Extracellular Glutamate Levels Research Articles

Ibuprofen treatment ameliorates memory deficits in rats with collagen-induced arthritis by normalizing aberrant MAPK/NF-κB and glutamatergic pathways

Many studies have indicated that the risk of cognitive impairment is higher in patients with rheumatoid arthritis (RA). Additionally, patients with RA may have a lower incidence of cognitive impairment with long-term use of ibuprofen. This study was aimed at investigating the impacts of RA on memory function and the mechanisms that ibuprofen may exhibit to improve memory function in rats with collagen-induced arthritis (CIA). Ibuprofen (30 mg/kg) was given twice daily to CIA rats for two weeks starting from Day 18 following the first immunization. Memory function was measured by the Morris water maze (MWM) test and long-term potentiation (LTP). The proinflammatory cytokine levels and downstream signaling pathways, including mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), were examined. Furthermore, the glutamatergic system, including glutamate transporters/receptors and brain extracellular levels of glutamate, was investigated. The results showed that the impaired learning memory in CIA rats, examined by the MWM test and LTP, can be ameliorated by ibuprofen treatment. Along with the improvement in memory deficits, ibuprofen attenuated both neuroinflammation and the associated elevated levels of phosphorylated p38, JNK, and p65 in the hippocampus of CIA rats. In addition, the decreased excitatory amino acid transporter 2 level, the increased extracellular glutamate, and the upregulated hippocampal NMDA receptor 2B of CIA rats were all normalized by ibuprofen treatment. These findings suggest that the effect of ibuprofen on the memory improvement in CIA rats is associated with the normalization of the activated MAPK and NF-κB pathways and the aberrant glutamatergic system.

Clock gene-dependent glutamate dynamics in the bean bug brain regulate photoperiodic reproduction.

Animals adequately modulate their physiological status and behavior according to the season. Many animals sense photoperiod for seasonal adaptation, and the circadian clock is suggested to play an essential role in photoperiodic time measurement. However, circadian clock-driven neural signals in the brain that convey photoperiodic information remain unclear. Here, we focused on brain extracellular dynamics of a classical neurotransmitter glutamate, which is widely used for brain neurotransmission, and analyzed its involvement in photoperiodic responses using the bean bug Riptortus pedestris that shows clear photoperiodism in reproduction. Extracellular glutamate levels in the whole brain were significantly higher under short-day conditions, which cause a reproductive diapause, than those under long-day conditions. The photoperiodic change in glutamate levels was clearly abolished by knockdown of the clock gene period. We also demonstrated that genetic modulation of glutamate dynamics by knockdown of glutamate-metabolizing enzyme genes, glutamate oxaloacetate transaminase (got) and glutamine synthetase (gs), attenuated photoperiodic responses in reproduction. Further, we investigated glutamate-mediated photoperiodic modulations at a cellular level, focusing on the pars intercerebralis (PI) neurons that photoperiodically change their neural activity and promote oviposition. Electrophysiological analyses showed that L-Glutamate acts as an inhibitory signal to PI neurons via glutamate-gated chloride channel (GluCl). Additionally, combination of electrophysiology and genetics revealed that knockdown of got, gs, and glucl disrupted cellular photoperiodic responses of the PI neurons, in addition to reproductive phenotypes. Our results reveal that the extracellular glutamate dynamics are photoperiodically regulated depending on the clock gene and play an essential role in the photoperiodic control of reproduction via inhibitory pathways.

N-acetylcysteine in the treatment of compulsive sexual behavior disorder: A case series

Although compulsive sexual behavior disorder (CSBD) is a relatively common disorder with an estimated prevalence of at least 3%, clinicians currently have limited evidence and no FDA-approved drugs to guide their treatment of this condition. N-acetylcysteine (NAC), an amino acid that seems to restore extracellular levels of glutamate in the nucleus accumbens, has previously demonstrated efficacy in treating multiple psychiatric disorders, including substance use disorders and putative behavioral addictions. However, no study has assessed the use of NAC (or any other glutamatergic agent, for that matter) in the treatment of CSBD. Here, we present data from a case series of 8 male patients with CSBD treated with NAC in routine clinical practice in a specialty outpatient clinic. Of these 8 patients, all of whom had previously been treated for CSBD with medications, therapy, or both, 5 had marked clinical improvement (>35% improvement on a modified version of Yale–Brown Obsessive Compulsive Scale) on NAC, and 3 showed minimal or no improvement (<15%). These findings suggest that NAC may be a potentially promising, well-tolerated treatment option for patients with CSBD, including those who have failed more traditional therapies.

