Glutamate Transporter Proteins Research Articles

Diverse glutamatergic inputs target spines expressing M1 muscarinic receptors in the basolateral amygdala: An ultrastructural analysis

Although it is known that acetylcholine acting through M1 muscarinic receptors (M1Rs) is essential for memory consolidation in the anterior basolateral nucleus of the amygdala (BLa), virtually nothing is known about the circuits involved. In the hippocampus M1R activation facilitates long-term potentiation (LTP) by potentiating NMDA glutamate receptor (NMDAR) currents. The majority of NMDAR+ profiles in the BLa are spines. Since about half of dendritic spines of BLa pyramidal neurons (PNs) receiving glutamatergic inputs are M1R-immunoreactive (M1R+) it is possible that the role of M1Rs in BLa mnemonic functions also involves potentiation of NMDAR currents in spines. However, the finding that only about half of BLa spines are M1R+ suggests that this proposed mechanism may only apply to a subset of glutamatergic inputs. As a first step in the identification of differential glutamatergic inputs to M1R+ spines in the BLa, the present electron microscopic study used antibodies to two different vesicular glutamate transporter proteins (VGluTs) to label two different subsets of glutamatergic inputs to M1R+ spines. These inputs are largely complimentary with VGluT1+ inputs arising mainly from cortical structures and the basolateral nucleus, and VGluT2+ inputs arising mainly from the thalamus. It was found that about one-half of the spines that were postsynaptic to VGluT1+ or VGluT2+ terminals were M1R+. In addition, a subset of the VGluT1+ or VGluT2+ axon terminals were M1R+, including those that synapsed with M1R+ spines. These results suggest that acetylcholine can modulate glutamatergic inputs to BLa spines by presynaptic as well as postsynaptic M1R-mediated mechanisms.

Changes in the glutamate biomarker expression in rats vulnerable or resistant to the rewarding effects of cocaine and their reversal by ceftriaxone

Literature data show diverse vulnerability to the rewarding effects of cocaine in human as well as in laboratory animals. The molecular mechanisms of these differences have not been discovered yet. While the initial effects of cocaine depend primarily on the dopamine system, numerous studies have shown that adaptation within the glutamatergic system is responsible for the development of addiction.In this paper, we used the unbiased conditioned place preference (CPP) to identify rats showing a vulnerable or resistant phenotype to the rewarding effects of cocaine. Next, we investigated the expression of membrane glutamate transporter proteins: GLT-1 and xCT in selected brain structures in the above-mentioned groups of rats. Moreover, we determined the nuclear level of NF-κB and Nrf2 to verify whether changes in GLT-1 and xCT expression correlate with NF-κB and Nrf2 levels, respectively. In addition, we determined GLT-1, NF-κB, xCT and Nrf2 mRNA levels to verify the involvement of transcriptional mechanisms. We also analyzed the ability of the β-lactam antibiotic, ceftriaxone, to attenuate the persistence of CPP after a cocaine-free period in animals showing vulnerability to cocaine rewarding effects, and furthermore we determined GLT-1, xCT, NF-κB and Nrf2 protein expression.Our findings demonstrated molecular and neurochemical differences in the response to cocaine administration that are characteristic of the phenotype vulnerable or resistant to the rewarding effects of cocaine. Moreover, repeated administrations of ceftriaxone during cocaine-free perios attenuated CPP persistence and normalized GLT-1 level in the NAc. The results suggest the a lack of NF-κB involvement in the regulation of GLT-1 expression by ceftriaxone in the NAc. Additionally, we are the first to report that ceftriaxone strongly upregulates the GLT-1 in the HIP in a transcriptional mechanism involving the Nf-κB transcription factor. Future experiments may resolve the question concerning whether modulation exclusively of the GLT-1 expression in the HIP may attenuate cocaine-induced place preference or relapse.

Millisecond Time Resolution by HS-AFM Line Scanning of Fast GltPh Dynamics

Mammalian glutamate transporter proteins play the crucial role in removing excitatory neurotransmitters from the synaptic cleft. Malfunction of Glutamate transporters is identified in various neurological disorders such as cerebral stroke, epilepsy, Alzheimer's disease, dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS) and malignant glioma. During the past decade, our understanding of the structure and function of these proteins have evolved profoundly from studying a prokaryotic glutamate transporter homolog (GltPh). Here, we report the development and use of novel high-speed atomic force microscopy (HS-AFM) based techniques that reach millisecond time resolution, and explored previously undetectable short-lived transport states in unlabeled membrane reconstituted GltPh. We find that GltPh exhibits much faster dynamics than previously thought.

