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The influence of neuroinflammation on glutamate uptake by glial cells was examined after exposing primary cultures of rat astrocytes to conditioned culture medium from lipopolysaccharide-activated microglia. While such treatment triggered an inflammatory response in astrocytes, as revealed by the induction of cytokine expression, a significant decrease in GLAST expression and activity was observed after 72 h. This regulation of glutamate transporter was not observed with medium from naive microglia, but was mimicked by direct addition of tumor necrosis factor-alpha (TNF-alpha), a major cytokine released from activated microglia. Hence, on its own, TNF-alpha also triggered inflammation in astrocyte cultures, highlighting complex cross-talk between astrocytes and microglia in inflammatory conditions. This putatively detrimental regulation of GLAST in response to inflammation was also studied in cells exposed to dibutyryl cAMP, recognized as a model of astrocytes exhibiting a typical differentiated or activated phenotype. In this model, the conditioned culture medium from activated microglia, as well as TNF-alpha, were found to increase glutamate uptake capacity. Consistently, both of these treatments caused only modest induction of an inflammatory response in dibutyryl cAMP-matured astrocytes as compared to undifferentiated astrocytes. Together, these results suggest that differentiated/activated astrocytes are endowed with the capacity to confront inflammatory insults and that drugs influencing the astrocytes phenotype would deserve further consideration in the treatment of neurological disorders.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.

Excitatory transmission in the CNS necessitates the existence of dynamic controls of the glutamate uptake achieved by astrocytes, both in physiological conditions and under pathological circumstances characterized by gliosis. In this context, this study was aimed at evaluating the involvement of group I metabotropic glutamate receptors (mGluR) in the regulation of glutamate transport in a model of rat astrocytes undergoing in vitro activation using a cocktail of growth factors (G5 supplement). The vast majority of the cells were found to take up aspartate, mainly through the glutamate/aspartate transporter (GLAST), and at least 60% expressed functional mGluR5a. When exposed for 15 s to the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine, reactive astrocytes showed a significant increase in their capacity to take up aspartate. This effect was confirmed at the single-cell level, since activation of mGluRs significantly increased the initial slope of aspartate-dependent Na+ entry associated with the activity of glutamate transporters. This up-regulation was inhibited by an antagonist of mGluR5 and, more importantly, was sensitive to a specific glutamate transporter 1 (GLT-1) blocker. The acute influence of mGluR5 on aspartate uptake was phospholipase C- and protein kinase C-dependent, and was mimicked by phorbol esters. We conclude that mGluR5a contributes to a dynamic control of GLT-1 function in activated astrocytes, acting as a glial sensor of the extracellular glutamate concentration in order to acutely regulate the excitatory transmission.

Impaired glial glutamate uptake is commonly involved in neuronal damages observed in acute and chronic nervous disorders. As nervous insults are frequently associated with local inflammation involving microglia, this study aims at exploring the link between activated microglia and altered glutamate uptake in astrocytes. The regulation of the expression and activity of type 1 glutamate transporter (GLT-1) was examined after exposing cultures of rat astrocytes to conditioned medium from lipopolysaccharide-activated microglia cultures. Significant increases in GLT-1 mRNA expression and dihydrokainate sensitive uptake of aspartate were observed after 72h of treatment. These effects were reproduced by direct exposure of the astrocyte cultures to tumor necrosis factor alpha, a major cytokine released by activated microglia. The regulation of GLT-1 activity in response to inflammatory stimuli was also evidenced in cells exposed to dibutyryl cAMP, recognised as a model of reactive astrocytes in which the expression of this glutamate transporter is constitutively enhanced. Taken together, these results suggest that the GLT-1-dependent control of glutamate neurotransmission by either naive or chemically activated astrocytes is influenced by microglia-mediated inflammation.

Oxidative stress, neuroinflammation, and excitotoxicity are frequently considered distinct but common hallmarks of several neurological disorders, including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease. Although neuron degeneration and death are the ultimate consequences of these pathological processes, it is now widely accepted that alterations in the function of surrounding glial cells are key features in the progression of these diseases. In response to alteration in their local environment, microglia, commonly considered the resident immune cells of the nervous parenchyma, become activated and release a variety of soluble factors. Among these, proinflammatory cytokines and free radicals actively participate in the degenerative insults. In addition, excitotoxic neuronal damage resulting from excessive glutamate is frequently associated with impaired handling of extracellular glutamate by gliotic astrocytes. Although several research projects have focused on the biochemical mechanisms of the regulation of glial glutamate transporters, a relationship between activation of microglia and modulation of astrocytic glutamate uptake is now suggested. The aim of this review is to summarize and discuss the data showing an influence of inflammatory mediators and related free radicals on the expression and activity of glial glutamate transporters.

To explore the impact of neuroinflammation on the control of glutamate transmission, we studied the metabotropic glutamate receptor 5 (mGluR5) expression in cultured astrocytes exposed to conditioned medium from lipopolysaccharide-activated microglia. This treatment caused profound changes in astrocyte phenotype that correlated with altered expression of GFAP and vimentin. This putative astrogliosis was accompanied by a down-regulation of mGluR5 protein and mRNA expression, with a maximal effect after 48 h treatment (up to 50% decrease). Such regulation was not observed with medium from naive microglia but was mimicked by direct addition of tumor necrosis factor, a major cytokine released from activated microglia. Conversely, treatment with prostaglandin E2 and induction of nitric oxide production resulted in a significant up-regulation of mGluR5. These results suggest that complex crosstalks between microglia and astrocytes during neuroinflammatory insults would influence glutamate-dependent responses in astrocytes.