RTg(TauP301L)4510 mice exhibit increased VGlut1 in hippocampal presynaptic glutamatergic vesicles and increased extracellular glutamate release.

The molecular pathways that contribute to the onset of symptoms in tauopathy models, including Alzheimer’s disease (AD), are difficult to distinguish because multiple changes can happen simultaneously at different stages of disease progression. Understanding early synaptic alterations and their supporting molecular pathways is essential to develop better pharmacological targets to treat AD. Here, we focus on an early onset rTg(TauP301L)4510 tauopathy mouse model that exhibits hyperexcitability in hippocampal neurons of adult mice that is correlated with presynaptic changes and increased extracellular glutamate levels. However, it is not clear if increased extracellular glutamate is caused by presynaptic changes alone, or if presynaptic changes are a contributing factor among other factors. To determine whether pathogenic tau alters presynaptic function and glutamate release, we studied cultured hippocampal neurons at 14–18 days in vitro (DIV) from animals of both sexes to measure presynaptic changes in tauP301L positive mice. We observed that presynaptic vesicles exhibit increased vesicular glutamate transporter 1 (VGlut1) using immunohistochemistry of fixed cells and an established pH-sensitive green fluorescent protein approach. We show that tauP301L positive neurons exhibit a 40% increase in VGlut1 per vesicle compared to tauP301L negative littermates. Further, we use the extracellular glutamate reporter iGluSnFR to show that increased VGlut1 per vesicle directly translates into a 40% increase in extracellular glutamate. Together, these results show that increased extracellular glutamate levels observed in tauP301L mice are not caused by increased vesicle exocytosis probability but rather are directly related to increased VGlut1 transporters per synaptic vesicle.

The role of glutamine synthetase expression and cystine/glutamate transporter (SLC7A11, xCT) in epilepsy pathogenesis of patients with supratentorial brain gliomas

Background. Epileptic seizures represent one of the leading clinical manifestations of glial brain tumors that develop on average in 51% of cases. In gliomas, epileptogenesis is quite complex and multifactorial. Objective: to study a role of the expressed glutamine synthetase enzyme, as well as cystine/glutamate transporter (xCT system, SLC7A11) in the pathogenesis of epilepsy developing in patients with cerebral gliomas. Material and methods. The study included 32 patients with supratentorial gliomas. The average age of the disease onset (diagnosis) was 50.69±18.01 years. All patients underwent inpatient examination and treatment at the Neurosurgery and Nervous Diseases Clinics of Kirov Military Medical Academy from 2018 to 2020. In all cases, a biopsy of tumor tissue was collected. Histological and immunohistochemical studies were performed (expression of glutamine synthetase and cystine/ glutamate transporter (SLC7A11, xCT)). Results. Significant (ANOVA p=0.027, Mann–Whitney U-test p=0.033) differences in the expression of cystine/glutamate transporter (xCT system, SLC7A11) were revealed, by showing the following median values (lower quartile; upper quartile): 50% (45; 75) in the group of patients with seizures, 40% (40; 50) in the seizure-free group. The expression level of glutamine synthetase did not significantly differ in the groups with and without seizures. Conclusion. The data obtained confirmed that one of the pathogenetic mechanisms for epilepsy development in patients with gliomas was related to highly expressed cystine/glutamate transporter (xCT system, SLC7A11) and, as a consequence, an increase in the extracellular glutamate level.