Astrocytes migrate from human neural stem cell grafts and functionally integrate into the injured rat spinal cord

Neural stem cells (NSCs) can differentiate into both neurons and glia after transplantation into spinal cord injury (SCI) sites. The neuronal component of stem cell grafts has the potential to form functional synaptic relays across the lesion site. The glial component may reform a blood-spinal cord barrier, support neuronal function, and contribute to remyelination. We performed a long-term, 1.5-year time course study focused on astrocyte migration, differentiation, integration, and safety following human NSC transplantation into C5 hemisection sites in immunodeficient rats. NSCs that adopted a neuronal fate did not migrate from the lesion site. In contrast, transplanted cells that adopted astrocyte fates exhibited long distance migration from the lesion site through host white matter in rostrocaudal directions. These cells migrated slowly at a mean rate of 2–3 mm/month, divided as they migrated, and gradually differentiated into astrocytes. After 1.5 years, human astrocytes migrated nine spinal cord segments, caudally to the mid-thoracic level, and rostrally into the brainstem. The migrated human astrocytes joined the endogenous population of astrocytes in the host spinal cord, extended perivascular endfeet towards host pericytes and endothelium, formed interspecies and intraspecies perivascular astrocytic networks connected by gap junctions, and expressed glutamate transporter proteins in perisynaptic processes, suggesting structural and functional integration. No adverse consequences of this extended glial migration were detected. Adjacent to the lesion site, chronic host astrocytic upregulation was significantly attenuated by NSC grafts. Thus, human astrocytes can migrate long distances from sites of SCI and safely integrate into the host central nervous system. Significance statementNeural stem cell (NSC) grafts are a candidate therapy for spinal cord injury (SCI). Here we report an 18-month study of astrocyte survival and migration from sites of SCI in immunodeficient rats. NSC grafts significantly attenuate host astrocyte reactivity at the lesion/host interface. Intra-graft astrocytes integrate into the host blood-spinal cord barrier (BSCB) and widely express glutamate transporter proteins characteristic of neurotransmitter regulation. Notably, astrocytic components of NSC grafts exhibit gradual yet extensive migration after implantation into the mid-cervical injury site; neurons do not migrate at all. This extensive astrocyte migration is not detectably associated with adverse outcomes anatomically or behaviorally.

Amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is caused by selective and progressive loss of spinal, bulbar and cortical motoneurons and leads to irreversible paralysis, loss of speech, inability to swallow and respiratory malfunctions with the eventual death of the affected individual in a rapid disease course. Several suggested molecular pathways are reviewed including SOD1 gene mutation, protein nitrosylation, phosphorylation and oxidative stress, excitotoxicity, glutamate transporter deprivation, mitochondrial involvement, protein aggregation and motor neuron trophic factors. The role of insulin and its receptor in the brain is described. It is very possible that in 90% of the sporadic ALS cases, the cause of the motor neuron degeneration is different or that multiple mechanisms are involved that would need drugs with multiple mechanisms or action. Several marketed drugs have been selected for clinical trials. Only two drugs have been approved by the FDA as showing positive effect in ALS: Riluzole and Edaravone. Two other drugs that have a significant benefit in ALS are Talampanel and Tamoxifen. The results for modulation of the neurotrophic factor Insulin Growth Factor-1 (IGF1) as a potential treatment are inconclusive. Several compounds are discussed that show a positive effect in the mouse model but which have failed in clinical trials. New approaches using different modalities such as peptides, proteins and stem cells are promising. Our ability to design better drugs would be enhanced by investigating the endogenous factors in neuron death, protein aggregation and oxidative stress that would improve our understanding of the potential pathways that result in neurodegeneration.

