2) stimulates glutamate receptor-dependent astrocyte neuromodulation in cultured hippocampal cells.

Recent Ca(2+) imaging studies in cell culture and in situ have shown that Ca(2+) elevations in astrocytes stimulate glutamate release and increase neuronal Ca(2+) levels, and that this astrocyte-neuron signaling can be stimulated by prostaglandin E(2) (PGE(2)). We investigated the electrophysiological consequences of the PGE(2)-mediated astrocyte-neuron signaling using whole-cell recordings on cultured rat hippocampal cells. Focal application of PGE(2) to astrocytes evoked a Ca(2+) elevation in the stimulated cell by mobilizing internal Ca(2+) stores, which further propagated as a Ca(2+) wave to neighboring astrocytes. Whole-cell recordings from neurons revealed that PGE(2) evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca(2+) wave and was mediated through both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE(2)-evoked Ca(2+) elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors.     

Astrocyte influences on ischemic neuronal death

Glutamate excitotoxicity, oxidative stress, and acidosis are primary mediators of neuronal death during ischemia and reperfusion. Astrocytes influence these processes in several ways. Glutamate uptake by astrocytes normally prevents excitotoxic glutamate elevations in brain extracellular space, and this process appears to be a critical determinant of neuronal survival in the ischemic penumbra. Conversely, glutamate efflux from astrocytes by reversal of glutamate uptake, volume sensitive organic ion channels, and other routes may contribute to extracellular glutamate elevations. Glutamate activation of neuronal N-methyl-D-aspartate (NMDA) receptors is modulated by glycine and D-serine: both of these neuromodulators are transported by astrocytes, and D-serine production is localized exclusively to astrocytes. Astrocytes influence neuronal antioxidant status through release of ascorbate and uptake of its oxidized form, dehydroascorbate, and by indirectly supporting neuronal glutathione metabolism. In addition, glutathione in astrocytes can serve as a sink for nitric oxide and thereby reduce neuronal oxidant stress during ischemia. Astrocytes probably also influence neuronal survival in the post-ischemic period. Reactive astrocytes secrete nitric oxide, TNFalpha, matrix metalloproteinases, and other factors that can contribute to delayed neuronal death, and facilitate brain edema via aquaporin-4 channels localized to the astrocyte endfoot-endothelial interface. On the other hand erythropoietin, a paracrine messenger in brain, is produced by astrocytes and upregulated after ischemia. Erythropoietin stimulates the Janus kinase-2 (JAK-2) and nuclear factor-kappaB (NF-kB) signaling pathways in neurons to prevent programmed cell death after ischemic or excitotoxic stress. Astrocytes also secrete several angiogenic and neurotrophic factors that are important for vascular and neuronal regeneration after stroke.     

Excitotoxicity triggered by Neurobasal culture medium

Neurobasal defined culture medium has been optimized for survival of rat embryonic hippocampal neurons and is now widely used for many types of primary neuronal cell culture. Therefore, we were surprised that routine medium exchange with serum- and supplement-free Neurobasal killed as many as 50% of postnatal hippocampal neurons after a 4 h exposure at day in vitro 12–15. Minimal Essential Medium (MEM), in contrast, produced no significant toxicity. Detectable Neurobasal-induced neuronal death occurred with as little as 5 min exposure, measured 24 h later. D-2-Amino-5-phosphonovalerate (D-APV) completely prevented Neurobasal toxicity, implicating direct or indirect N-methyl-D-aspartate (NMDA) receptor-mediated neuronal excitotoxicity. Whole-cell recordings revealed that Neurobasal but not MEM directly activated D-APV-sensitive currents similar in amplitude to those gated by 1 µM glutamate. We hypothesized that L-cysteine likely mediates the excitotoxic effects of Neurobasal incubation. Although the original published formulation of Neurobasal contained only 10 µM L-cysteine, commercial recipes contain 260 µM, a concentration in the range reported to activate NMDA receptors. Consistent with our hypothesis, 260 µM L-cysteine in bicarbonate-buffered saline gated NMDA receptor currents and produced toxicity equivalent to Neurobasal. Although NMDA receptor-mediated depolarization and Ca²⁺ influx may support survival of young neurons, NMDA receptor agonist effects on development and survival should be considered when employing Neurobasal culture medium.     

