Cultured Rat Cortical Astrocytes Research Articles

Ketamine Reduces the Surface Density of the Astroglial Kir4.1 Channel and Inhibits Voltage-Activated Currents in a Manner Similar to the Action of Ba2+ on K+ Currents.

A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K+ channel Kir4.1, the principal regulator of K+ buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls (p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5'-monophosphate, 1 mM) or [K+]o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba2+, known to block Kir4.1 channels.

Distinct Effects of Escitalopram and Vortioxetine on Astroglial L-Glutamate Release Associated with Connexin43

It has been established that enhancement of serotonergic transmission contributes to improvement of major depression; however, several post-mortem studies and experimental depression rodent models suggest that functional abnormalities of astrocytes play important roles in the pathomechanisms/pathophysiology of mood disorders. Direct effects of serotonin (5-HT) transporter inhibiting antidepressants on astroglial transmission systems has never been assessed in this context. Therefore, to explore the effects of antidepressants on transmission associated with astrocytes, the present study determined the effects of the selective 5-HT transporter inhibitor, escitalopram, and the 5-HT partial agonist reuptake inhibitor, vortioxetine, on astroglial L-glutamate release through activated hemichannels, and the expression of connexin43 (Cx43), type 1A (5-HT1AR) and type 7 (5-HT7R) 5-HT receptor subtypes, and extracellular signal-regulated kinase (ERK) in astrocytes using primary cultured rat cortical astrocytes in a 5-HT-free environment. Both escitalopram and 5-HT1AR antagonist (WAY100635) did not affect basal astroglial L-glutamate release or L-glutamate release through activated hemichannels. Subchronic (for seven days) administrations of vortioxetine and the 5-HT7R inverse agonist (SB269970) suppressed both basal L-glutamate release and L-glutamate release through activated hemichannels, whereas 5-HT1AR agonist (BP554) inhibited L-glutamate release through activated hemichannels, but did not affect basal L-glutamate release. In particular, WAY100635 did not affect the inhibitory effects of vortioxetine on L-glutamate release. Subchronic administration of vortioxetine, BP554 and SB269970 downregulated 5-HT1AR, 5-HT7R and phosphorylated ERK in the plasma membrane fraction, but escitalopram and WAY100635 did not affect them. Subchronic administration of SB269970 decreased Cx43 expression in the plasma membrane but did not affect the cytosol; however, subchronic administration of BP554 increased Cx43 expression in the cytosol but did not affect the plasma membrane. Subchronic vortioxetine administration increased Cx43 expression in the cytosol and decreased it in the plasma membrane. WAY100635 prevented an increased Cx43 expression in the cytosol induced by vortioxetine without affecting the reduced Cx43 expression in the plasma membrane. These results suggest that 5-HT1AR downregulation probably increases Cx43 synthesis, but 5-HT7R downregulation suppresses Cx43 trafficking to the plasma membrane. These results also suggest that the subchronic administration of therapeutic-relevant concentrations of vortioxetine inhibits both astroglial L-glutamate and Cx43 expression in the plasma membrane via 5-HT7R downregulation but enhances Cx43 synthesis in the cytosol via 5-HT1AR downregulation. This combination of the downregulation of 5-HT1AR, 5-HT7R and Cx43 in the astroglial plasma membrane induced by subchronic vortioxetine administration suggest that astrocytes is possibly involved in the pathophysiology of depression.

Novel small chemical compound with NFAT modulatory properties alleviates synaptic dysfunction and improves cognition in mouse models of amyloid pathology