Adeno-associated viral overexpression of neuroligin 2 in the mouse hippocampus enhances GABAergic synapses and impairs hippocampal-dependent behaviors

The cell adhesion molecule neuroligin2 (NLGN2) regulates GABAergic synapse development, but its role in neural circuit function in the adult hippocampus is unclear. We investigated GABAergic synapses and hippocampus-dependent behaviors following viral-vector-mediated overexpression of NLGN2. Transducing hippocampal neurons with AAV-NLGN2 increased neuronal expression of NLGN2 and membrane localization of GABAergic postsynaptic proteins gephyrin and GABAARγ2, and presynaptic vesicular GABA transporter protein (VGAT) suggesting trans-synaptic enhancement of GABAergic synapses. In contrast, glutamatergic postsynaptic density protein-95 (PSD-95) and presynaptic vesicular glutamate transporter (VGLUT) protein were unaltered. Moreover, AAV-NLGN2 significantly increased parvalbumin immunoreactive (PV+) synaptic boutons co-localized with postsynaptic gephyrin+ puncta. Furthermore, these changes were demonstrated to lead to cognitive impairments as shown in a battery of hippocampal-dependent mnemonic tasks and social behaviors.

Ceftriaxone Treatment Preserves Cortical Inhibitory Interneuron Function via Transient Salvage of GLT-1 in a Rat Traumatic Brain Injury Model.

Traumatic brain injury (TBI) results in a decrease in glutamate transporter-1 (GLT-1) expression, the major mechanism for glutamate removal from synapses. Coupled with an increase in glutamate release from dead and dying neurons, this causes an increase in extracellular glutamate. The ensuing glutamate excitotoxicity disproportionately damages vulnerable GABAergic parvalbumin-positive inhibitory interneurons, resulting in a progressively worsening cortical excitatory:inhibitory imbalance due to a loss of GABAergic inhibitory tone, as evidenced by chronic post-traumatic symptoms such as epilepsy, and supported by neuropathologic findings. This loss of intracortical inhibition can be measured and followed noninvasively using long-interval paired-pulse transcranial magnetic stimulation with mechanomyography (LI-ppTMS-MMG). Ceftriaxone, a β-lactam antibiotic, is a potent stimulator of the expression of rodent GLT-1 and would presumably decrease excitotoxic damage to GABAergic interneurons. It may thus be a viable antiepileptogenic intervention. Using a rat fluid percussion injury TBI model, we utilized LI-ppTMS-MMG, quantitative PCR, and immunohistochemistry to test whether ceftriaxone treatment preserves intracortical inhibition and cortical parvalbumin-positive inhibitory interneuron function after TBI in rat motor cortex. We show that neocortical GLT-1 gene and protein expression are significantly reduced 1 week after TBI, and this transient loss is mitigated by ceftriaxone. Importantly, whereas intracortical inhibition declines progressively after TBI, 1 week of post-TBI ceftriaxone treatment attenuates the loss of inhibition compared to saline-treated controls. This finding is accompanied by significantly higher parvalbumin gene and protein expression in ceftriaxone-treated injured rats. Our results highlight prospects for ceftriaxone as an intervention after TBI to prevent cortical inhibitory interneuron dysfunction, partly by preserving GLT-1 expression.

Anxiolytic activity of paraoxon is associated with alterations in rat brain glutamatergic system

Exposure to organophosphate (OP) compounds leads to behavioral alterations. To determine whether paraoxon has effects on anxiety, anxiety-like behaviors were assessed in paraoxon-exposed rats. Protein expression of glutamate transporters has also been measured in hippocampus and prefrontal cortex. Three doses of paraoxon (0.3, 0.7, or 1 mg/kg) or corn oil (vehicle) were intraperitoneally injected to adult male rats. At 14 or 28 days after exposure, behavioral tests were done using elevated plus-maze (EPM) or open field tests. Thereafter, animals were sacrificed and both hippocampi and prefrontal cortices were extracted for cholinesterase assay and western blotting. Animals treated with convulsive doses of paraoxon (0.7 and 1 mg/kg) showed an increase in percentage of time spent in open arms and percentage of open arm entries in the EPM. In the open field test, an increase in the time spent in central area was observed in rats treated with the same doses of paraoxon. These effects of paraoxon were independent of any changes in locomotor activity. There was an increase in both astrocytic glutamate transporter proteins (GLAST and GLT-1) in the hippocampus of animals treated with 0.7 and 1 mg/kg of paraoxon. In the prefrontal cortex, protein levels of the GLAST and GLT-1 increased in 0.7 and decreased in 1 mg/kg groups. Only a significant decrease in EAAC1 protein was observed in the prefrontal cortex at 14 days following exposure to 1 mg/kg of paraoxon. Collectively, this study showed that exposure to convulsive doses of paraoxon induced anxiolytic-like behaviors in both behavioral tests. This effect may be attributed to alterations of glutamate transporter proteins in the rat hippocampus and prefrontal cortex.

