Extracellular Glutamate Levels Research Articles

Ketamine metabolism via hepatic CYP450 isoforms contributes to its sustained antidepressant actions

(R,S)-ketamine (ketamine) has rapid and sustained antidepressant (AD) efficacy at sub-anesthetic doses in depressed patients. A metabolite of ketamine, including (2R,6R)-hydroxynorketamine ((6)-HNKs) has been reported to exert antidepressant actions in rodent model of anxiety/depression. To further understand the specific role of ketamine's metabolism in the AD actions of the drug, we evaluated the effects of inhibiting hepatic cytochrome P450 enzymes on AD responses. We assessed whether pre-treatment with fluconazole (10 and 20 mg/kg, i. p.) 1 h prior to ketamine or HNKs (10 mg/kg, i. p.) administration would alter behavioral and neurochemical actions of the drugs in male BALB/cJ mice with a highly anxious phenotype. Extracellular microdialysate levels of glutamate and GABA (Gluext, GABAext) were also measured in the medial prefrontal cortex (mPFC). Pre-treatment with fluconazole altered the pharmacokinetic profile of ketamine, by increasing both plasma and brain levels of ketamine and (R,S)-norketamine, while robustly reducing those of (6)-HNKs. At 24 h post-injection (t24 h), fluconazole prevented the sustained AD-like response of ketamine responses in the forced swim test and splash test, as well as the enhanced cortical GABA levels produced by ketamine. A single (2R,6R)-HNK administration resulted in prevention of the effects of fluconazole on the antidepressant-like activity of ketamine in mice. Overall, these findings are consistent with an essential contribution of (6)-HNK to the sustained antidepressant-like effects of ketamine and suggest potential interactions between pharmacological CYPIs and ketamine during antidepressant treatment in patients.

MGluR5 positive allosteric modulation prevents MK-801 induced increases in extracellular glutamate in the rat medial prefrontal cortex

Potentiation of metabotropic glutamate receptor subtype 5 (mGluR5) function produces antipsychotic-like and pro-cognitive effects in animal models of schizophrenia and can reverse cognitive deficits induced by N-methyl-D-aspartate type glutamate receptor (NMDAR) antagonists. However, it is currently unknown if mGluR5 positive allosteric modulators (PAMs) can modulate NMDAR antagonist-induced alterations in extracellular glutamate levels in regions underlying these cognitive and behavioral effects, such as the medial prefrontal cortex (mPFC). We therefore assessed the ability of the mGluR5 PAM, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), to reduce elevated extracellular glutamate levels induced by the NMDAR antagonist, dizocilpine (MK-801), in the mPFC. Male Sprague-Dawley rats were implanted with a guide cannula aimed at the mPFC and treated for ten consecutive days with MK-801 and CDPPB or their corresponding vehicles. CDPPB or vehicle was administered thirty minutes before MK-801 or vehicle each day. On the final day of treatment, in vivo microdialysis was performed, and samples were collected every thirty minutes to analyze extracellular glutamate levels. Compared to animals receiving only vehicle, administration of MK-801 alone significantly increased extracellular levels of glutamate in the mPFC. This effect was not observed in animals administered CDPPB before MK-801, nor in those administered CDPPB alone, indicating that CDPPB decreased extracellular glutamate release stimulated by MK-801. Results indicate that CDPPB attenuates MK-801 induced elevations in extracellular glutamate in the mPFC. This effect of CDPPB may underlie neurochemical adaptations associated with the pro-cognitive effects of mGluR5 PAMs in rodent models of schizophrenia.

Fluorocitrate inhibition of astrocytes reduces nicotine self-administration and alters extracellular levels of glutamate and dopamine within the nucleus accumbens in male wistar rats

Emerging evidence suggests an important role of astrocytes in mediating behavioral and molecular effects of commonly misused drugs. Passive exposure to nicotine alters molecular, morphological, and functional properties of astrocytes. However, a potential involvement of astrocytes in nicotine reinforcement remains largely unexplored. The overall hypothesis tested in the current study is that astrocytes play a critical role in nicotine reinforcement. Protein levels of the astrocyte marker glial fibrillary acidic protein (GFAP) were examined in key mesocorticolimbic regions following chronic nicotine intravenous self-administration. Fluorocitrate, a metabolic inhibitor of astrocytes, was tested for its effects on behaviors related to nicotine reinforcement and relapse. Effects of fluorocitrate on extracellular neurotransmitter levels, including glutamate, GABA, and dopamine, were determined with microdialysis. Chronic nicotine intravenous self-administration increased GFAP expression in the nucleus accumbens core (NACcr), but not other key mesocorticolimbic regions, compared to saline intravenous self-administration. Both intra-ventricular and intra-NACcr microinjection of fluorocitrate decreased nicotine self-administration. Intra-NACcr fluorocitrate microinjection also inhibited cue-induced reinstatement of nicotine seeking. Local perfusion of fluorocitrate decreased extracellular glutamate levels, elevated extracellular dopamine levels, but did not alter extracellular GABA levels in the NACcr. Fluorocitrate did not alter basal locomotor activity. These results indicate that nicotine reinforcement upregulates the astrocyte marker GFAP expression in the NACcr, metabolic inhibition of astrocytes attenuates nicotine reinforcement and relapse, and metabolic inhibition of astrocytes disrupts extracellular dopamine and glutamate transmission. Overall, these findings suggest that astrocytes play an important role in nicotine reinforcement and relapse, potentially through regulation of extracellular glutamate and dopamine neurotransmission.