AbstractBackgroundHyperactivity of the protein phosphatase calcineurin (CN) in brain directly corresponds to key neuropathological and clinical features of Alzheimer’s disease (AD). While inhibition of CN ameliorates pathophysiologic and cognitive changes in mice with AD‐like pathology, concerns over adverse effects have slowed the transition of common CN‐inhibiting drugs to the clinic for treatment of AD and AD‐related disorders. Targeting substrates of CN, like the nuclear factor of activated T‐cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that modulates NFAT signaling, without inhibiting CN activity.Method Cell culture studies — Rat cortical astrocyte cultures were treated with a variety of pathogenic factors in the presence of Q134R or the CN inhibitor, cyclosporine. NFAT‐dependent luciferase activity and dephosphorylation of non‐NFAT substrates were assessed. Intact mouse studies — Q134R was delivered (via oral gavage) to APP/PS1 mice at different ages, or to wild type (WT) mice infused with oligomeric amyloid‐beta peptides. Cognition was assessed on a Y maze along with several postmortem measures including NFAT4 activity, glial activation (GFAP and Iba‐1 expression), amyloid deposition, long‐term survival, and synaptic function and plasticity.ResultIn primary astrocytes, Q134R significantly inhibited NFAT activation in a concentration‐dependent manner, but did not inhibit the CN‐dependent dephosphorylation of non‐NFAT substrates in vivo or in vitro. Short‐term treatment of APP/PS1 mice with Q134R inhibited the nuclear translocation of NFAT4 in hippocampal astrocytes while causing a significant drop in GFAP volume, a mild reduction in Iba1, and no change in amyloid pathology. Short‐term treatment with Q134R also improved cognitive function in APP/PS1 mice and amyloid‐infused WT mice. Long‐term oral delivery of Q134R over many months promoted survival in WT mice and improved synaptic function and plasticity in APP/PS1 mice.ConclusionQ134R has novel CN‐independent, NFAT modulatory properties and improves key neurologic function parameters in mouse models of AD‐like pathology. The results suggest that Q134R is a promising drug for treating AD and AD‐related disorders.

Lysophosphatidic acid induces thrombospondin-1 production in primary cultured rat cortical astrocytes.

Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study, the effects of LPA on astrocyte-derived synaptogenesis factor thrombospondins (TSPs) production were examined by real-time PCR and western blotting, and the mechanism underlying this event was examined by pharmacological approaches in primary cultured rat cortical astrocytes. Treatment of astrocytes with LPA increased TSP-1 mRNA, and TSP-2 mRNA, but not TSP-4 mRNA expression. TSP-1 protein expression and release were also increased by LPA. LPA-induced TSP-1 production were inhibited by AM966 a LPA1 receptor antagonist, and Ki16425, LPA1/3 receptors antagonist, but not by H2L5146303, LPA2 receptor antagonist. Pertussis toxin, Gi/o inhibitor, but not YM-254890, Gq inhibitor, and NF499, Gs inhibitor, inhibited LPA-induced TSP-1 production, indicating that LPA increases TSP-1 production through Gi/o-coupled LPA1 and LPA3 receptors. LPA treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). LPA-induced TSP-1 mRNA expression was inhibited by U0126, MAPK/ERK kinase (MEK) inhibitor, but not SB202190, p38 MAPK inhibitor, or SP600125, JNK inhibitor. However, LPA-induced TSP-1 protein expression was diminished with inhibition of all three MAPKs, indicating that these signaling molecules are involved in TSP-1 protein production. Treatment with antidepressants, which bind to astrocytic LPA1 receptors, increased TSP-1 mRNA and protein production. The current findings show that LPA/LPA1/3 receptors signaling increases TSP-1 production in astrocytes, which could be important in the pathogenesis of affective disorders and could potentially be a target for the treatment of affective disorders.

Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms.

A major risk factor for major depressive disorder (MDD) is stress. Stress leads to the release of high-mobility group box-1 (HMGB1), which in turn leads to neuroinflammation, a potential pathophysiological basis of MDD. The mechanism underlying stress-induced HMGB1 release is not known, but stress-associated glucocorticoids could be involved. To test this, rat primary cultured cortical astrocytes, the most abundant cell type in the central nervous system (CNS), were treated with corticosterone and HMGB1 release was assessed by Western blotting and ELISA. Significant HMGB1 was released with treatment with either corticosterone or dexamethasone, a synthetic glucocorticoid. HMGB1 translocated from the nucleus to the cytoplasm following corticosterone treatment. HMGB1 release was significantly attenuated with glucocorticoid receptor blocking. In addition, inhibition of pannexin-1, and P2X7 receptors led to a significant decrease in corticosterone-induced HMGB1 release. Taken together, corticosterone stimulates astrocytic glucocorticoid receptors and triggers cytoplasmic translocation and extracellular release of nuclear HMGB1 through a mechanism involving pannexin-1 and P2X7 receptors. Thus, under conditions of stress, glucocorticoids induce astrocytic HMGB1 release, leading to a neuroinflammatory state that could mediate neurological disorders such as MDD.

