Ketamine triggers rapid antidepressant effects by modulating synaptic plasticity in a new depressive-like mouse model based on astrocyte glutamate transporter GLT-1 knockdown in infralimbic cortex.

OBJECTIVE: Recently, we reported on a new MDD-like mouse model based on a regionally selective knockdown of astroglial glutamate transporters, GLAST/GLT-1, in infralimbic cortex (IL) which evokes widespread changes in mouse brain associated with the typical alterations found in MDD patients. To further characterize this new MDD-like mouse model, here we examine some transcriptional elements of glutamatergic/GABAergic neurotransmission and neuroplasticity in forebrain regions in the GLT-1 knockdown mice. Furthermore, we assess the acute ketamine effects on these transcriptional processes. MATERIAL AND METHODS: We used a small interfering RNA (siRNA) pool targeting GLT-1 mRNA to disrupt the GLT-1 transcription in mouse IL. Histological assays were performed to examine postsynaptic density protein-95 (PSD95), neuritin (NRN), glutamine acid descarboxilase-65 (GAD65), and GLT-1 mRNA expression in IL and hippocampus. RESULTS: Knockdown of GLT-1 in mouse IL leads to decreased expression of PSD95 and NRN neuroplasticity mRNAs in IL and hippocampus, which was reversed by an acute dose of ketamine antidepressant. Likewise, a single dose of ketamine also increased the mRNA levels of GAD65 and GLT-1 in IL of GLT-1 knockdown mice, reaching the basal values of control mice. CONCLUSIONS: The glutamatergic neuronal hyperactivity and deficits in the GABA system resulting from siRNA-induced astroglial glutamate transporter knockdown in IL can compromise the integrity/plasticity of neurocircuits affected in MDD. Suitable depressive-like animal models to address the neurobiological changes in MDD are an unmet need and the development of the GLAST/GLT-1 knockdown mouse model may represent a better option to understand the rapid-acting antidepressant effects of ketamine.

Ceftriaxone regulates glutamate production and vesicular assembly in presynaptic terminals through GLT-1 in APP/PS1 mice.

Perturbations in the glutamate-glutamine cycle and glutamate release from presynaptic terminals have been involved in the development of cognitive deficits in Alzheimer's disease (AD) patients and mouse models. Glutamate transporter-1 (GLT-1) removes glutamate from the synaptic cleft and transports it into astrocytes, where it is used as substrate for the glutamate-glutamine cycle. Ceftriaxone has been reported to improve cognitive deficits in AD mice by increasing GLT-1 expression, glutamate transformation to glutamine, and glutamine efflux from astrocytes. However, the impact of ceftriaxone on glutamine metabolism in neurons is unknown. The present study aimed to investigate whether ceftriaxone regulated the production and vesicular assembly of glutamate in the presynaptic terminals of neurons and to determine GLT-1 involvement in this process. We used the amyloid precursor protein (APP)/presenilin-1 (PS1) AD mouse model and GLT-1 knockdown APP/PS1 (GLT-1+/-/APP/PS1) mice. The expression levels of sodium-coupled neutral amino-acid transporter 1 (SNAT1) and vesicular glutamate transporters 1 and 2 (VGLUT1/2) were analyzed by immunofluorescence and immunohistochemistry staining as well as by Western blotting. Glutaminase activity was assayed by fluorometry. Ceftriaxone treatment significantly increased SNAT1 expression and glutaminase activity in neurons in APP/PS1 mice. Similarly, VGLUT1/2 levels were increased in the presynaptic terminals of APP/PS1 mice treated with ceftriaxone. The deletion of one GLT-1 allele in APP/PS1 mice prevented the ceftriaxone-induced upregulation of SNAT1 and VGLUT1/2 expression, indicating that GLT-1 played an important role in ceftriaxone effect. Based on the role of SNAT1, glutaminase, and VGLUT1/2 in the glutamate-glutamine cycle in neurons, the present results suggested that ceftriaxone improved the production and vesicular assembly of glutamate as a neurotransmitter in presynaptic terminals by acting on GLT-1 in APP/PS1 mice.

Mild hypothermia protects rat cortical neurons against oxygen-glucose deprivation/reoxygenation injury via the PI3K/Akt pathway.