Effect of ACEA 1021 on Neurotoxicity Induced by Dexamethasone — Initial Behavioral Study

Considerable evidence suggests that glucocorticoids (GCs) play an important role in neurodegeneration. Chronic elevated levels of GCs can result in neuronal degeneration of the hippocampal piramidal neurons, which are paralleled by cognitive deficits. Moreover, GCs potentiate stress or ischemia-induced accumulation of excitatory amino acids (EAA) in the extracellular space of hippocampus. ACEA 1021 (licostinel), a selective antagonist of the N-methyl-D-aspartate (NMDA) receptor, has been reported to prevent the excitotoxic action of high extracellular glutamate levels. The aim of this study was to investigate the effect of ACEA 1021 on neurotoxic effect of dexamethasone (DEX - a synthetic GCs receptor agonist). The experiments were carried out on Albino Swiss mice (25-30 g). ACEA 1021, at the doses: 1.25 and 2.5 mg/kg/day, ip, was administered 15 min before DEX (16 mg/kg/day, ip). The long-term memory acquisition (passive avoidance test) and the motor performance (“chimney” test) were evaluated 14 days after the drugs administration. The prolongation of climbing time in the “chimney” test and decrease of the retention time in the memory task of mice treated with DEX for 14 days. In mice treated with DEX for 14 days, ACEA 1021 at the both doses reduced the climbing time in the “chimney” test, at the dose of 1.25 mg/kg improved memory acquisition. The above findings suggest that ACEA 1021 could prevent the neurotoxic effects induced by DEX, but further study needs to be carried out to explain this effect.

Anesthetic Sevoflurane Induces Enlargement of Dendritic Spine Heads in Mouse Neurons via Tau-Dependent Mechanisms.

Sevoflurane induces neuronal dysfunction and cognitive impairment. However, the underlying mechanism remains largely to be determined. Tau, cyclophilin D, and dendritic spine contribute to cognitive function. But whether changes in dendritic spines are involved in the effects of sevoflurane and the potential association with tau and cyclophilin D is not clear. We harvested hippocampal neurons from wild-type mice, tau knockout mice, and cyclophilin D knockout mice. We treated these neurons with sevoflurane at day in vitro 7 and measured the diameter of dendritic spine head and the number of dendritic spines. Moreover, we determined the effects of sevoflurane on the expression of excitatory amino acid transporter 3 (EAAT3), extracellular glutamate levels, and miniature excitatory postsynaptic currents (mEPSCs). Finally, we used lithium, cyclosporine A, and overexpression of EAAT3 in the interaction studies. Sevoflurane-induced tau phosphgorylation increased the diameter of dendritic spine head and decreased the number of dendritic spines in neurons harvested from wild-type and cyclophilin D knockout mice, but not tau knockout mice. Sevoflurane decreased the expression of EAAT3, increased extracellular glutamate levels, and decreased the frequency of mEPSCs in the neurons. Overexpression of EAAT3 mitigated the effects of sevoflurane on dendritic spines. Lithium, but not cyclosporine A, attenuated the effects of sevoflurane on dendritic spines. Lithium also inhibited the effects of sevoflurane on EAAT3 expression and mEPSCs. These data suggest that sevoflurane induces a tau phosphorylation-dependent demtrimental effect on dendritic spine via decreasing EAAT3 expression and increasing extracellular glutamate levels, leading to neuronal dysfunction.