Itaconate modulates tricarboxylic acid and redox metabolism to mitigate reperfusion injury

ObjectivesCerebral ischemia/reperfusion (IR) drives oxidative stress and injurious metabolic processes that lead to redox imbalance, inflammation, and tissue damage. However, the key mediators of reperfusion injury remain unclear, and therefore, there is considerable interest in therapeutically targeting metabolism and the cellular response to oxidative stress. MethodsThe objective of this study was to investigate the molecular, metabolic, and physiological impact of itaconate treatment to mitigate reperfusion injuries in in vitro and in vivo model systems. We conducted metabolic flux and bioenergetic studies in response to exogenous itaconate treatment in cultures of primary rat cortical neurons and astrocytes. In addition, we administered itaconate to mouse models of cerebral reperfusion injury with ischemia or traumatic brain injury followed by hemorrhagic shock resuscitation. We quantitatively characterized the metabolite levels, neurological behavior, markers of redox stress, leukocyte adhesion, arterial blood flow, and arteriolar diameter in the brains of the treated/untreated mice. ResultsWe demonstrate that the “immunometabolite” itaconate slowed tricarboxylic acid (TCA) cycle metabolism and buffered redox imbalance via succinate dehydrogenase (SDH) inhibition and induction of anti-oxidative stress response in primary cultures of astrocytes and neurons. The addition of itaconate to reperfusion fluids after mouse cerebral IR injury increased glutathione levels and reduced reactive oxygen/nitrogen species (ROS/RNS) to improve neurological function. Plasma organic acids increased post-reperfusion injury, while administration of itaconate normalized these metabolites. In mouse cranial window models, itaconate significantly improved hemodynamics while reducing leukocyte adhesion. Further, itaconate supplementation increased survival in mice experiencing traumatic brain injury (TBI) and hemorrhagic shock. ConclusionsWe hypothesize that itaconate transiently inhibits SDH to gradually “awaken” mitochondrial function upon reperfusion that minimizes ROS and tissue damage. Collectively, our data indicate that itaconate acts as a mitochondrial regulator that controls redox metabolism to improve physiological outcomes associated with IR injury.

TNFα increases STAT3-mediated expression of glutaminase isoform KGA in cultured rat astrocytes

Glutamate related excitotoxicity and excess of cerebral levels of tumor necrosis factor alpha (TNFα) are interrelated and well documented abnormalities noticed in many central nervous system diseases. Contribution of kidney type glutaminase (KGA) and shorter alternative splicing form (GAC) to glutamine degradation in astrocytes has been recently a matter of dispute and extensive study but the regulation of the GLS isoforms by inflammatory factors is still not well known. Here we show that treatment of cultured rat cortical astrocytes with pathophysiologically relevant (50 ng/ml) concentration of TNFα specifically increases the expression of KGA but not GAC and increases activity of GLS. No changes in the expression of either of two GLS isoforms were observed following treatment with other tested cytokines IL-1β and IL-6. The TNFα mediated KGA expression was associated with increased phosphorylation of signal transducer and activator of transcription 3 (STAT3). Stimulatory effect of TNF-α on KGA expression was reduced by selective inhibition of (STAT3) but not by inhibition of STAT1 nor nuclear transcription factor kappa. Additionally, the role of miRNA in TNFα–induced expression of KGA in astrocytes was excluded, since the expression of miR-23a/b and miR-200c, potential regulators of KGA expression, was unchanged. This study documents increased KGA expression in the astrocytes under inflammatory stimulation, identifying TNFα as a cytokine mediating this response, and demonstrates the specific and selective involvement of STAT3.