Prostaglandin E2 stimulates glutamate receptor‐dependent astrocyte neuromodulation in cultured hippocampal cells

Recent Ca²⁺ imaging studies in cell culture and in situ have shown that Ca²⁺ elevations in astrocytes stimulate glutamate release and increase neuronal Ca²⁺ levels, and that this astrocyte‐neuron signaling can be stimulated by prostaglandin E₂ (PGE₂). We investigated the electrophysiological consequences of the PGE₂‐mediated astrocyte‐neuron signaling using whole‐cell recordings on cultured rat hippocampal cells. Focal application of PGE₂ to astrocytes evoked a Ca²⁺ elevation in the stimulated cell by mobilizing internal Ca²⁺ stores, which further propagated as a Ca²⁺ wave to neighboring astrocytes. Whole‐cell recordings from neurons revealed that PGE₂ evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca²⁺ wave and was mediated through both N‐methyl‐D ‐aspartate (NMDA) and non‐NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE₂‐evoked Ca²⁺ elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 221–229, 1999     

Glia Modulate NMDA-Mediated Signaling in Primary Cultures of Cerebellar Granule Cells

Abstract: Excessive activation of N-methyl-d -aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR-mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte-rich and astrocyte-poor cultures. In astrocyte-poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte-rich neuronal cultures demonstrated three significant differences, compared with astrocyte-poor cultures: (a) Neuronal viability was increased; (b) glutamate-mediated neurotoxicity was decreased, consistent with the presence of a sodium-coupled glutamate transport system in astrocytes; and (c) NMDA- but not kainate-mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT-PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR-mediated changes in intracellular Ca²⁺ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte-rich and astrocyte-poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA-mediated toxicity in astrocyte-poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR-mediated toxicity.     

Inflammatory neurodegeneration mediated by nitric oxide,glutamate, and mitochondria

In inflammatory, infectious, ischemic, and neurodegenerative pathologies of th central nervous system (CNS) glia become “activated” by inflammatory mediators, and express new proteins such as the inducible isoform of nitric oxide synthase (iNOS). Although these activated glia have beneficial roles, in vitro they potently kill cocultured neurons, and there is increasing evidence that they contribute to pathology in vivo. Nitric oxide (NO) from iNOS appears to be a key mediator of such glial-induced neuronal death. The high sensitivity of neurons to NO is partly due to NO causing inhibition of respiration, rapid glutamate release from both astrocytes and neurons, and subsequent excitotoxic death of the neurons. NO is a potent inhibitor of mitochondrial respiration, due to reversible binding of NO to cytochrome oxidase in competition with oxygen, resulting in inhibition of energy production and sensitization to hypoxia. Activated astrocytes or microglia cause a potent inhibition of respiration in cocultured neurons due to glial NO inhibiting cytochrome oxidase within the neurons, resulting in ATP depletion and glutamate release. In some conditions, glutamate-induced neuronal death can itself be mediated by N-methyl-d-aspartate (NMDA)-receptor activation of the neuronal isoform of NO synthase (nNOS) causing mitochondrial damage. In addition NO can be converted to a number of reactive derivatives such as peroxynitrite, NO₂, N₂O₃, and S-nitrosothiols that can kill cells in part by inhibiting mitochondrial respiration or activation of mitochondrial permeability transition, triggering neuronal apoptosis or necrosis.     

Differential Response of Neural Cells to Trauma-Induced Swelling In Vitro

Brain edema and the associated increase in intracranial pressure are major consequences of traumatic brain injury (TBI) that accounts for most early deaths after TBI. We recently showed that acute severe trauma to cultured astrocytes results in cell swelling. We further examined whether trauma induces cell swelling in neurons and microglia. We found that severe trauma also caused cell swelling in cultured neurons, whereas no swelling was observed in microglia. While severe trauma caused cell swelling in both astrocytes and neurons, mild trauma to astrocytes, neurons, and microglia failed to cell swelling. Since extracellular levels of glutamate are increased in brain post-TBI and microglia are known to release cytokine, and direct exposure of astrocytes to these molecules are known to stimulate cell swelling, we examined whether glutamate or cytokines have any additive effect on trauma-induced cell swelling. Exposure of cultured astrocytes to trauma caused cell swelling, and such swelling was potentiated by the exposure of traumatized astrocytes to glutamate and cytokines. Conditioned medium (CM) from traumatized astrocytes had no effect on neuronal swelling post-trauma, while CM from traumatized neurons and microglia potentiated the effect of trauma on astrocyte swelling. Further, trauma significantly increased the Na–K–Cl co-transporter (NKCC) activity in neurons, and that inhibition of NKCC activity diminished the trauma-induced neuronal swelling. Our results indicate that a differential sensitivity to trauma-induced cell swelling exists in neural cells and that neurons and microglia are likely to be involved in the potentiation of the astrocyte swelling post-trauma.     