Ceftriaxone Improves Cognitive Function and Upregulates GLT-1-Related Glutamate-Glutamine Cycle in APP/PS1 Mice.

Alzheimer's disease (AD) is characterized by progressive impairment of learning, memory, and cognitive deficits. Glutamate is the major excitatory neurotransmitter in the central nervous system and plays an important role in learning, memory, and cognition. The homeostasis and reutilization of glutamate are dependent on astrocytic uptake by glutamate transporter-1 (GLT-1) and the subsequent glutamate-glutamine cycle. Increasing evidence showed impairments in GLT-1 expression and uptake activity and glutamate-glutamine cycle in AD. Ceftriaxone (Cef) has been reported to upregulate the expression and uptake of GLT-1. Therefore, the present study was undertaken to explore whether Cef can improve cognitive deficits of APP/PS1 mice in early stage of AD by upregulating GLT-1 expression, and then promoting the glutamate-glutamine cycle. It was shown that Cef treatment significantly alleviated the cognitive deficits measured by Morris water maze test and upregulated GLT-1 protein expression in the hippocampus of APP/PS1 mice. Particularly, the activity of glutamine synthetase (GS) and the protein expression of system N glutamine transporter 1 (SN1), which are the key factors involved in the glutamate-glutamine cycle, were significantly upregulated as well after the Cef treatment. Furthermore, inhibition of GLT-1 uptake activity by dihydrokainic acid, an inhibitor of GLT-1, blocked the Cef-induced improvement on the cognitive deficits, GS activity, and SN1 expression. The above results suggested that Cef could improve cognitive deficits of APP/PS1 mice in early stage of AD by upregulating the GLT-1 expression, GS activity, and SN1 expression, which would lead to stimulating the glutamate-glutamine cycle.

Selective deletion of glutamine synthetase in the mouse cerebral cortex induces glial dysfunction and vascular impairment that precede epilepsy and neurodegeneration

Glutamate-ammonia ligase (glutamine synthetase; Glul) is enriched in astrocytes and serves as the primary enzyme for ammonia detoxification and glutamate inactivation in the brain. Loss of astroglial Glul is reported in hippocampi of epileptic patients, but the mechanism by which Glul deficiency might cause disease remains elusive. Here we created a novel mouse model by selectively deleting Glul in the hippocampus and neocortex. The Glul deficient mice were born without any apparent malformations and behaved unremarkably until postnatal week three. There were reductions in tissue levels of aspartate, glutamate, glutamine and GABA and in mRNA encoding glutamate receptor subunits GRIA1 and GRIN2A as well as in the glutamate transporter proteins EAAT1 and EAAT2. Adult Glul-deficient mice developed progressive neurodegeneration and spontaneous seizures which increased in frequency with age. Importantly, progressive astrogliosis occurred before neurodegeneration and was first noted in astrocytes along cerebral blood vessels. The responses to CO2-provocation were attenuated at four weeks of age and dilated microvessels were observed histologically in sclerotic areas of cKO. Thus, the abnormal glutamate metabolism observed in this model appeared to cause epilepsy by first inducing gliopathy and disrupting the neurovascular coupling.

GLT-1 mediates exercise-induced fatigue through modulation of glutamate and lactate in rats.

Glutamate has been implicated in exercise-induced fatigue, but the underlying mechanism is unknown. This study aimed to determine whether glutamate transporter-1 (GLT-1) has a key role in the regulation of exercise-induced fatigue. The expressions of GLT-1 and glial fibrillary acidic protein (GFAP) in the supplementary motor area of rats exposed to exhaustion were analyzed by immunohistochemistry. The expression of GLT-1 was further confirmed by Western blotting. The effects of GLT-1 on extracellular levels of glutamate and lactate and on exercise endurance were studied by microdialysis. GLT-1 expression was decreased in rats subjected to exercise-induced fatigue. Functional inhibition of GLT-1 using dihydrokainate and transcriptional inhibition of GLT-1 using antisense oligodeoxynucleotides led to a decrease in exercise endurance with a subsequent increase in extracellular glutamate concentration and decrease in extracellular lactate concentration. The expression of GLT-1 in the supplementary motor area is reduced after strenuous exercise, resulting in an increased extracellular glutamate concentration and decreased extracellular lactate level that may be responsible for the development of fatigue. GLT-1-mediated uptake of glutamate ameliorates exercise-induced fatigue in rat models.