Amyloid β Peptide Specifically Promotes Phosphorylation and Nuclear Translocation of the Extracellular Signal-Regulated Kinase in Cultured Rat Cortical Astrocytes

Amyloid β Peptide Specifically Promotes Phosphorylation and Nuclear Translocation of the Extracellular Signal-Regulated Kinase in Cultured Rat Cortical Astrocytes

Galanin Protects Rat Cortical Astrocyte from Oxidative Stress: Involvement of GalR2 and pERK1/2 Signal Pathway.

The neuropeptide galanin and its receptors have been found to have protective effects on neurons. However, the role of galanin on astrocytes is still unclear. The present study is aimed at investigating the effects of galanin on the viability of cultured rat cortical astrocytes after oxidative stress induced by H2O2 and possible receptor and signaling mechanisms involved. Treatment of galanin had significant protective effects against H2O2-induced toxicity in the cultured cortical astrocytes. H2O2 induced an upregulation of phosphorylated extracellular signal-related kinase1/2 (pERK1/2) in astrocytes, which was suppressed by coapplication of galanin, suggesting an involvement of the pERK1/2 signal pathway in the protective effects of galanin. GalR2 has higher expression levels than GalR1 and GalR3 in the cultured cortical astrocytes, and GalR2 agonist AR-M1896 mimicked galanin effects on the astrocytes, implying that galanin protective effects mainly mediated by GalR2. Meanwhile, galanin had no effect on the A1-type transformation of rat cortical astrocytes. All those results suggest that galanin protects rat cortical astrocytes from oxidative stress by suppressing H2O2-induced upregulation of pERK1/2, mainly through GalR2.

Delta Opioid Peptide [d-Ala2, d-Leu5] Enkephalin (DADLE) Exerts a Cytoprotective Effect in Astrocytes Exposed to Oxygen-Glucose Deprivation by Inducing Autophagy

Astrocytes protection and functional regulation are important strategies to protect against neuronal damage caused by ischemia. Activation of the delta opioid receptor (DOR) could reduce astrocytes damage, although the mechanism remains unclear. The present study aimed to test the effect of DOR activation on autophagy in astrocytes exposed to oxygen-glucose deprivation (OGD), and to further investigate whether this effect has a protective effect on astrocytes. Primary cultured rat cortical astrocytes were treated with various doses of [d-Ala2, d-Leu5]-Enkephalin (DADLE, a selective DOR agonist) followed by 6 h OGD. Cell viability was evaluated by CCK-8 assay and lactate dehydrogenase release. Autophagic vacuole was analyzed with LC3 immunofluorescent staining. The levels of autophagy and apoptosis-related proteins were analyzed by western blot. Results demonstrated that treatment with 10 nM DADLE was sufficient to increase cell viability and induced autophagy in astrocytes. The DADLE-induced autophagy displayed a cytoprotective effect on astrocytes. Inhibition of autophagy by 3-methyladenine (3-MA, an autophagy inhibitor) reversed the protective effect of DADLE. Naltrindole (a DOR antagonist) only partially antagonized the role of DADLE, which indicated that DADLE might have a cytoprotective mechanism independent of DOR. Further results showed that DADLE significantly enhanced the level of Bcl-2 protein and reduced the level of Bax protein in astrocytes exposed to OGD. Our results suggest a novel mechanism in which DADLE induces autophagy in astrocytes and exerts cytoprotective effects by inhibiting apoptosis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin promotes migration ability of primary cultured rat astrocytes via aryl hydrocarbon receptor

Emerging evidence showed that 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) could induce expression of certain reactivation-associated genes in astrocytes, however, the consequent cellular effects and molecular mechanisms are still unclear. During the process of astrocyte reactivation, migration is a critical cellular event. In the present study, we employed wound-healing assay and Transwell® motility assay to explore the effects of TCDD on cell migration in primary cultured rat cortical astrocytes. We found that upon TCDD treatments at relative low concentrations (10-10 and/or 10-9 mol/L), the ability of primary astrocytes to migrate horizontally and vertically was promoted. In line with this cellular effect, the mRNA expression of two pro-migratory genes, including cell division cycle 42 (CDC42) and matrix metalloproteinase 2 (MMP2) was induced by TCDD treatment. Dioxin exerts its toxic effects mainly through aryl hydrocarbon receptor (AhR) pathway. So the role of AhR pathway in the pro-migratory effects of TCDD was examined using an AhR antagonist, CH223191. We found that application of CH223191 significantly reversed the pro-migratory effects of TCDD. Interestingly, the basal ability of horizontal migration as well as basal levels of CDC42 and MMP2 expression were dramatically reduced suggesting a possible physiological role of AhR in maintaining the endogenous migration ability of the primary astrocytes. These findings support the notion that dioxin promotes astrocyte reactivation at molecular and cellular levels.