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.     

Glutamate/glutamine metabolism coupling between astrocytes and glioma cells: Neuroprotection and inhibition of glioma growth

Glioma glutamate release has been shown to promote the growth of glioma cells and induce neuronal injuries from epilepsy to neuronal death. However, potential counteractions from normal astrocytes against glioma glutamate release have not been fully evaluated. In this study, we investigated the glutamate/glutamine cycling between glioma cells and astrocytes and their impact on neuronal function. Co-cultures of glioma cells with astrocytes (CGA) in direct contact were established under different mix ratio of astrocyte/glioma. Culture medium conditioned in these CGAs were sampled for HPLC measurement, for neuronal ratiometric calcium imaging, and for neuronal survival assay. We found: (1) High levels of glutaminase expression in glioma cells, but not in astrocytes, glutaminase enables glioma cells to release large amount of glutamate in the presence of glutamine. (2) Glutamate levels in CGAs were directly determined by the astrocyte/glioma ratios, indicating a balance between glioma glutamate release and astrocyte glutamate uptake. (3) Culture media from CGAs of higher glioma/astrocyte ratios induced stronger neuronal Ca²⁺ response and more severe neuronal death. (4) Co-culturing with astrocytes significantly reduced the growth rate of glioma cells. These results indicate that normal astrocytes in the brain play pivotal roles in glioma growth inhibition and in reducing neuronal injuries from glioma glutamate release. However, as tumor growth, the protective role of astrocytes gradually succumb to glioma cells.     

Neurotoxicity of Methylmercury in Isolated Astrocytes and Neurons: the Cytoskeleton as a Main Target

In the present work, we focused on mechanisms of methylmercury (MeHg) toxicity in primary astrocytes and neurons of rats. Cortical astrocytes and neurons exposed to 0.5–5 μM MeHg present a link among morphological alterations, glutathione (GSH) depletion, glutamate dyshomeostasis, and cell death. Disrupted neuronal cytoskeleton was assessed by decreased neurite length and neurite/neuron ratio. Astrocytes presented reorganization of actin and glial fibrillary acidic protein (GFAP) networks and reduced cytoplasmic area. Glutamate uptake and Na⁺K⁺ATPase activity in MeHg-treated astrocytes were preserved; however, downregulated EAAC1-mediated glutamate uptake was associated with impaired Na⁺K⁺ATPase activity in neurons. Oxidative imbalance was found in astrocytes and neurons through increased 2′7′-dichlorofluorescein (DCF) production and misregulated superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GPX) activities. Glutathione (GSH) levels were downregulated in both astrocytes and neurons. MeHg reduced neuronal viability and induced caspase 3-dependent apoptosis together with downregulated PI3K/Akt pathway. In astrocytes, necrotic death was associated with increased TNF-α and JNK/MAPK activities. Cytoskeletal remodeling and cell death were fully prevented in astrocytes and neurons by GSH, but not melatonin or Trolox supplementation. These findings support a role for depleted GSH in the cytotoxicity of MeHg leading to disruption of the cytoskeleton and cell death. Moreover, in neurons, glutamate antagonists also prevented cytoskeletal disruption and neuronal death. We propose that cytoskeleton is an end point in MeHg cytotoxicity. Oxidative imbalance and glutamate mechanisms mediate MeHg cytoskeletal disruption and apoptosis in neurons. Otherwise, redox imbalance and glutamate-independent mechanisms disrupted the cytoskeleton and induced necrosis in MeHg-exposed astrocyte.     

Involvement of Bcl-2 Family and Caspase-3-Like Protease in NO-Mediated Neuronal Apoptosis

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N ^G-monomethyl- l -arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of BCl-2 and up-regulation of Bax protein levels.     