Metabolic Dysregulation in Acute SARS-CoV-2 Infection

The global pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), mostly presented with mild to moderate or no symptoms and patients having pre-existing metabolic disorders like diabetes, cardiovascular diseases, and obesity are at risk for severe and critical cases of infection. The metabolic landscape of COVID-19 and its association with disease severity has urged the need to understand how metabolic reprogramming occurs during the acute SARS-CoV-2 infection with the ultimate goal toward therapeutic intervention. Viral replication is dependent on extracellular carbon sources such as glucose and glutamine and induces metabolic alterations in host cell including host central carbon metabolism, nucleotide, fatty acids, and lipid synthesis that modulate viral pathogenesis and host response. SARSCoV-2 dysregulation of PI3K/Akt/mTOR and hypoxia-inducible factor 1 (HIF-1) signaling pathways in infected cells and affect mitochondrial functions. These pathways regulate glycolysis by altering glucose transporters (GLUTs) across cell membranes. The altered extracellular glucose, mannose, and glutamate levels could be due to dysregulated carbohydrate metabolism and mitochondrial function. Host cellular response following SARSCoV-2 infection identified a strong acute metabolic adaptation in the lung epithelial cells (Calu-3) by modulating central carbon metabolism and indicative of mitochondrial dysfunction that is also observed in severe COVID-19 patients. Glycolysis and glutaminolysis can be essential for virus replication, and hostbased metabolic strategies to inhibit viruses to weaken the viral replication by metabolic intervention could be an attractive antiviral therapy. Targeting these pathways with inhibitors such as MK2206 (Akt inhibitor) or 2-deoxy-D-glucose (2-DG; glycolysis inhibitor) can lower the viral burden in the cells in vitro.

Depolarization time and extracellular glutamate levels aggravate ultraearly brain injury after subarachnoid hemorrhage

Early brain injury after aneurysmal subarachnoid hemorrhage (SAH) worsens the neurological outcome. We hypothesize that a longer duration of depolarization and excessive release of glutamate aggravate neurological outcomes after SAH, and that brain hypothermia can accelerate repolarization and inhibit the excessive release of extracellular glutamate and subsequent neuronal damage. So, we investigated the influence of depolarization time and extracellular glutamate levels on the neurological outcome in the ultra-early phase of SAH using a rat injection model as Experiment 1 and then evaluated the efficacy of brain hypothermia targeting ultra-early brain injury as Experiment 2. Dynamic changes in membrane potentials, intracranial pressure, cerebral perfusion pressure, cerebral blood flow, and extracellular glutamate levels were observed within 30 min after SAH. A prolonged duration of depolarization correlated with peak extracellular glutamate levels, and these two factors worsened the neuronal injury. Under brain hypothermia using pharyngeal cooling after SAH, cerebral perfusion pressure in the hypothermia group recovered earlier than that in the normothermia group. Extracellular glutamate levels in the hypothermia group were significantly lower than those in the normothermia group. The early induction of brain hypothermia could facilitate faster recovery of cerebral perfusion pressure, repolarization, and the inhibition of excessive glutamate release, which would prevent ultra-early brain injury following SAH.

Disruption of Glutamate Homeostasis in the Brain of Rat Offspring Induced by Prenatal and Early Postnatal Exposure to Maternal High-Sugar Diet.

A high-calorie diet has contributed greatly to the prevalence of overweight and obesity worldwide for decades. These conditions also affect pregnant women and have a negative impact on the health of both the woman and the fetus. Numerous studies indicate that an unbalanced maternal diet, rich in sugars and fats, can influence the in utero environment and, therefore, the future health of the child. It has also been shown that prenatal exposure to an unbalanced diet might permanently alter neurotransmission in offspring. In this study, using a rat model, we evaluated the effects of a maternal high-sugar diet on the level of extracellular glutamate and the expression of key transporters crucial for maintaining glutamate homeostasis in offspring. Glutamate concentration was assessed in extracellular fluid samples collected from the medial prefrontal cortex and hippocampus of male and female offspring. Analysis showed significantly increased glutamate levels in both brain structures analyzed, regardless of the sex of the offspring. These changes were accompanied by altered expression of the EAAT1, VGLUT1, and xc− proteins in these brain structures. This animal study further confirms our previous findings that a maternal high-sugar diet has a detrimental effect on the glutamatergic system.

Group I mGluRs in Therapy and Diagnosis of Parkinson's Disease: Focus on mGluR5 Subtype.

Metabotropic glutamate receptors (mGluRs; members of class C G-protein-coupled receptors) have been shown to modulate excitatory neurotransmission, regulate presynaptic extracellular glutamate levels, and modulate postsynaptic ion channels on dendritic spines. mGluRs were found to activate myriad signalling pathways to regulate synapse formation, long-term potentiation, autophagy, apoptosis, necroptosis, and pro-inflammatory cytokines release. A notorious expression pattern of mGluRs has been evident in several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and schizophrenia. Among the several mGluRs, mGluR5 is one of the most investigated types of considered prospective therapeutic targets and potential diagnostic tools in neurodegenerative diseases and neuropsychiatric disorders. Recent research showed mGluR5 radioligands could be a potential tool to assess neurodegenerative disease progression and trace respective drugs’ kinetic properties. This article provides insight into the group I mGluRs, specifically mGluR5, in the progression and possible therapy for PD.