In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5.

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM–10 μM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.

CCL2 Induces the Production of β2 Adrenergic Receptors and Modifies Astrocytic Responses to Noradrenaline.

The decline in brain noradrenaline levels is associated with the progression of certain neurodegenerative diseases. This seems to be due, at least in part, to the ability of noradrenaline to limit glial activation and to reduce the damage associated with it. Our previous studies of the mechanisms involved in this process indicate that noradrenaline induces the production of the chemokine CCL2 in astrocytes. While CCL2 can protect neurons against certain injuries, its overproduction has also proven to be harmful and to prevent noradrenaline neuroprotective effects. Therefore, in this study, we analyze if the modifications caused to astrocytes by an excessive production of CCL2 may alter their response to noradrenaline. Using primary cultures of rat cortical astrocytes, we observed that CCL2 enhances the production of beta 2 adrenergic receptors in these cells. While this potentiates noradrenaline signaling through cAMP, the activation of the transcription factor CREB is inhibited by CCL2. Furthermore, although CCL2 potentiates noradrenaline induction of glycogenolysis, this does not translate into an augmented release of lactate, one of the processes through which astrocytes help support neurons. Additionally, other neuroprotective actions of noradrenaline, such as the production of brain derived neurotrophic factor and the inhibition of the inducible nitric oxide synthase in astrocytes were modified by CCL2. These data suggest that some of the central nervous system alterations related to CCL2 could be due to its effects on adrenergic receptors and its interference with noradrenaline signaling.

Unconjugated bilirubin induces pyroptosis in cultured rat cortical astrocytes

BackgroundBilirubin-induced neurological dysfunction (BIND), a severe complication of extreme neonatal hyperbilirubinemia, could develop into permanent neurodevelopmental impairments. Several studies have demonstrated that inflammation and nerve cell death play important roles in bilirubin-induced neurotoxicity; however, the underlying mechanism remains unidentified.MethodsThe present study was intended to investigate whether pyroptosis, a highly inflammatory form of programmed cell death, participated in the bilirubin-mediated toxicity on cultured rat cortical astrocytes. Further, VX-765, a potent and selective competitive drug, was used to inhibit the activation of caspase-1. The effects of VX-765 on astrocytes treated with bilirubin, including the cell viability, morphological changes of the cell membrane and nucleus, and the production of pro-inflammation cytokines, were observed.ResultsStimulation of the astrocytes with unconjugated bilirubin (UCB) at the conditions mimicking those of jaundiced newborns significantly increased the activation of caspase-1. Further, caspase-1 activation was inhibited by treatment with VX-765. Compared with UCB-treated astrocytes, the relative cell viability of VX-765-pretreated astrocytes was improved; meanwhile, the formation of plasma membrane pores was prevented, as measured by lactate dehydrogenase release, trypan blue staining, and ethidium bromide (EtBr) uptake. Moreover, DNA fragmentation was partly attenuated and the release of IL-1β and IL-18 was apparently decreased.ConclusionPyroptosis is involved in the process of UCB-induced rat cortical astrocytes’ injury in vitro and may be the missing link of cell death and inflammatory response exacerbating UCB-related neurotoxicity. More importantly, the depression of caspase-1 activation, the core link of pyroptosis, attenuated UCB-induced cellular dysfunction and cytokine release, which might shed light on a new therapeutic approach to BIND.

Inhibition of Gap Junction Elevates Glutamate Uptake in Cultured Astrocytes.