Ischemic stroke is the most frequent cause of long-term morbidity and mortality in the elderly worldwide. Mild hypothermia (32-35°C) has been found to have a neuroprotective effect against ischemic stroke. However, the protective mechanisms remain unclear. In the present study, we explore the neuroprotective effect of mild hypothermia in neuron-astrocyte cocultures by oxygen-glucose deprivation/reoxygenation (OGD/R) as well as the underlying mechanisms. Thionin staining was performed and cell viability, extracellular glutamate concentration and the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway-related proteins were detected after OGD/R. The results indicated that mild hypothermia significantly alleviated damage to Nissl bodies and increased the viability of neurons, which alleviated OGD/R-triggered neuronal injury. Furthermore, mild hypothermia significantly enhanced the phosphorylation of Akt (pAkt) and glutamate transporter-1 (GLT-1) and reduced extracellular glutamate concentration after OGD/R. When the PI3K inhibitor LY294002 was added, neuronal viability and the expression of pAkt and GLT-1 decreased, and extracellular glutamate concentration increased. The protective effect of mild hypothermia was counteracted by LY294002. There was no significant change in neuronal viability or the expression of pAkt and GLT-1 in the group treated with dihydrokainate, an inhibitor of GLT-1-function, compared with the mild hypothermia + OGD/R (HOGD) group, but extracellular glutamate concentration was increased. Consequently, mild hypothermia promoted glutamate clearance by regulating GLT-1 expression via the PI3K/Akt pathway, providing a neuroprotective effect against OGD/R injury.

Astroglial glutamate transporter 1 and glutamine synthetase of the nucleus accumbens are involved in the antidepressant-like effects of allopregnanolone in learned helplessness rats.

Clinical studies have demonstrated that allopregnanolone (3α5α-tetrahydroprogesterone, ALLO) has antidepressant-like effects on patients with depression. Previous studies have shown alteration of the astroglial glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) in depression, and ALLO is known to modulate glutamate release. The present study aimed to investigate whether astroglial GLT-1 and GS are indeed involved in the antidepressant-like effects of ALLO in learned helplessness (LH) rats, a validated animal model of depression. The results of this study showed that bilateral microinjection of ALLO into the lateral ventricles could normalize the levels of GLT-1 and GS in the nucleus accumbens (NAc) and of GS in the hippocampal CA1 region of LH rats. These results suggest a certain connection between the antidepressant-like effects of ALLO and the astroglial GLT-1/GS system of the NAc in LH rats.

Effects of Combined Bushen Zhichan Recipe and Levodopa in a Rodent Model of Parkinson Disease: Potential Mechanisms.

BACKGROUND Parkinson disease is characterized by the loss of neurons in the substantia nigra, and under pathological conditions, glutamate can produce excitotoxic effects on nerve cells. The astrocytic excitatory amino acid transporter (EAAT) 1 can be functionally upregulated and targeted to functional compartments, resulting in reduced excitotoxicity. levodopa is the gold standard for the treatment of Parkinson disease, but prolonged levodopa treatment often leads to the development of abnormal involuntary movements. Numerous studies suggest the potential beneficial effects of traditional Chinese medicine on Parkinson disease. MATERIAL AND METHODS We validated the efficacy of a Bushen Zhichan recipe combined with levodopa in a rodent Parkinson disease model and explored its possible mechanisms. RESULTS Rats in the combined levodopa and Bushen Zhichan recipe group performed significantly better than the control group in the open field and forelimb function experiments. The number of midbrain dopaminergic neurons in rats in the levodopa and Bushen Zhichan recipe group was greater compared to controls. The levodopa and Bushen Zhichan recipe group exhibited decreased glutamate receptors and increased γ-aminobutyric acid receptors in the striatum. At the same time, EAAT1 was increased and EAAT2 was synchronized with the number of glutamate receptors. CONCLUSIONS Our results indicate that levodopa combined with Bushen Zhichan recipe significantly improves behavior and protects dopaminergic neurons in a rodent Parkinson disease model, and suggest that the mechanism involves the decrease of excitatory amino acid toxicity and the increase in the expression of EAAT1.

Fluoxetine and Riluzole Mitigates Manganese-Induced Disruption of Glutamate Transporters and Excitotoxicity via Ephrin-A3/GLAST-GLT-1/Glu Signaling Pathway in Striatum of Mice.