Metabolomics Based Identification of SIRT5 and Protein Kinase C Epsilon Regulated Pathways in Brain.

The role of Sirtuins in brain function is emerging, yet little is known about SIRT5 in this domain. Our previous work demonstrates that protein kinase C epsilon (PKCε)-induced protection from focal ischemia is lost in SIRT5−/− mice. Thus, metabolic regulation by SIRT5 contributes significantly to ischemic tolerance. The aim of this study was to identify the SIRT5-regulated metabolic pathways in the brain and determine which of those pathways are linked to PKCε. Our results show SIRT5 is primarily expressed in neurons and endothelial cells in the brain, with mitochondrial and extra-mitochondrial localization. Pathway and enrichment analysis of non-targeted primary metabolite profiles from Sirt5−/− cortex revealed alterations in several pathways including purine metabolism (urea, adenosine, adenine, xanthine), nitrogen metabolism (glutamic acid, glycine), and malate-aspartate shuttle (malic acid, glutamic acid). Additionally, perturbations in β-oxidation and carnitine transferase (pentadecanoic acid, heptadecanoic acid) and glutamate transport and glutamine synthetase (urea, xylitol, adenine, adenosine, glycine, glutamic acid) were predicted. Metabolite changes in SIRT5−/− coincided with alterations in expression of amino acid (SLC7A5, SLC7A7) and glutamate (EAAT2) transport proteins as well as key enzymes in purine (PRPS1, PPAT), fatty acid (ACADS, HADHB), glutamine-glutamate (GAD1, GLUD1), and malate-aspartate shuttle (MDH1) metabolic pathways. Moreover, PKCε activation induced alternations in purine metabolites (urea, glutamine) that overlapped with putative SIRT5 pathways in WT but not in SIRT5−/− mice. Finally, we found that purine metabolism is a common metabolic pathway regulated by SIRT5, PKCε and ischemic preconditioning. These results implicate Sirt5 in the regulation of pathways central to brain metabolism, with links to ischemic tolerance.

Histamine H3 Heteroreceptors Suppress Glutamatergic and GABAergic Synaptic Transmission in the Rat Insular Cortex.

Histamine H3 receptors are autoreceptors that regulate histamine release from histaminergic neuronal terminals. The cerebral cortex, including the insular cortex (IC), expresses abundant H3 receptors; however, the functions and mechanisms of H3 receptors remain unknown. The aim of this study was to elucidate the functional roles of H3 in synaptic transmission in layer V of the rat IC. Unitary excitatory and inhibitory postsynaptic currents (uEPSCs and uIPSCs) were obtained through paired whole-cell patch-clamp recording in cerebrocortical slice preparations. The H3 receptor agonist, R-α-methylhistamine (RAMH), reduced the uEPSC amplitude obtained from pyramidal cell to pyramidal cell or GABAergic interneuron connections. Similarly, RAMH reduced the uIPSC amplitude in GABAergic interneuron to pyramidal cell connections. RAMH-induced decreases in both the uEPSC and uIPSC amplitudes were accompanied by increases in the failure rate and paired-pulse ratio. JNJ 5207852 dihydrochloride or thioperamide, H3 receptor antagonists, inhibited RAMH-induced suppression of uEPSCs and uIPSCs. Unexpectedly, thioperamide alone increased the uIPSC amplitude, suggesting that thioperamide was likely to act as an inverse agonist. Miniature EPSC or IPSC recordings support the hypothesis that the activation of H3 receptors suppresses the release of glutamate and GABA from presynaptic terminals. The colocalization of H3 receptors and glutamate decarboxylase or vesicular glutamate transport protein 1 in presynaptic axon terminals was confirmed through double pre-embedding microscopy, using a combination of pre-embedding immunogold and immunoperoxidase techniques. The suppressive regulation of H3 heteroreceptors on synaptic transmission might mediate the regulation of sensory information processes, such as gustation and visceral sensation, in the IC.

MGluR2/3 activation of the SIRT1 axis preserves mitochondrial function in diabetic neuropathy.