Riluzole prevents stress-induced spine plasticity in the hippocampus but mimics it in the amygdala

Stress elicits divergent patterns of structural plasticity in the amygdala and hippocampus. Despite these contrasting effects, at least one of the immediate consequences of stress – elevated levels of extracellular glutamate – is similar in both brain areas. This raises the possibility that the contrasting effects of stress on neuronal plasticity is shaped by differences in astrocytic glutamate clearance in these two brain areas. Although astrocytes play a key role in glutamate reuptake, past analyses of, and interventions against, stress-induced plasticity have focused largely on neurons. Hence, we tested the impact of riluzole, which potentiates glutamate clearance by astrocytic glutamate transporters, on principal neurons and astrocytes in the basal amygdala (BA) and hippocampal area CA1. Chronic immobilization stress reduced spine-density on CA1 pyramidal neurons of male rats. Riluzole, administered in the drinking water during chronic stress, prevented this decrease; but, the drug by itself had no effect. In contrast, the same chronic stress enhanced spine-density on BA principal neurons, and this effect, unlike area CA1, was not reversed by riluzole. Strikingly, riluzole treatment alone also caused spinogenesis in the BA. Thus, the same riluzole treatment that prevented the effect of stress on spines in the hippocampus, mimicked its effect in the amygdala. Further, chronic stress and riluzole alone decreased the neuropil volume occupied by astrocytes in both the BA and CA1 area. Riluzole treatment in stressed animals, however, did not reverse or further add to this reduction in either region. Thus, while the effects on astrocytes were similar, neuronal changes were distinct between the two areas following stress, riluzole and the two together. Therefore, similar to the impact of repeated stress, pharmacological potentiation of glutamate clearance, with or without stress, also leads to differential effects on dendritic spines in principal neurons of the amygdala and hippocampus. This highlights differences in the astrocytic glutamate reuptake machinery that are likely to have important functional consequences for stress-induced dysfunction, and its reversal, in two brain areas implicated in stress-related psychiatric disorders.

Disulfiram Produces Potent Anxiolytic-Like Effects Without Benzodiazepine Anxiolytics-Related Adverse Effects in Mice.

Disulfiram is an FDA approved drug for the treatment of alcoholism. The drug acts by inhibiting aldehyde dehydrogenase, an enzyme essential to alcohol metabolism. However, a recent study has demonstrated that disulfiram also potently inhibits the cytoplasmic protein FROUNT, a common regulator of chemokine receptor CCR2 and CCR5 signaling. Several studies have reported that chemokine receptors are associated with the regulation of emotional behaviors in rodents, such as anxiety. Therefore, this study was performed to clarify the effect of disulfiram on emotional behavior in rodents. The anxiolytic-like effects of disulfiram were investigated using an elevated plus-maze (EPM) test, a typical screening model for anxiolytics. Disulfiram (40 or 80 mg/kg) significantly increased the amount of time spent in the open arms of the maze and the number of open arm entries without affecting the total open arms entries. Similar results were obtained in mice treated with a selective FROUNT inhibitor, disulfiram-41 (10 mg/kg). These disulfiram-associated behavioral changes were similar to those observed following treatment with the benzodiazepine anxiolytic diazepam (1.5 mg/kg). Moreover, disulfiram (40 mg/kg) significantly and completely attenuated increased extracellular glutamate levels in the prelimbic-prefrontal cortex (PL-PFC) during stress exposure on the elevated open-platform. However, no effect in the EPM test was seen following administration of the selective aldehyde dehydrogenase inhibitor cyanamide (40 mg/kg). In contrast to diazepam, disulfiram caused no sedation effects in the open-field, coordination disorder on a rotarod, or amnesia in a Y-maze. This is the first report suggesting that disulfiram produces anxiolytic-like effects in rodents. We found that the presynaptic inhibitory effects on glutaminergic neurons in the PL-PFC may be involved in its underlying mechanism. Disulfiram could therefore be an effective and novel anxiolytic drug that does not produce benzodiazepine-related adverse effects, such as amnesia, coordination disorder, or sedation, as found with diazepam. We propose that the inhibitory activity of disulfiram against FROUNT function provides an effective therapeutic option in anxiety.