Manganese (Mn) is an essential element required for many biological processes and systems in the human body. Mn intoxication increases brain glutamate (Glu) levels causing neuronal damage. Recent studies have reported that ephrin-A3 regulates this glutamate transporter. However, none has explored the role of this crucial molecule in Mn-induced excitotoxicity. The present study investigated whether ephrin-A3/GLAST-GLT-1/Glu signaling pathway participates in Mn-induced excitotoxicity using astrocytes and Kunming mice. The mechanisms were explored using fluoxetine (ephrin-A3 inhibitor) and riluzole (a Glu release inhibitor). Firstly, we demonstrated that Mn exposure (500 µM or 50 mg/kg MnCl2) significantly increased Mn, ephrin-A3, and Glu levels, and inhibited Na+-K+ ATPase activity, as well as mRNA and protein levels of GLAST and GLT-1. Secondly, we found that astrocytes and mice pretreated with fluoxetine (100 µM or 15 mg/kg) and riluzole (100 µM or 32 µmol/kg) prior to Mn exposure had lower ephrin-A3 and Glu levels, but higher Na+-K+ ATPase activity, expression levels of GLAST and GLT-1 than those exposed to 500 µM or 50 mg/kg MnCl2. Moreover, the morphology of cells and the histomorphology of mice striatum were injured. Results showed that pretreatment with fluoxetine and riluzole attenuated the Mn-induced motor dysfunctions. Together, these results suggest that the ephrin-A3/GLAST-GLT-1/Glu signaling pathway participates in Mn-induced excitotoxicity, and fluoxetine and riluzole can mitigate the Mn-induced excitotoxicity in mice brain.

Effects of Cocaine Exposure on Astrocytic Glutamate Transporters and Relapse-Like Ethanol-Drinking Behavior in Male Alcohol-Preferring Rats.

AIM: Glutamate has been considered as neurotransmitter that is critical in triggering relapse to drugs of abuse, including ethanol and cocaine. Extracellular glutamate concentrations are tightly regulated by several mechanisms, including reuptake through glutamate transporters. Glutamate transporter type 1 (GLT-1) is responsible for clearing the majority of extracellular glutamate. The astrocytic cystine/glutamate antiporter (xCT) regulates also glutamate homeostasis. In this study, we investigated the effects of cocaine exposure and ampicillin/sulbactam (AMP/SUL), a ß-lactam antibiotic known to upregulate GLT-1 and xCT, on relapse-like ethanol intake and the expression of astrocytic glutamate transporters in mesocorticolimbic brain regions. METHODS: Male alcohol-preferring (P) rats had free access to ethanol for 5 weeks. On Week 6, rats were exposed to either cocaine (20 mg/kg, i.p.) or saline for 12 consecutive days. Ethanol bottles were then removed for 7 days; during the last 5 days, either AMP/SUL (100 or 200 mg/kg, i.p.) or saline was administered to the P rats. Ethanol bottles were reintroduced, and ethanol intake was measured for 4 days. RESULTS: Cocaine exposure induced an alcohol deprivation effect (ADE), which was associated in part by a decrease in the expression of GLT-1 and xCT in the nucleus accumbens (NAc) core. AMP/SUL (100 mg/kg, i.p.) attenuated the ADE, while AMP/SUL (200 mg/kg, i.p.) reduced ethanol intake during 4 days of ethanol re-exposure and upregulated GLT-1 and xCT expression in the NAc core, NAc shell and dorsomedial prefrontal cortex (dmPFC). CONCLUSION: This study suggests that these astrocytic glutamate transporters might be considered as potential targets for the treatment of polysubstance abuse.

Minocycline induces the expression of intra-accumbal glutamate transporter-1 in the morphine-dependent rats.

Glial glutamate transporters (GLT-1) is responsible for glutamate homeostasis. GLT-1 expression and glutamate uptake can be affected by addictive drugs and can be used as a target in addiction pharmacotherapy. It has been shown that minocycline, an antibiotic with anti-inflammatory, and neuroprotective properties, can upregulate the expression of GLT-1. In the present study, in morphine-dependent rats, the effect of minocycline on expression of GLT-1 in nucleus accumbens was investigated by immunohistochemistry. The expression of GLT-1 significantly increased in minocycline treated animals. In line with other studies, our findings showed that restoring GLT-1 expression with minocycline might be considered as a potential target for correcting pre-clinical and clinical manifestations of drug addiction.

