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The interactions between astrocytes and neurons in the context of stroke play crucial roles in the disease's progression and eventual outcomes. After a stroke, astrocytes undergo significant changes in their morphology, molecular profile, and function, together termed reactive astrogliosis. Many of these changes modulate how astrocytes relate to neurons, inducing mechanisms both beneficial and detrimental to stroke recovery. For example, excessive glutamate release and astrocytic malfunction contribute to excitotoxicity in stroke, eventually causing neuronal death. Astrocytes also provide essential metabolic support and neurotrophic signals to neurons after stroke, ensuring homeostatic stability and promoting neuronal survival. Furthermore, several astrocyte-secreted molecules regulate synaptic plasticity in response to stroke, allowing for the rewiring of neural circuits to compensate for damaged areas. In this chapter, we highlight the current understanding of the interactions between astrocytes and neurons in response to stroke, explaining the varied mechanisms contributing to injury progression and the potential implications for future therapeutic interventions., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate α-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[(3)H]glutamate and L-[(3)H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of (3)H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.

In our previous work, we found that perfusion of the rat cerebral cortex with hypo-osmotic medium triggers massive release of the excitatory amino acid L-glutamate but decreases extracellular levels of L-glutamine (R. E. Haskew-Layton et al., PLoS ONE, 3: e3543). The release of glutamate was linked to activation of volume-regulated anion channels, whereas mechanism(s) responsible for alterations in extracellular glutamine remained unclear. When mannitol was added to the hypo-osmotic medium to reverse reductions in osmolarity, changes in microdialysate levels of glutamine were prevented, indicating an involvement of cellular swelling. As the main source of brain glutamine is astrocytic synthesis and export, we explored the impact of hypo-osmotic medium on glutamine synthesis and transport in rat primary astrocyte cultures. In astrocytes, a 40% reduction in medium osmolarity moderately stimulated the release of L-[(3) H]glutamine by ∼twofold and produced no changes in L-[(3) H]glutamine uptake. In comparison, hypo-osmotic medium stimulated the release of glutamate (traced with D-[(3) H]aspartate) by more than 20-fold. In whole-cell enzymatic assays, we discovered that hypo-osmotic medium caused a 20% inhibition of astrocytic conversion of L-[(3) H]glutamate into L-[(3) H]glutamine by glutamine synthetase. Using an HPLC assay, we further found a 35% reduction in intracellular levels of endogenous glutamine. Overall, our findings suggest that cellular swelling (i) inhibits astrocytic glutamine synthetase activity, and (ii) reduces substrate availability for this enzyme because of the activation of volume-regulated anion channels. These combined effects likely lead to reductions in astrocytic glutamine export in vivo and may partially explain occurrence of hyperexcitability and seizures in human hyponatremia., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Glutamate is the most abundant neurotransmitter of the central nervous system, as the majority of neurons use glutamate as neurotransmitter. It is also well known that this neurotransmitter is not restricted to synaptic clefts, but found in the extrasynaptic regions as ambient glutamate. Extrasynaptic glutamate originates from spillover of synaptic release, as well as from astrocytes and microglia. Its concentration is magnitudes lower than in the synaptic cleft, but receptors responding to it have higher affinity for it. Extrasynaptic glutamate receptors can be found in neuronal somatodendritic location, on astroglia, oligodendrocytes or microglia. Activation of them leads to changes of neuronal excitability with different amplitude and kinetics. Extrasynaptic glutamate is taken up by neurons and astrocytes mostly via EAAT transporters, and astrocytes, in turn metabolize it to glutamine. Extrasynaptic glutamate is involved in several physiological phenomena of the central nervous system. It regulates neuronal excitability and synaptic strength by involving astroglia; contributing to learning and memory formation, neurosecretory and neuromodulatory mechanisms, as well as sleep homeostasis.The extrasynaptic glutamatergic system is affected in several brain pathologies related to excitotoxicity, neurodegeneration or neuroinflammation. Being present in dementias, neurodegenerative and neuropsychiatric diseases or tumor invasion in a seemingly uniform way, the system possibly provides a common component of their pathogenesis. Although parts of the system are extensively discussed by several recent reviews, in this review I attempt to summarize physiological actions of the extrasynaptic glutamate on neuronal excitability and provide a brief insight to its pathology for basic understanding of the topic. [ABSTRACT FROM AUTHOR]