Activation of Extrasynaptic Kainate Receptors Drives Hilar Mossy Cell Activity.

Mossy cells (MCs) of the dentate gyrus are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation, and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission, and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.SIGNIFICANCE STATEMENT Hilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability in vivo and may also promote dentate gyrus hyperexcitability and epileptogenesis.

Blood Glutamate Scavenging With Pyruvate as a Novel Preventative and Therapeutic Approach for Depressive-Like Behavior Following Traumatic Brain Injury in a Rat Model.

Depression is a common and serious complication following traumatic brain injury (TBI). Both depression and TBI have independently been associated with pathologically elevated extracellular brain glutamate levels. In the setting of TBI, blood glutamate scavenging with pyruvate has been widely shown as an effective method to provide neuroprotection by reducing blood glutamate and subsequent brain glutamate levels. Here we evaluate pyruvate as a novel approach in the treatment and prevention of post-TBI depression-like behavior in a rat model. Rats were divided into five groups: (1) sham-operated control with pyruvate, (2) sham-operated control with placebo, (3) post-TBI with placebo, (4) post-TBI given preventative pyruvate, and (5) post-TBI treated with pyruvate. These groups had an equal number of females and males. Rats were assessed for depressive-like behavior, neurological status, and glutamate levels in the blood and brain. Post-TBI neurological deficits with concurrent elevations in glutamate levels were demonstrated, with peak glutamate levels 24 h after TBI. Following TBI, the administration of either prophylactic or therapeutic pyruvate led to reduced glutamate levels, improved neurologic recovery, and improved depressive-like behavior. Glutamate scavenging with pyruvate may be an effective prophylactic and therapeutic option for post-TBI depression by reducing associated elevations in brain glutamate levels.

TNF\u03b1-mediated necroptosis in brain endothelial cells as a potential mechanism of increased seizure susceptibility in mice following systemic inflammation

BackgroundSystemic inflammation is a potent contributor to increased seizure susceptibility. However, information regarding the effects of systemic inflammation on cerebral vascular integrity that influence neuron excitability is scarce. Necroptosis is closely associated with inflammation in various neurological diseases. In this study, necroptosis was hypothesized to be involved in the mechanism underlying sepsis-associated neuronal excitability in the cerebrovascular components (e.g., endothelia cells).MethodsLipopolysaccharide (LPS) was used to induce systemic inflammation. Kainic acid intraperitoneal injection was used to measure the susceptibility of the mice to seizure. The pharmacological inhibitors C87 and GSK872 were used to block the signaling of TNFα receptors and necroptosis. In order to determine the features of the sepsis-associated response in the cerebral vasculature and CNS, brain tissues of mice were obtained for assays of the necroptosis-related protein expression, and for immunofluorescence staining to identify morphological changes in the endothelia and glia. In addition, microdialysis assay was used to assess the changes in extracellular potassium and glutamate levels in the brain.ResultsSome noteworthy findings, such as increased seizure susceptibility and brain endothelial necroptosis, Kir4.1 dysfunction, and microglia activation were observed in mice following LPS injection. C87 treatment, a TNFα receptor inhibitor, showed considerable attenuation of increased kainic acid-induced seizure susceptibility, endothelial cell necroptosis, microglia activation and restoration of Kir4.1 protein expression in LPS-treated mice. Treatment with GSK872, a RIP3 inhibitor, such as C87, showed similar effects on these changes following LPS injection.ConclusionsThe findings of this study showed that TNFα-mediated necroptosis induced cerebrovascular endothelial damage, neuroinflammation and astrocyte Kir4.1 dysregulation, which may coalesce to contribute to the increased seizure susceptibility in LPS-treated mice. Pharmacologic inhibition targeting this necroptosis pathway may provide a promising therapeutic approach to the reduction of sepsis-associated brain endothelia cell injury, astrocyte ion channel dysfunction, and subsequent neuronal excitability.

DNA Methylation-Dependent Gene Expression Regulation of Glutamate Transporters in Cultured Radial Glial Cells.