Novel Positive Allosteric Modulators of Glutamate Transport Have Neuroprotective Properties in an in Vitro Excitotoxic Model.

Dysfunction of excitatory amino acid transporters (EAATs) has been implicated in the pathogenesis of various neurological disorders, such as stroke, brain trauma, epilepsy, and several neurodegenerative disorders. EAAT2 is the main transporter subtype responsible for glutamate clearance in the brain, and plays a key role in regulating neurotransmission and preventing excitotoxicity. Therefore, compounds that increase the activity of EAAT2 have therapeutic potential for neuroprotection. In previous studies, we used virtual screening approaches to identify novel positive allosteric modulators (PAMs) of EAAT2. These compounds were shown to selectively increase the activity of EAAT2 and increase Vmax of transport, without changing substrate affinity. In this work, our major effort was to investigate whether increasing the activity of EAAT2 by allosteric modulation would translate to neuroprotection in in vitro primary culture models of excitotoxicity. To investigate potential neuroprotective effects of one EAAT2 PAM, GT949, we subjected cultures to acute and prolonged excitotoxic insults by exogenous application of glutamate, or oxidative stress by application of hydrogen peroxide. GT949 administration did not result in neuroprotection in the oxidative stress model, likely due to damage of the glutamate transporters. However, GT949 displayed neuroprotective properties after acute and prolonged glutamate-mediated excitotoxicity. We propose that this compound prevents excess glutamate signaling by increasing the rate of glutamate clearance by EAAT2, thereby preventing excitotoxic damage and cell death. This novel class of compounds is therefore an innovative approach for neuroprotection with potential for translation in in vivo animal models of excitotoxicity.

The role of astrocytic glutamate transporters GLT-1 and GLAST in neurological disorders: Potential targets for neurotherapeutics.

Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS) which initiates rapid signal transmission in the synapse before its re-uptake into the surrounding glia, specifically astrocytes. The astrocytic glutamate transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) and their human homologs excitatory amino acid transporter 1 (EAAT1) and 2 (EAAT2), respectively, are the major transporters which take up synaptic glutamate to maintain optimal extracellular glutamic levels, thus preventing accumulation in the synaptic cleft and ensuing excitotoxicity. Growing evidence has shown that excitotoxicity is associated with various neurological disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), manganism, ischemia, schizophrenia, epilepsy, and autism. While the mechanisms of neurological disorders are not well understood, the dysregulation of GLAST/GLT-1 may play a significant role in excitotoxicity and associated neuropathogenesis. The expression and function of GLAST/GLT-1 may be dysregulated at the genetic, epigenetic, transcriptional or translational levels, leading to high levels of extracellular glutamate and excitotoxicity. Consequently, understanding the regulatory mechanisms of GLAST/GLT-1 has been an area of interest in developing therapeutics for the treatment of neurological disorders. Pharmacological agents including ß-lactam antibiotics, estrogen/selective estrogen receptor modulators (SERMs), growth factors, histone deacetylase inhibitors (HDACi), and translational activators have shown significant efficacy in enhancing the expression and function of GLAST/GLT-1 and glutamate uptake both in vitro and in vivo. This comprehensive review will discuss the regulatory mechanisms of GLAST/GLT-1, their association with neurological disorders, and the pharmacological agents which mediate their expression and function. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Increased cocaine self-administration in rats lacking the serotonin transporter: a role for glutamatergic signaling in the habenula.

Serotonin (5-HT) and the habenula (Hb) contribute to motivational and emotional states such as depression and drug abuse. The dorsal raphe nucleus, where 5-HT neurons originate, and the Hb are anatomically and reciprocally interconnected. Evidence exists that 5-HT influences Hb glutamatergic transmission. Using serotonin transporter knockout (SERT-/- ) rats, which show depression-like behavior and increased cocaine intake, we investigated the effect of SERT reduction on expression of genes involved in glutamate neurotransmission under both baseline conditions as well as after short-access or long-access cocaine (ShA and LgA, respectively) intake. In cocaine-naïve animals, SERT removal led to reduced baseline Hb mRNA levels of critical determinants of glutamate transmission, such as SLC1A2, the main glutamate transporter and N-methyl-D-aspartate (Grin1, Grin2A and Grin2B) as well as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (Gria1 and Gria2) receptor subunits, with no changes in the scaffolding protein Dlg4. In response to ShA and LgA cocaine intake, SLC1A2 and Grin1 mRNA levels decreased in SERT+/+ rats to levels equal of those of SERT-/- rats. Our data reveal that increased extracellular levels of 5-HT modulate glutamate neurotransmission in the Hb, serving as critical neurobiological substrate for vulnerability to cocaine addiction.