Key points The volume-regulated anion channel (VRAC) is a swelling-activated chloride channel that is permeable to inorganic anions and a variety of small organic molecules., VRAC is formed via heteromerization of LRRC8 proteins, among which LRRC8A is essential, while LRRC8B/C/D/E serve as exchangeable complementary partners., We used an RNAi approach and radiotracer assays to explore which LRRC8 isoforms contribute to swelling-activated release of diverse organic osmolytes in rat astrocytes., Efflux of uncharged osmolytes ( myo-inositol and taurine) was suppressed by deletion of LRRC8A or LRRC8D, but not by deletion of LRRC8C+LRRC8E., Conversely, release of charged osmolytes ( d-aspartate) was strongly reduced by deletion of LRRC8A or LRRC8C+LRRC8E, but largely unaffected by downregulation of LRRC8D., Our findings point to the existence of multiple heteromeric VRACs in the same cell type: LRRC8A/D-containing heteromers appear to dominate release of uncharged osmolytes, while LRRC8A/C/E, with the additional contribution of LRRC8D, creates a conduit for movement of charged molecules., Abstract The volume-regulated anion channel (VRAC) is the ubiquitously expressed vertebrate Cl−/anion channel that is composed of proteins belonging to the LRRC8 family and activated by cell swelling. In the brain, VRAC contributes to physiological and pathological release of a variety of small organic molecules, including the amino acid neurotransmitters glutamate, aspartate and taurine. In the present work, we explored the role of all five LRRC8 family members in the release of organic osmolytes from primary rat astrocytes. Expression of LRRC8 proteins was modified using an RNAi approach, and amino acid fluxes via VRAC were quantified by radiotracer assays in cells challenged with hypoosmotic medium (30% reduction in osmolarity). Consistent with our prior work, knockdown of LRRC8A potently and equally suppressed the release of radiolabelled d-[14C]aspartate and [3H]taurine. Among other LRRC8 subunits, downregulation of LRRC8D strongly inhibited release of the uncharged osmolytes [3H]taurine and myo-[3H]inositol, without major impact on the simultaneously measured efflux of the charged d-[14C]aspartate. In contrast, the release of d-[14C]aspartate was preferentially sensitive to deletion of LRRC8C+LRRC8E, but unaffected by downregulation of LRRC8D. Finally, siRNA knockdown of LRRC8C+LRRC8D strongly inhibited the release of all osmolytes. Overall, our findings suggest the existence of at least two distinct heteromeric VRACs in astroglial cells. The LRRC8A/D-containing permeability pathway appears to dominate the release of uncharged osmolytes, while an alternative channel (or channels) is composed of LRRC8A/C/D/E and responsible for the loss of charged molecules. [ABSTRACT FROM AUTHOR]

The immune privileged nature of the CNS can make it vulnerable to chronic and latent infections. Little is known about the effects of lifelong brain infections, and thus inflammation, on the neurological health of the host. Toxoplasma gondii is a parasite that can infect any mammalian nucleated cell with average worldwide seroprevalence rates of 30%. Infection by Toxoplasma is characterized by the lifelong presence of parasitic cysts within neurons in the brain, requiring a competent immune system to prevent parasite reactivation and encephalitis. In the immunocompetent individual, Toxoplasma infection is largely asymptomatic, however many recent studies suggest a strong correlation with certain neurodegenerative and psychiatric disorders. Here, we demonstrate a significant reduction in the primary astrocytic glutamate transporter, GLT-1, following infection with Toxoplasma. Using microdialysis of the murine frontal cortex over the course of infection, a significant increase in extracellular concentrations of glutamate is observed. Consistent with glutamate dysregulation, analysis of neurons reveal changes in morphology including a reduction in dendritic spines, VGlut1 and NeuN immunoreactivity. Furthermore, behavioral testing and EEG recordings point to significant changes in neuronal output. Finally, these changes in neuronal connectivity are dependent on infection-induced downregulation of GLT-1 as treatment with the ß-lactam antibiotic ceftriaxone, rescues extracellular glutamate concentrations, neuronal pathology and function. Altogether, these data demonstrate that following an infection with T. gondii, the delicate regulation of glutamate by astrocytes is disrupted and accounts for a range of deficits observed in chronic infection. [ABSTRACT FROM AUTHOR]