ObjectivesThere is a critical need to develop effective treatments for diabetic neuropathy. This study determined if a selective mGluR2/3 receptor agonist prevented or treated experimental diabetic peripheral neuropathy (DPN) through glutamate recycling and improved mitochondrial function.MethodsAdult male streptozotocin treated Sprague‐Dawley rats with features of type 1 diabetes mellitus (T1DM) or Low Capacity Running (LCR) rats with insulin resistance or glucose intolerance were treated with 3 or 10 mg/kg/day LY379268. Neuropathy end points included mechanical allodynia, nerve conduction velocities (NCV), and intraepidermal nerve fiber density (IENFD). Markers of oxidative stress, antioxidant response, glutamate recycling pathways, and mitochondrial oxidative phosphorylation (OXPHOS) associated proteins were measured in dorsal root ganglia (DRG).ResultsIn diabetic rats, NCV and IENFD were decreased. Diabetic rats treated with an mGluR2/3 agonist did not develop neuropathy despite remaining diabetic. Diabetic DRG showed increased levels of oxidized proteins, decreased levels of glutathione, decreased levels of mitochondrial DNA (mtDNA) and OXPHOS proteins. In addition, there was a 20‐fold increase in levels of glial fibrillary acidic protein (GFAP) and the levels of glutamine synthetase and glutamate transporter proteins were decreased. When treated with a specific mGluR2/3 agonist, levels of glutathione, GFAP and oxidized proteins were normalized and levels of superoxide dismutase 2 (SOD2), SIRT1, PGC‐1α, TFAM, glutamate transporter proteins, and glutamine synthetase were increased in DRG neurons.InterpretationActivation of glutamate recycling pathways protects diabetic DRG and this is associated with activation of the SIRT1‐PGC‐1α–TFAM axis and preservation of mitochondrial OXPHOS function.

Regulation of glutamate transporter 1 (GLT-1) gene expression by cocaine self-administration and withdrawal

Downregulation of the astroglial glutamate transporter GLT-1 is observed in the nucleus accumbens (NAc) following administration of multiple drugs of abuse. The decrease in GLT-1 protein expression following cocaine self-administration is dependent on both the amount of cocaine self-administered and the length of withdrawal, with longer access to cocaine and longer withdrawal periods leading to greater decreases in GLT-1 protein. However, the mechanism(s) by which cocaine downregulates GLT-1 protein remains unknown. We used qRT-PCR to examine gene expression of GLT-1 splice isoforms (GLT-1A, GLT-1B) in the NAc, prelimbic cortex (PL) and basolateral amygdala (BLA) of rats, following two widely used models of cocaine self-administration: short-access (ShA) self-administration, and the long-access (LgA) self-administration/incubation model. While downregulation of GLT-1 protein is observed following ShA cocaine self-administration and extinction, this model did not lead to a change in GLT-1A or GLT-1B gene expression in any brain region examined. Forced abstinence following ShA cocaine self-administration also was without effect. In contrast, LgA cocaine self-administration and prolonged abstinence significantly decreased GLT-1A gene expression in the NAc and BLA, and significantly decreased GLT-1B gene expression in the PL. No change was observed in NAc GLT-1A gene expression one day after LgA cocaine self-administration, indicating withdrawal-induced decreases in GLT-1A mRNA. In addition, LgA cocaine self-administration and withdrawal induced hypermethylation of the GLT-1 gene in the NAc. These results indicate that a decrease in NAc GLT-1 mRNA is only observed after extended access to cocaine combined with protracted abstinence, and that epigenetic mechanisms likely contribute to this effect.

Valproate and sodium butyrate attenuate manganese-decreased locomotor activity and astrocytic glutamate transporters expression in mice