Effects of glial glutamate transporter activator in formalin-induced pain behaviour in mice.

BACKGROUND: Nociceptive pain remains a prevalent clinical problem and often poorly responsive to the currently available analgesics. Previous studies have shown that astroglial glutamate transporter-1 (GLT-1) in the hippocampus and anterior cingulate cortex (ACC) is critically involved in pain processing and modulation. However, the role of astroglial GLT-1 in nociceptive pain involving the hippocampus and ACC remains unknown. We investigated the role of 3-[[(2-Methylphenyl) methyl]thio]-6-(2-pyridinyl)-pyridazine (LDN-212320), a GLT-1 activator, in nociceptive pain model and hippocampal-dependent behavioural tasks in mice. METHODS: We evaluated the effects of LDN-212320 in formalin-induced nociceptive pain model. In addition, formalin-induced impaired hippocampal-dependent behaviours were measured using Y-maze and object recognition test. Furthermore, GLT-1 expression and extracellular signal-regulated kinase phosphorylation (pERK1/2) were measured in the hippocampus and ACC using Western blot analysis and immunohistochemistry. RESULTS: The LDN-212320 (10 or 20 mg/kg, i.p) significantly attenuated formalin-evoked nociceptive behaviour. The antinociceptive effects of LDN-212320 were reversed by systemic administration of DHK (10 mg/kg, i.p), a GLT-1 antagonist. Moreover, LDN-212320 (10 or 20 mg/kg, i.p) significantly reversed formalin-induced impaired hippocampal-dependent behaviour. In addition, LDN-212320 (10 or 20 mg/kg, i.p) increased GLT-1 expressions in the hippocampus and ACC. On the other hand, LDN-212320 (20 mg/kg, i.p) significantly reduced formalin induced-ERK phosphorylation, a marker of nociception, in the hippocampus and ACC. CONCLUSION: These results suggest that the GLT-1 activator LDN-212320 prevents nociceptive pain by upregulating astroglial GLT-1 expression in the hippocampus and ACC. Therefore, GLT-1 activator could be a novel drug candidate for nociceptive pain. SIGNIFICANCE: The present study provides new insights and evaluates the role of GLT-1 activator in the modulation of nociceptive pain involving hippocampus and ACC. Here, we provide evidence that GLT-1 activator could be a potential therapeutic utility for the treatment of nociceptive pain.

ß-Lactamase inhibitor, clavulanic acid, attenuates ethanol intake and increases glial glutamate transporters expression in alcohol preferring rats.

Studies from our laboratory showed that upregulation of glutamate transporter 1 (GLT-1) and cystine-glutamate exchanger (xCT) expression with ceftriaxone, ß-lactam antibiotic, in the brain was associated with attenuation of ethanol consumption. In this study, we tested clavulanic acid, which is another ß-lactam compound with negligible antimicrobial activity, on ethanol consumption and expression of GLT-1, xCT and glutamate aspartate transporter (GLAST) in male alcohol-preferring (P) rats. Clavulanic acid has the central ß-lactam pharmacophore that is critical for the upregulation of GLT-1 and xCT expression. We found that clavulanic acid, at 5mg/kg (i.p.) dose, significantly attenuated ethanol consumption and ethanol preference in P rats as compared to vehicle-treated group. This effect was associated with a significant increase in water intake in clavulanic acid treated group. Importantly, we found that clavulanic acid increased the expression of GLT-1 and xCT in nucleus accumbens. However, there was no effect of clavulanic acid on GLAST expression in the nucleus accumbens. Clavulanic acid treatment did not upregulate the expression of GLT-1, xCT and GLAST in prefrontal cortex. These findings revealed that clavulanic acid at 20-40 fold lower dose than ceftriaxone can attenuate ethanol consumption, in part through upregulation of GLT-1 and xCT expression in the nucleus accumbens. Thus, we suggest that clavulanic acid might be used as an alternative option to ceftriaxone to attenuate ethanol drinking behavior.