The volume-regulated anion channel (VRAC) is a ubiquitously expressed yet highly enigmatic member of the superfamily of chloride/anion channels. It is activated by cellular swelling and mediates regulatory cell volume decrease in a majority of vertebrate cells, including those in the central nervous system (CNS). In the brain, besides its crucial role in cellular volume regulation, VRAC is thought to play a part in cell proliferation, apoptosis, migration, and release of physiologically active molecules. Although these roles are not exclusive to the CNS, the relative significance of VRAC in the brain is amplified by several unique aspects of its physiology. One important example is the contribution of VRAC to the release of the excitatory amino acid neurotransmitters glutamate and aspartate. This latter process is thought to have impact on both normal brain functioning (such as astrocyte-neuron signaling) and neuropathology (via promoting the excitotoxic death of neuronal cells in stroke and traumatic brain injury). In spite of much work in the field, the molecular nature of VRAC remained unknown until less than 2 years ago. Two pioneer publications identified VRAC as the heterohexamer formed by the leucine-rich repeat-containing 8 (LRRC8) proteins. These findings galvanized the field and are likely to result in dramatic revisions to our understanding of the place and role of VRAC in the brain, as well as other organs and tissues. The present review briefly recapitulates critical findings in the CNS and focuses on anticipated impact on the LRRC8 discovery on further progress in neuroscience research. [ABSTRACT FROM AUTHOR]

In mammals, cellular swelling activates release of small organic osmolytes, including the excitatory amino acids (EAA) glutamate and aspartate, via a ubiquitously expressed volume-regulated chloride/anion channel (VRAC). Pharmacological evidence suggests that VRAC plays plural physiological and pathological roles, including excitotoxic release of glutamate in stroke. However, the molecular identity of this pathway was unknown. Two recent studies discovered that LRRC8 gene family members encode heteromeric VRAC composed of LRRC8A plus LRRC8B-E, which mediate swelling-activated Cl- currents and taurine release in human non-neural cells (Z. Qiu et al. Cell 157: 447, 2014; F.K. Voss et al. Science 344: 634, 2014). Here, we tested the contribution of LRRC8A to the EAA release in brain glia. We detected and quantified expression levels of LRRC8A-E in primary rat astrocytes with quantitative RT-PCR and then downregulated LRRC8A with gene-specific siRNAs. In astrocytes exposed to hypo-osmotic media, LRRC8A knockdown dramatically reduced swelling-activated release of the EAAtracer D-[³H]aspartate. In parallelHPLCassays, LRRC8AsiRNAprevented hypo-osmotic media-induced loss of the endogenous intracellular L-glutamate and taurine. Furthermore, downregulation of LRRC8A completely ablated the ATP-stimulated release of D-[³H]aspartate and [14C]taurine from non-swollen astrocytes. Overall, these data indicate that LRRC8A is an indispensable component of a permeability pathway that mediates both swelling-activated and agonist-induced amino acid release in brain glial cells. [ABSTRACT FROM AUTHOR]

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na+/H+ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H+ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1–5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na+ and Ca2+ overload. The latter was mediated by reversal of Na+/Ca2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1–24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H+ homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na+ and Ca2+ homeostasis, which reduces Na+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI. [ABSTRACT FROM AUTHOR]

Astrocytes, a major class of glial cells, regulate neurotransmitter systems, synaptic processing, ion homeostasis, antioxidant defenses and energy metabolism. Astrocyte cultures derived from rodent brains have been extensively used to characterize astrocytes' biochemical, pharmacological and morphological properties. The aims of this study were to develop a protocol for routine preparation and to characterize a primary astrocyte culture from the brains of adult (90 days old) Wistar rats. For this we used enzymatic digestion (trypsin and papain) and mechanical dissociation. Medium exchange occurred from 24 h after obtaining a culture and after, twice a week up to reach the confluence (around the 4th to 5th week). Under basal conditions, adult astrocytes presented a polygonal to fusiform and flat morphology. Furthermore, approximately 95% the cells were positive for the main glial markers, including GFAP, glutamate transporters, glutamine synthetase and S100B. Moreover, the astrocytes were able to take up glucose and glutamate. Adult astrocytes were also able to respond to acute H2O2 exposure, which led to an increase in reactive oxygen species (ROS) levels and a decrease in glutamate uptake. The antioxidant compound resveratrol was able to protect adult astrocytes from oxidative damage. A response of adult astrocytes to an inflammatory stimulus with LPS was also observed. Changes in the actin cytoskeleton were induced in stimulated astrocytes, most likely by a mechanism dependent on MAPK and Rho A signaling pathways. Taken together, these findings indicate that the culture model described in this study exhibits the biochemical and physiological properties of astrocytes and may be useful for elucidating the mechanisms related to the adult brain, exploring changes between neonatal and adult astrocytes, as well as investigating compounds involved in cytotoxicity and cytoprotection. [ABSTRACT FROM AUTHOR]