Glutamate uptake is a main function of astrocytes to keep extracellular glutamate levels low and protect neurons against glutamate-induced excitotoxicity. On the other hand, astrocyte networks formed by gap junctions, which are consisted with connexins and connecting neighboring cells, are reported to play a critical role in maintaining the homeostasis in the brain. In the present study, we examined the effects of gap junction inhibitors on the glutamate uptake activity in cultured rat cortical astrocytes. At first, we confirmed the effects of gap junction inhibitors, 1-octanol and carbenoxolone, on cell-cell communication by the scrape-loading assay using a fluorescent dye Lucifer yellow. Both of 1-octanol and carbenoxolone treatments for 20min in cultured astrocytes significantly suppressed the cell-cell communication assessed as the distance of dye-spreading. 1-octanol and carbenoxolone increased the glutamate uptake by astrocytes and glutamate aspartate transporter (GLAST) expression on the cell membrane. These results suggest that gap junction inhibitors increase the glutamate uptake activity through the increase of GLAST proteins located on the cell membrane. The regulation of gap junction in astrocytes might protect neurons against glutamate-induced excitotoxicity.

Yokukansan, a Traditional Japanese Medicine, Enhances the Glutamate Transporter GLT-1 Function in Cultured Rat Cortical Astrocytes.

Astrocytes carry two glutamate transporters—GLAST and GLT-1—the latter of which is responsible for >90% of glutamate uptake activity in the brain; however, under culture conditions, the GLT-1 expression in astrocytes is exceedingly low, as is the glutamate uptake activity mediated by GLT-1. This study aimed to elucidate the effects of yokukansan (YKS) in relation to the GLT-1-mediated regulation of extracellular glutamate concentrations. Thus, we treated cultured astrocytes with tumor necrosis factor-α (TNF-α) and dibutyryl-cAMP (dBcAMP) (hereinafter, referred to as “TA”) to increase GLT-1 expression and then functionally examined how YKS would affect glutamate uptake ability derived from GLT-1. Contrary to expectations, although the TA treatments did not affect the uptake activity, YKS significantly augmented it. Conversely, GLAST-derived glutamate uptake was significantly reduced by TA treatments but was unaffected by YKS. Subsequently, we analyzed the GLT-1 protein and mRNA levels and found that TA treatments had significantly increased them, which were then further augmented by YKS. These findings suggest that YKS enhances GLT-1-derived glutamate transport functions in TA-treated cultured astrocytes and that this process entails increased GLT-1 protein and mRNA levels. This type of mechanism may contribute to the YKS-mediated regulation of extracellular glutamate concentrations.

The protective effect of 2-(2-benzonfu-ranyl)-2-imidazoline against oxygen-glucose deprivation in cultured rat cortical astrocytes

Astrocytes play a pivotal role in neuronal survival in the setting of post-ischemic brain inflammation, but the astrocyte-derived mediators of ischemic brain injury remain to be defined. 2-(2-Benzofu-ranyl)-2-imidazoline (2-BFI) is a newly discovered ligand for high-affinity imidazoline I2 receptors (I2Rs) mainly located on the mitochondrial outer membrane in astrocytes. We previously reported that in a rat model of cerebral ischemia-reperfusion injury, 2-BFI limits infarct volume, reduces neurological impairment scores, and inhibits neuronal apoptosis in the ischemic penumbra. This study was performed to clarify the underlying mechanism in an astrocyte oxygen-glucose deprivation (OGD) model. The results show that 2-BFI reduces lipid peroxidation and inhibits mitochondria apoptotic pathways.

Dopamine cross-reacts with adrenoreceptors in cortical astrocytes to induce BDNF expression, CREB signaling and morphological transformation.