Manganese (Mn) is an essential trace element, but chronic overexposure to this metal, either environmentally or occupationally may cause manganism, a disease analogous to Parkinson’s disease. Inhibitors of histone deacetylases, such as valproic acid (VPA) and sodium butyrate (NaB) exert neuroprotective effects in various animal models of neurological disorders. Thus, the present study investigated whether VPA or NaB prevent Mn-induced neurotoxicity by assessing locomotor activities and expression of astrocytic glutamate transporters, glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST), in C57BL/6 mice. C57BL/6 mice were pretreated with VPA (200mg/kg, i.p.) or NaB (1200mg/kg, i.p.) prior to intranasal instillation of Mn (30mg/kg) continually for 21days, followed by open-field and rota-rod behavioral tests and analyses of astrocytic glutamate transporters GLT-1 and GLAST protein/mRNA levels. The results showed that Mn significantly decreased locomotor activity as determined by total distance travelled, stereotypic and ambulatory counts. Mn also significantly decreased rota-rod activity reflecting altered motor coordination. Pretreatment with VPA and NaB with Mn reversed the effects of Mn on the locomotor activity and motor coordination. VPA and NaB also attenuated the Mn-induced decrease in GLT-1 and GLAST mRNA and protein levels in the cerebral cortical and cerebellar regions of mice. These results suggest that VPA and NaB exert protective effects against Mn toxicity seem in vitro are also shown in vivo. VPA and NaB pretreatment in mice enhancing astrocytic glutamate transporter GLT-1 expression as well as locomotor activities. Future research endeavors are warranted to determine if the therapeutic potential of VPA and NaB is via common molecular mechanism, namely, inhibition of histone deacetylases.

Contrasting the Role of xCT and GLT-1 Upregulation in the Ability of Ceftriaxone to Attenuate the Cue-Induced Reinstatement of Cocaine Seeking and Normalize AMPA Receptor Subunit Expression.

Long-term treatment with ceftriaxone attenuates the reinstatement of cocaine seeking while increasing the function of the glutamate transporter 1 (GLT-1) and system xC- (Sxc) in the nucleus accumbens core (NAc). Sxc contributes the majority of nonsynaptic extracellular glutamate in the NAc, while GLT-1 is responsible for the majority of glutamate uptake. Here we used antisense to decrease the expression of GLT-1 and xCT (a catalytic subunit of Sxc) to determine the relative importance of both proteins in mediating the ability of ceftriaxone to prevent cue-induced reinstatement of cocaine seeking and normalize glutamatergic proteins in the NAc of rats. Intra-NAc xCT knockdown prevented ceftriaxone from attenuating reinstatement and from upregulating GLT-1 and resulted in increased surface expression of AMPA receptor subunits GluA1 and GluA2. Intra-NAc GLT-1 knockdown also prevented ceftriaxone from attenuating reinstatement and from upregulating xCT expression, without affecting GluA1 and GluA2 expression. In the absence of cocaine or ceftriaxone treatment, xCT knockdown in the NAc increased the expression of both GluA1 and GluA2 without affecting GLT-1 expression while GLT-1 knockdown had no effect. PCR and immunoprecipitation of GLT-1 revealed that ceftriaxone does not upregulate GLT-1 and xCT through a transcriptional mechanism, and their coregulation by ceftriaxone is not mediated by physical interaction. These data support important and distinct roles for xCT and GLT-1 in the actions of ceftriaxone and add to a body of literature finding evidence for coregulation of these transporters. Our results also point to xCT expression and subsequent basal glutamate levels as being a key mediator of AMPA receptor expression in the NAc.SIGNIFICANCE STATEMENT Ceftriaxone attenuates the reinstatement of cocaine, alcohol, and heroin seeking. The mechanism of action of this behavioral effect has been attributed to glutamate transporter 1 (GLT-1) and xCT (a catalytic subunit of Sxc)/Sxc upregulation in the nucleus accumbens core. Here we used an antisense strategy to knock down GLT-1 or xCT in the nucleus accumbens core and examined the behavioral and molecular consequences. While upregulation of both xCT and GLT-1 are essential to the ability of ceftriaxone to attenuate cue-induced reinstatement of cocaine seeking, each protein uniquely affects the expression of other glutamate receptor and transporter proteins. We also report that reducing basal glutamate levels through the manipulation of xCT expression increases the surface expression of AMPA receptor subunits, providing insight to the mechanism by which cocaine alters AMPA surface expression.

Aquaporin 4 Forms a Macromolecular Complex with Glutamate Transporter 1 and Mu Opioid Receptor in Astrocytes and Participates in Morphine Dependence.