The astrocytic glutamate transporters (GLT-1, GLAST) are critical for removing excess glutamate from synaptic sites, thereby maintaining glutamate homeostasis within the brain. 17β-Estradiol (E2) is one of the most active estrogen hormones possessing neuroprotective effects both in in vivo and in vitro models, and it has been shown to enhance astrocytic glutamate transporter function (Liang et al. in J Neurochem 80:807-814, ; Pawlak et al. in Brain Res Mol Brain Res 138:1-7, ). However, E2 is not clinically optimal for neuroprotection given its peripheral feminizing and proliferative effects; therefore, brain selective estrogen receptor modulators (neuro SERMs) (Zhao et al. in Neuroscience 132:299-311, ) that specifically target estrogenic mechanisms, but lack the systemic estrogen side effects offer more promising therapeutic modality for the treatment of conditions associated with excessive synaptic glutamate levels. This review highlights recent studies from our laboratory showing that E2 and SERMs effectively reverse glutamate transport inhibition in a manganese (Mn)-induced model of glutamatergic deregulation. Specifically, we discuss mechanisms by which E2 restores the expression and activity of glutamate uptake. We advance the hypothesis that E2 and related compounds, such as tamoxifen may offer a potential therapeutic modality in neurodegenerative disorders, which are characterized by altered glutamate homeostasis. [ABSTRACT FROM AUTHOR]

Accumulating evidence indicates a significant astrocytic involvement in cerebral ischemia neuropathology, but little is known about the immediate astrocytic responses to ischemia insults in terms of electrophysiology and their pathologic implications. We show that astrocytes in acute rat hippocampal slices responded reversibly to more than 30 mins oxygen and glucose deprivation (OGD) treatment with depolarized membrane potentials (Vm) in whole-cell current clamp recording. This depolarization was multiphasic, showing an initial ∼11 mins small-amplitude depolarization plateau, followed by a 6-mins accelerated depolarization, and then a second plateau. Oxygen and glucose deprivation-induced astrocyte Vm depolarization was only marginally inhibited, ∼10%, by inhibition of ionotropic glutamate, γ-aminobutyric acid, purinergic receptors, and glutamate transporters presumed to be present on astrocytes in situ, suggesting increase in extracellular [K+] was primarily responsible for the astrocytic Vm change. The Vm depolarization was five-fold greater when glycolysis was inhibited by iodoacetate in a short 8 mins OGD treatment, suggesting glycolytic ATP is critical in maintaining extracellular K+ homeostasis in the early phase of OGD. Addition of oxidative metabolism inhibitors had much less effect. Cessation of OGD was always followed by a rapid and transient 9 mV astrocyte Vm hyperpolarization relative to the control Vm that was inhibited by ouabain, indicating a reactively enhanced Na+/K+-ATPase activity in post-OGD reperfusion. Altogether, hippocampal astrocytes appear to be electrophysiologically more resistant to acute ischemia insults as compared with neurons, and this should allow astrocytes to rescue endangered neurons in the face of acute ischemia insults via their various homeostatic functions.Journal of Cerebral Blood Flow & Metabolism (2008) 28, 456–467; doi:10.1038/sj.jcbfm.9600545; published online 22 August 2007 [ABSTRACT FROM AUTHOR]

Examines the regulatory activity of adenosine triphosphate (ATP) in the excitatory amino acid (EAA) release from astrocytes. Mechanism of ATP release in the brain; Activation of EAA release by exogenous ATP; Degradation of the ATP effect by osmolarity elevation.