Expression of brain-derived neurotrophic factor (BDNF) is induced in cultured rat cortical astrocytes by catecholamines norepinephrine and dopamine as well as selective α1 and β adrenergic agonists. However, it has remained unknown which receptors mediate dopamine-induced BDNF upregulation in astrocytes. Here, we demonstrate that β adrenoreceptors are the main mediators of this effect in cultured cortical astrocytes, while α1 adrenoreceptors and D1 dopamine receptors contribute to a lesser extent. We show that in cortical astrocytes BDNF exon IV and exon VI containing mRNAs are induced by dopamine and norepinephrine via CREB-dependent signaling and that this regulation is mediated by a mechanism that is distinct from activity-dependent CREB-mediated activation of BDNF transcription in neurons. We also show that regulation of BDNF promoters IV and VI by catecholamines requires a distal regulatory element in the BDNF locus. Finally, we demonstrate that dopamine-induced astrocyte stellation and induction of CREB signaling are mediated by cross-reaction of dopamine with β adrenoreceptors.

The β-lactam clavulanic acid mediates glutamate transport-sensitive pain relief in a rat model of neuropathic pain.

Following nerve injury, down-regulation of astroglial glutamate transporters (GluTs) with subsequent extracellular glutamate accumulation is a key factor contributing to hyperexcitability within the spinal dorsal horn. Some β-lactam antibiotics can up-regulate GluTs, one of which, ceftriaxone, displays analgesic effects in rodent chronic pain models. Here, the antinociceptive actions of another β-lactam clavulanic acid, which possesses negligible antibiotic activity, were compared with ceftriaxone in rats with chronic constriction injury (CCI)-induced neuropathic pain. In addition, the protein expression of glutamate transporter-1 (GLT1), its splice variant GLT1b and glutamate-aspartate transporter (GLAST) was measured in the spinal cord of CCI rats. Finally, protein expression of the same GluTs was evaluated in cultured astrocytes obtained from rodents and humans. Repeated injection of ceftriaxone or clavulanic acid over 10days alleviated CCI-induced mechanical hypersensitivity, whilst clavulanic acid was additionally able to affect the thermal hypersensitivity. In addition, clavulanic acid up-regulated expression of GLT1b within the spinal cord of CCI rats, whereas ceftriaxone failed to modulate expression of any GluTs in this model. However, both clavulanic acid and ceftriaxone up-regulated GLT1 expression in rat cortical and human spinal astrocyte cultures. Furthermore, clavulanic acid increased expression of GLT1b and GLAST in rat astrocytes in a dose-dependent manner. Thus, clavulanic acid up-regulates GluTs in cultured rodent- and human astroglia and alleviates CCI-induced hypersensitivity, most likely through up-regulation of GLT1b in spinal dorsal horn. Chronic dosing of clavulanic acid alleviates neuropathic pain in rats and up-regulates glutamate transporters both invitro and in vivo. Crucially, a similar up-regulation of glutamate transporters in human spinal astrocytes by clavulanic acid supports the development of novel β-lactam-based analgesics, devoid of antibacterial activity, for the clinical treatment of chronic pain.

Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves hepatic production of acute-phase proteins, including serum amyloid A (SAA). Extrahepatically, SAA immunoreactivity is found in axonal myelin sheaths of cortex in Alzheimer’s disease and multiple sclerosis (MS), although its cellular origin is unclear. We examined the responses of cultured rat cortical astrocytes, microglia and oligodendrocyte precursor cells (OPCs) to master pro-inflammatory cytokine tumour necrosis factor (TNF)-α and lipopolysaccaride (LPS). TNF-α time-dependently increased Saa1 (but not Saa3) mRNA expression in purified microglia, enriched astrocytes, and OPCs (as did LPS for microglia and astrocytes). Astrocytes depleted of microglia were markedly less responsive to TNF-α and LPS, even after re-addition of microglia. Microglia and enriched astrocytes showed complementary Saa1 expression profiles following TNF-α or LPS challenge, being higher in microglia with TNF-α and higher in astrocytes with LPS. Recombinant human apo-SAA stimulated production of both inflammatory mediators and its own mRNA in microglia and enriched, but not microglia-depleted astrocytes. Co-ultramicronized palmitoylethanolamide/luteolin, an established anti-inflammatory/ neuroprotective agent, reduced Saa1 expression in OPCs subjected to TNF-α treatment. These last data, together with past findings suggest that co-ultramicronized palmitoylethanolamide/luteolin may be a novel approach in the treatment of inflammatory demyelinating disorders like MS.