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes and provides a mechanism by which water permeability of the plasma membrane can be regulated. Evidence suggests that AQP4 is associated with glutamate transporter-1 (GLT-1) for glutamate clearance and contributes to morphine dependence. Previous studies show that AQP4 deficiency changed the mu opioid receptor expression and opioid receptors' characteristics as well. In this study, we focused on whether AQP4 could form macromolecular complex with GLT-1 and mu opioid receptor (MOR) and participates in morphine dependence. By using immunofluorescence staining, fluorescence resonance energy transfer, and co-immunoprecipitation, we demonstrated that AQP4 forms protein complexes with GLT-1 and MOR in both brain tissue and primary cultured astrocytes. We then showed that the C-terminus of AQP4 containing the amino acid residues 252 to 323 is the site of interaction with GLT-1. Protein kinase C, activated by morphine, played an important role in regulating the expression of these proteins. These findings may help to reveal the mechanism that AQP4, GLT-1, and MOR form protein complex and participate in morphine dependence, and deeply understand the reason that AQP4 deficiency maintains extracellular glutamate homeostasis and attenuates morphine dependence, moreover emphasizes the function of astrocyte in morphine dependence.

Ligands of peroxisome proliferator-activated receptor-alpha promote glutamate transporter-1 endocytosis in astrocytes

Astrocytes, a stellate-shape glial population in the central nervous system (CNS), maintain glutamate homeostasis in adult CNS by undergoing glutamate uptake at the synapse through their glutamate transporter-1 (GLT-1). Peroxisome proliferator-activated receptor-α (PPARα) can be activated by endogenous saturated fatty acids to regulate astrocytic lipid metabolism and functions. However, it is unclear if PPARα can exert the regulatory action on GLT-1 expression in astrocytes. This study showed that treatment with palmitic acid (PA) and the other two PPARα agonists (GW 7647 and WY 14,643) caused no change in the morphology of astrocytes, whereas membranous GLT-1 protein levels in astrocytes were significantly decreased by PA and PPARα agonists. Through lentivirus-mediated overexpression of GLT-1 tagged with red fluorescent protein (GLT-1-RFP), we also observed that GLT-1-RFP puncta in the processes of astrocytes were inhibited by the PPARα agonists. This reduction was prevented by the addition of the PPARα antagonist, GW6471. GLT-1-RFP was co-localized to the early endosome marker–EEA1 in astrocytes treated with the PPARα agonists. Moreover, PPARα-induced inhibition in membranous GLT-1 expression was abolished by the addition of dynamin inhibitor (dynasore). Furthermore, the co-treatment of astrocytes with PPARα agonists and dynasore, or with PPARα agonists and protein kinase C (PKC) inhibitor bis-indolylmaleimide 1 (BIS1), prevented the endocytosis of GLT-1-RFP. Based on the results, we conclude that the PPARα agonists increased GLT-1 endocytosis in astrocytes possibly through the PKC signaling pathway. In addition, our findings provide important information of PPARα involvement in the downregulation of astrocytic glutamate uptake via the promoted GLT-1 endocytosis.

Characterization of the cystine/glutamate antiporter in cultured Bergmann glia cells

Glutamate, the major excitatory transmitter in the vertebrate brain is a potent neurotoxin through the over-stimulation of its specific membrane receptors. In accordance, a tight regulation of its extracellular levels by plasma membrane transporters is present. A family of excitatory amino acid transporters is expressed in neurons and glia cells and is responsible of the removal of the neurotransmitter from the synaptic cleft. Glial transporters account for more than 80% of the brain uptake activity. The cystine/glutamate antiporter is another plasma membrane-bound protein critically involved in glutamatergic transmission. Upon oxidative stress, it begins to pump out glutamate in exchange for cystine, mostly needed for glutathione production. Taking into consideration that all of these glutamate transporter proteins are present in glia cells that surround glutamatergic synapses, we reasoned that a functional coupling of them should exist to prevent an excitotoxic insult to the neighboring neuronal cells. To this end, we used the established model of chick cerebellar Bergmann glia cultures. Once we could establish the expression of the cystine/glutamate antiporter in our system, we characterized its kinetic properties and started to gain insight into its regulation and plausible coupling to other transporters. Exposure to glutamate reduces the uptake activity and favors a physical interaction with the excitatory amino acid transporter 1 and the Na+-dependent neutral amino acids transporter 3. In contrast, treatment of the cultured cells with a nitric oxide donor such as sodium nitroprussiate augments the exchanger activity. Longer sodium nitroprussiate exposure periods down-regulates the cystine/glutamate protein levels. These results suggest that a coordinated interplay between glutamate transporters and exchangers takes place in glia cells to prevent excitotoxic insults.