Different pathways for iNOS-mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression

Co-localization of activated microglia and damaged neurones seen in brain injury suggests microglia-induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co-cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)-derived NO aloneis responsible for neuronal death from interferon gamma (IFNgamma) +lipopolysaccharide (LPS)-activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO-mediated toxicity to immature neurones, MK801 rescues 60-70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO-donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO-mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.     

In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells

In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in some other properties between Epo produced by these astrocyte cell lines and that by CHO cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spatio-temporal spread of neuronal death after focal photolysis of caged glutamate in neuron/astrocyte co-cultures

Glutamate-mediated excitotoxicity is now accepted as a major mechanism of ischemic neuronal damage. In the infarct core region, massive neuronal death is observed, but neurons in the surroundings of the core (ischemic penumbra) seem viable at the time of stroke. Several hours or days after a stroke, however, many neurons in the penumbra will undergo delayed neuronal death (DND). The mechanisms responsible for such DND are not fully understood. In this study, we investigated whether and how glutamate-mediated localized excitotoxic neuronal death affects surrounding neurons and astrocytes. To induce spatially-restricted excitotoxic neuronal death, a caged glutamate was focally photolyzed by a UV flash in neuron/astrocyte co-cultures. Uncaging of the glutamate resulted in acute neuronal death in the flashed area. After that, DND was observed in the surroundings of the flashed area late after the uncaging. In contrast, DND was not observed in neuron-enriched cultures, suggesting that functional changes in astrocytes, not neurons, after focal acute neuronal death were involved in the induction of DND. The present in vitro study showed that the spatially-restricted excitotoxic neuronal death resulted in DND in the surroundings of the flashed area, and suggested that the nitric oxide (NO)-induced reduction in the expression of astrocytic GLT-1 was responsible for the occurrence of the DND.     

A Cytotoxic,Co-operative Interaction Between Energy Deprivation and Glutamate Release From System xc? Mediates Aglycemic Neuronal Cell Death

The astrocyte cystine/glutamate antiporter (system x_c⁻) contributes substantially to the excitotoxic neuronal cell death facilitated by glucose deprivation. The purpose of this study was to determine the mechanism by which this occurred. Using pure astrocyte cultures, as well as, mixed cortical cell cultures containing both neurons and astrocytes, we found that neither an enhancement in system x_c⁻ expression nor activity underlies the excitotoxic effects of aglycemia. In addition, using three separate bioassays, we demonstrate no change in the ability of glucose-deprived astrocytes—either cultured alone or with neurons—to remove glutamate from the extracellular space. Instead, we demonstrate that glucose-deprived cultures are 2 to 3 times more sensitive to the killing effects of glutamate or N-methyl-D-aspartate when compared with their glucose-containing controls. Hence, our results are consistent with the weak excitotoxic hypothesis such that a bioenergetic deficiency, which is measureable in our mixed but not astrocyte cultures, allows normally innocuous concentrations of glutamate to become excitotoxic. Adding to the burgeoning literature detailing the contribution of astrocytes to neuronal injury, we conclude that under our experimental paradigm, a cytotoxic, co-operative interaction between energy deprivation and glutamate release from astrocyte system x_c⁻ mediates aglycemic neuronal cell death.     

星形胶质细胞的兴奋对神经元树突丝运动的调节机制

神经元树突上树突丝(filopodia)的形成及其运动,是神经元探索胞外环境、寻找突触前膜结构的一种方式.为研究星形胶质细胞的兴奋对神经元树突上树突丝运动的调节机制,在与神经元混合培养的星形胶质细胞中转染光敏感通道(channelrhodopsin-2).Channelrhodopsin-2是一种可表达于细胞膜表面的非选择性阳离子通道,可被特定模式的蓝光激活,导致大量钙离子内流并进一步诱发星形胶质细胞产生钙波,从而实现了选择性激活星形胶质细胞的目的.研究结果显示,在混合培养的神经元与星形胶质细胞模型中,激活的星形胶质细胞可以抑制神经元filopodia的运动,与外源性ATP、谷氨酸的作用效果一致.这表明星形胶质细胞激活后可能通过释放ATP和谷氨酸等递质来抑制神经元filopodia的运动.