Ceftriaxone reduces L-dopa-induced dyskinesia severity in 6-hydroxydopamine parkinson's disease model.

BACKGROUND: Increased extracellular glutamate may contribute to l-dopa induced dyskinesia, a debilitating side effect faced by Parkinson's disease patients 5 to 10 years after l-dopa treatment. Therapeutic strategies targeting postsynaptic glutamate receptors to mitigate dyskinesia may have limited success because of significant side effects. Increasing glutamate uptake may be another approach to attenuate excess glutamatergic neurotransmission to mitigate dyskinesia severity or prolong the time prior to onset. Initiation of a ceftriaxone regimen at the time of nigrostriatal lesion can attenuate tyrosine hydroxylase loss in conjunction with increased glutamate uptake and glutamate transporter GLT-1 expression in a rat 6-hydroxydopamine model. In this article, we examined if a ceftriaxone regimen initiated 1 week after nigrostriatal lesion, but prior to l-dopa, could reduce l-dopa-induced dyskinesia in an established dyskinesia model. METHODS: Ceftriaxone (200 mg/kg, intraperitoneal, once daily, 7 consecutive days) was initiated 7 days post-6-hydroxydopamine lesion (days 7-13) and continued every other week (days 21-27, 35-39) until the end of the study (day 39 postlesion, 20 days of l-dopa). RESULTS: Ceftriaxone significantly reduced abnormal involuntary movements at 5 time points examined during chronic l-dopa treatment. Partial recovery of motor impairment from nigrostriatal lesion by l-dopa was unaffected by ceftriaxone. The ceftriaxone-treated l-dopa group had significantly increased striatal GLT-1 expression and glutamate uptake. Striatal tyrosine hydroxylase loss in this group was not significantly different when compared with the l-dopa alone group. CONCLUSIONS: Initiation of ceftriaxone after nigrostriatal lesion, but prior to and during l-dopa, may reduce dyskinesia severity without affecting l-dopa efficacy or the reduction of striatal tyrosine hydroxylase loss. © 2017 International Parkinson and Movement Disorder Society.

Age and Alzheimer's disease gene expression profiles reversed by the glutamate modulator riluzole.

Alzheimer's disease (AD) and age-related cognitive decline represent a growing health burden and involve the hippocampus, a vulnerable brain region implicated in learning and memory. To understand the molecular effects of aging on the hippocampus, this study characterized the gene expression changes associated with aging in rodents using RNA-sequencing (RNA-seq). The glutamate modulator, riluzole, which was recently shown to improve memory performance in aged rats, prevented many of the hippocampal age-related gene expression changes. A comparison of the effects of riluzole in rats against human AD data sets revealed that many of the gene changes in AD are reversed by riluzole. Expression changes identified by RNA-Seq were validated by qRT-PCR open arrays. Riluzole is known to increase the glutamate transporter EAAT2's ability to scavenge excess glutamate, regulating synaptic transmission. RNA-seq and immunohistochemistry confirmed an increase in EAAT2 expression in hippocampus, identifying a possible mechanism underlying the improved memory function after riluzole treatment.

Neuroprotective Effects of the Glutamate Transporter Activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) following Traumatic Brain Injury in the Adult Rat.

Traumatic brain injury (TBI) in humans and in animals leads to an acute and sustained increase in tissue glutamate concentrations within the brain, triggering glutamate-mediated excitotoxicity. Excitatory amino acid transporters (EAATs) are responsible for maintaining extracellular central nervous system glutamate concentrations below neurotoxic levels. Our results demonstrate that as early as 5 min and up to 2 h following brain trauma in brain-injured rats, the activity (Vmax) of EAAT2 in the cortex and the hippocampus was significantly decreased, compared with sham-injured animals. The affinity for glutamate (KM) and the expression of glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST) were not altered by the injury. Administration of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a GLT-1 activator, beginning immediately after injury and continuing for 24 h, significantly decreased neurodegeneration, loss of microtubule-associated protein 2 and NeuN (+) immunoreactivities, and attenuated calpain activation in both the cortex and the hippocampus at 24 h after the injury; the reduction in neurodegeneration remained evident up to 14 days post-injury. In synaptosomal uptake assays, MS-153 up-regulated GLT-1 activity in the naïve rat brain but did not reverse the reduced activity of GLT-1 in traumatically-injured brains. This study demonstrates that administration of MS-153 in the acute post-traumatic period provides acute and long-term neuroprotection for TBI and suggests that the neuroprotective effects of MS-153 are related to mechanisms other than GLT-1 activation, such as the inhibition of voltage-gated calcium channels.