Exposure to xenobiotics has a significant impact in brain physiology that could be liked to an excitotoxic process induced by a massive release of the main excitatory neurotransmitter, L-glutamate. Overstimulation of extra-synaptic glutamate receptors, mainly of the N-methyl-D-aspartate subtype leads to a disturbance of intracellular calcium homeostasis that is critically involved in neuronal death. Hence, glutamate extracellular levels are tightly regulated through its uptake by glial glutamate transporters. It has been observed that glutamate regulates its own removal, both in the short-time frame via a transporter-mediated decrease in the uptake, and in the long-term through the transcriptional control of its gene expression, a process mediated by glutamate receptors that involves the Ca2+/diacylglycerol-dependent protein kinase and the transcription factor Ying Yang 1. Taking into consideration that this transcription factor is a member of the Polycomb complex and thus, part of repressive and activating chromatin remodeling factors, it might direct the interaction of DNA methyltransferases or dioxygenases of methylated cytosines to their target sequences. Here we explored the role of dynamic DNA methylation in the expression and function of glial glutamate transporters. To this end, we used the well-characterized models of primary cultures of chick cerebellar Bergmann glia cells and a human retina-derived Müller glia cell line. A time and dose-dependent increase in global DNA methylation was evident upon glutamate exposure. Under hypomethylation conditions, the glial glutamate transporter protein levels and uptake activity were increased. These results favor the notion that a dynamic DNA methylation program triggered by glutamate in glial cells modulates one of its major functions: glutamate removal.

Early Life Stress Induces Different Behaviors in Adolescence and Adulthood May Related With Abnormal Medial Prefrontal Cortex Excitation/Inhibition Balance.

Early life stress is thought to be a risk factor for emotional disorders, particularly depression and anxiety. Although the excitation/inhibition (E/I) imbalance has been implicated in neuropsychiatric disorders, whether early life stress affects the E/I balance in the medial prefrontal cortex at various developmental stages is unclear. In this study, rats exposed to maternal separation (MS) that exhibited a well-established early life stress paradigm were used to evaluate the E/I balance in adolescence (postnatal day P43–60) and adulthood (P82–100) by behavior tests, whole-cell recordings, and microdialysis coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. First, the behavioral tests revealed that MS induced both anxiety- and depressive-like behaviors in adolescent rats but only depressive-like behavior in adult rats. Second, MS increased the action potential frequency and E/I balance of synaptic transmission onto L5 pyramidal neurons in the prelimbic (PrL) brain region of adolescent rats while decreasing the action potential frequency and E/I balance in adult rats. Finally, MS increases extracellular glutamate levels and decreased the paired-pulse ratio of evoked excitatory postsynaptic currents (EPSCs) of pyramidal neurons in the PrL of adolescent rats. In contrast, MS decreased extracellular glutamate levels and increased the paired-pulse ratio of evoked EPSCs of pyramidal neurons in the PrL of adult rats. The present results reveal a key role of E/I balance in different MS-induced disorders may related to the altered probability of presynaptic glutamate release at different developmental stages.

Defects in Mouse Cortical Glutamate Uptake Can Be Unveiled In Vivo by a Two-in-One Quantitative Microdialysis.

Extracellular glutamate levels are maintained low by efficient transporters, whose dysfunction can cause neuronal hyperexcitability, excitotoxicity, and neurological disease. While many methods estimate glutamate uptake in vitro/ex vivo, a limited number of techniques address glutamate transport in vivo. Here, we used in vivo microdialysis in a two-in-one approach combining reverse dialysis of isotopic glutamate to measure uptake ability and zero-flow (ZF) methods to quantify extracellular glutamate levels. The complementarity of both techniques is discussed on methodological and anatomical basis. We used a transgenic mouse model of human disease, expressing low levels of the EAAT-2/GLT1 glutamate transporter, to validate our approach in a relevant animal model. As expected, isotopic analysis revealed an overall decrease in glutamate uptake, while the ZF method unveiled higher extracellular glutamate levels in these mice. We propose a sensitive and expedite two-in-one microdialysis approach that is sufficiently robust to reveal significant differences in neurotransmitter uptake and extracellular levels through the analysis of a relatively low number of animals.