Glutamate transporter splice variant expression in an enriched pyramidal cell population in schizophrenia.

Dysregulation of the glutamate transporters EAAT1 and EAAT2 and their isoforms have been implicated in schizophrenia. EAAT1 and EAAT2 expression has been studied in different brain regions but the prevalence of astrocytic glutamate transporter expression masks the more subtle changes in excitatory amino acid transporters (EAATs) isoforms in neurons in the cortex. Using laser capture microdissection, pyramidal neurons were cut from the anterior cingulate cortex of postmortem schizophrenia (n = 20) and control (n = 20) subjects. The messenger RNA (mRNA) levels of EAAT1, EAAT2 and the splice variants EAAT1 exon9skipping, EAAT2 exon9skipping and EAAT2b were analyzed by real time PCR (RT-PCR) in an enriched population of neurons. Region-level expression of these transcripts was measured in postmortem schizophrenia (n = 25) and controls (n = 25). The relationship between selected EAAT polymorphisms and EAAT splice variant expression was also explored. Anterior cingulate cortex pyramidal cell expression of EAAT2b mRNA was increased (P < 0.001; 67%) in schizophrenia subjects compared with controls. There was no significant change in other EAAT variants. EAAT2 exon9skipping mRNA was increased (P < 0.05; 38%) at region level in the anterior cingulate cortex with no significant change in other EAAT variants at region level. EAAT2 single-nucleotide polymorphisms were significantly associated with changes in EAAT2 isoform expression. Haloperidol decanoate-treated animals, acting as controls for possible antipsychotic effects, did not have significantly altered neuronal EAAT2b mRNA levels. The novel finding that EAAT2b levels are increased in populations of anterior cingulate cortex pyramidal cells further demonstrates a role for neuronal glutamate transporter splice variant expression in schizophrenia.

N-Acetylcysteine improves mitochondrial function and ameliorates behavioral deficits in the R6/1 mouse model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington's disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy.

Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains.

Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate-binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain. We identified a domain (purple star) in the glutamate transporter EAAT2 that is important for transport stimulation through a spider venom, and suggest a mechanism for enhanced transporter function through facilitated substrate translocation (arrow). Because the dysfunction of glutamate transporters is implicated in the pathogenesis of neurological disorders, understanding the mechanisms of enhanced transport could have therapeutic implications.

Stress rapidly dysregulates the glutamatergic synapse in the prefrontal cortex of cocaine-withdrawn adolescent rats.

Although several lines of evidence have shown that chronic cocaine use is associated with stress system dysregulation, the underlying neurochemical mechanisms are still elusive. To investigate whether the rapid stress-induced response of the glutamatergic synapse was influenced by a previous history of cocaine, rats were exposed to repeated cocaine injections during adolescence [from postnatal day (PND) 28-42], subjected to a single swim stress (5 minutes) three days later (PND 45) and sacrificed 15 minutes after the end of this stressor. Critical determinants of glutamatergic homeostasis were measured in the medial prefrontal cortex (mPFC) whereas circulating corticosterone levels were measured in the plasma. Exposure to stress in saline-treated animals did not show changes in the crucial determinants of the glutamatergic synapse. Conversely, in cocaine-treated animals, stress dynamically altered the glutamatergic synapse by: (1) enhancing the presynaptic vesicular mediators of glutamate release; (2) reducing the transporters responsible for glutamate clearance; (3) increasing the postsynaptic responsiveness of the N-methyl-D-aspartate subunit GluN1; and (4) causing hyperresponsive spines as evidenced by increased activation of the postsynaptic cdc42-Pak pathway. These findings indicate that exposure to cocaine during adolescence sensitizes mPFC glutamatergic synapses to stress. It is suggested that changes in glutamatergic signaling may contribute to the increased sensitivity to stress observed in cocaine users. Moreover, glutamatergic processes may play an important role in stress-induced reinstatement of cocaine seeking.