Long-term exposure to high glucose induces changes in the expression of AMPA receptor subunits and glutamate transmission in primary cultured cortical neurons

Hyperglycemia, which occurs under the diabetic conditions, induces serious diabetic complications. Diabetic encephalopathy has been defined as one of the major complications of diabetes, and is characterized by neurochemical and neurodegenerative changes. However, little is known about the effect of long-term exposure to high glucose on neuronal cells. In the present study, we showed that exposure to glutamate (100 mM) for 7 days induced toxicity in primary cortical neurons using the MTT assay. Additionally, high glucose increased the sensitivity of AMPA- or NMDA-induced neurotoxicity, and decreased extracellular glutamate levels in primary cortical neurons. In Western blot analyses, the protein levels of the GluA1 and GluA2 subunits of the AMPA receptor as well as synaptophysin in neurons treated with high glucose were significantly increased compared with the control (25 mM glucose). Therefore, long-term exposure to high glucose induced neuronal death through the disruption of glutamate homeostasis.

Astrocytes modulate extracellular neurotransmitter levels and excitatory neurotransmission in dorsolateral striatum via dopamine D2 receptor signaling.

Astrocytes provide structural and metabolic support of neuronal tissue, but may also be involved in shaping synaptic output. To further define the role of striatal astrocytes in modulating neurotransmission we performed in vivo microdialysis and ex vivo slice electrophysiology combined with metabolic, chemogenetic, and pharmacological approaches. Microdialysis recordings revealed that intrastriatal perfusion of the metabolic uncoupler fluorocitrate (FC) produced a robust increase in extracellular glutamate levels, with a parallel and progressive decline in glutamine. In addition, FC significantly increased the microdialysate concentrations of dopamine and taurine, but did not modulate the extracellular levels of glycine or serine. Despite the increase in glutamate levels, ex vivo electrophysiology demonstrated a reduced excitability of striatal neurons in response to FC. The decrease in evoked potentials was accompanied by an increased paired pulse ratio, and a reduced frequency of spontaneous excitatory postsynaptic currents, suggesting that FC depresses striatal output by reducing the probability of transmitter release. The effect by FC was mimicked by chemogenetic inhibition of astrocytes using Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) targeting GFAP, and by the glial glutamate transporter inhibitor TFB-TBOA. Both FC- and TFB-TBOA-mediated synaptic depression were inhibited in brain slices pre-treated with the dopamine D2 receptor antagonist sulpiride, but insensitive to agents acting on presynaptic glutamatergic autoreceptors, NMDA receptors, gap junction coupling, cannabinoid 1 receptors, µ-opioid receptors, P2 receptors or GABAA receptors. In conclusion, our data collectively support a role for astrocytes in modulating striatal neurotransmission and suggest that reduced transmission after astrocytic inhibition involves dopamine.

Reduction of glutamate neurotoxicity: A novel therapeutic approach for Niemann-Pick disease, type C1

Niemann-Pick disease, type C1 is a progressive, lethal, neurodegenerative disorder due to endolysosomal storage of unesterified cholesterol. Cerebellar ataxia, as a result of progressive loss of cerebellar Purkinje neurons, is a major symptom of Nieman-Pick disease, type C1. Comparing single cell RNAseq data from control (Npc1+/+) and mutant (Npc1−/−) mice, we observed significantly decreased expression of Slc1a3 in Npc1−/− astrocytes. Slc1a3 encodes a glutamate transporter (GLAST, EAAT1) which functions to decrease glutamate concentrations in the post synaptic space after neuronal firing. Glutamate is an excitatory neurotransmitter and elevated extracellular levels of glutamate can be neurotoxic. Impaired EAAT1 function underlies type-6 episodic ataxia, a rare disorder with progressive cerebellar dysfunction, thus suggesting that impaired glutamate uptake in Niemann-Pick disease, type C1 could contribute to disease progression. We now show that decreased expression of Slc1a3 in Npc1−/− mice has functional consequences that include decreased surface protein expression and decreased glutamate uptake by Npc1−/− astrocytes. To test whether glutamate neurotoxicity plays a role in Niemann-Pick disease, type C1 progression, we treated NPC1 deficient mice with ceftriaxone and riluzole. Ceftriaxone is a β-lactam antibiotic that is known to upregulate the expression of Slc1a2, an alternative glial glutamate transporter. Although ceftriaxone increased Slc1a2 expression, we did not observe a treatment effect in NPC1 mutant mice. Riluzole is a glutamate receptor antagonist that inhibits postsynaptic glutamate receptor signaling and reduces the release of glutamate. We found that treatment with riluzole increased median survival in Npc1−/− by 12%. Given that riluzole is an approved drug for the treatment of amyotrophic lateral sclerosis, repurposing of this drug may provide a novel therapeutic approach to decrease disease progression in Niemann-Pick disease type, C1 patients.