Induction of glioblastoma invasion triggered by system Xc−-mediated glutamate release

Abstract Backgrounds Glioblastoma (GBM) is a highly aggressive brain cancer associated with poor prognosis, primarily attributed to its profound invasive characteristics. Glutamate is the main cause of invasion, and invasion is promoted by system Xc− (cystine/glutamate antiporter), which is highly expressed in GBM. To date, no studies have examined the relationship between invasion and the specific downregulation of system Xc− (xCT or SLC7A11) using shRNA in GBM. Objective We aimed to determine the effect of a specific knockdown system, Xc−, in GBM using short hairpin RNA (shRNA) rather than pharmacological approaches. Results Invasion was inhibited in GBM cells treated with sulfasalazine, a system Xc− inhibitor. Our experiments validated a reduction in extracellular glutamate concentration following sulfasalazine treatment, without affecting GBM proliferation or calcium response. However, the efficacy of pharmacological methods is hindered by nonspecific effects and the prevalence of multiple side effects. Therefore, we specifically targeted the system Xc− molecule through shRNA. Downregulation using shRNA demonstrated decreased invasion and extracellular glutamate levels, without affecting the calcium response and proliferation. Conclusion The targeted inhibition of system Xc− using shRNA yields a notable reduction in GBM invasion.

Long-term intermittent theta burst stimulation enhanced hippocampus-dependent memory by regulating hippocampal theta oscillation and neurotransmitter levels in healthy rats

Intermittent theta burst stimulation (iTBS), an updated pattern of high-frequency repetitive transcranial magnetic stimulation, is a potential candidate for improving memory. The hippocampus has been shown to be involved in the memory-enhancing effect induced by iTBS. However, it remains largely unknown whether this effect is achieved by regulating hippocampal theta oscillation and neurotransmitters gamma-aminobutyric acid (GABA) and glutamate, which are strongly related to memory. Thus, we investigated the effect of 14 days of iTBS on hippocampus-dependent memory and further explored the roles of hippocampal theta oscillation and neurotransmitters GABA and glutamate in this effect. We found that compared to sham iTBS, real iTBS enhanced hippocampus-dependent memory measured by hole-board test and object place recognition test. Further, real iTBS increased the density of c-Fos positive neurons and normalized power of theta oscillation in the dorsal hippocampus (dHip) compared to sham iTBS. Interestingly, we observed a decrease in the level of extracellular GABA and an increase in the level of extracellular glutamate in the dHip after real iTBS. Our results suggest that long-term iTBS improved hippocampus-dependent memory, which may be attributed to the enhancement of theta oscillation and altered levels of extracellular GABA and glutamate in the dHip.

Cannabidiol Modifies the Glutamate Over-Release in Brain Tissue of Patients and Rats with Epilepsy: A Pilot Study.

Drug-resistant epilepsy (DRE) is associated with high extracellular levels of glutamate. Studies support the idea that cannabidiol (CBD) decreases glutamate over-release. This study focused on investigating whether CBD reduces the evoked glutamate release in cortical synaptic terminals obtained from patients with DRE as well as in a preclinical model of epilepsy. Synaptic terminals (synaptosomes) were obtained from the epileptic neocortex of patients with drug-resistant temporal lobe epilepsy (DR-TLE, n = 10) or drug-resistant extratemporal lobe epilepsy (DR-ETLE, n = 10) submitted to epilepsy surgery. Synaptosomes highly purified by Percoll-sucrose density gradient were characterized by confocal microscopy and Western blot. Synaptosomes were used to estimate the high KCl (33 mM)-evoked glutamate release in the presence of CBD at different concentrations. Our results revealed responsive tissue obtained from seven patients with DR-TLE and seven patients with DR-ETLE. Responsive tissue showed lower glutamate release (p < 0.05) when incubated with CBD at low concentrations (less than 100 µM) but not at higher concentrations. Tissue that was non-responsive to CBD (DR-TLE, n = 3 and DR-ELTE, n = 3) showed high glutamate release despite CBD exposure at different concentrations. Simultaneously, a block of the human epileptic neocortex was used to determine its viability through whole-cell and extracellular electrophysiological recordings. The electrophysiological evaluations supported that the responsive and non-responsive human epileptic neocortices used in the present study exhibited proper neuronal viability and stability to acquire electrophysiological responses. We also investigated whether the subchronic administration of CBD could reduce glutamate over-release in a preclinical model of temporal lobe epilepsy. Administration of CBD (200 mg/kg, p.o. every 24 h for 7 days) to rats with lithium-pilocarpine-evoked spontaneous recurrent seizures reduced glutamate over-release in the hippocampus. The present study revealed that acute exposure to low concentrations of CBD can reduce the glutamate over-release in synaptic terminals obtained from some patients with DRE. This effect is also evident when applied subchronically in rats with spontaneous recurrent seizures. An important finding was the identification of a group of patients that were non-responsive to CBD effects. Future studies are essential to identify biomarkers of responsiveness to CBD to control DRE.

Amelioration of morphine withdrawal syndrome by systemic and intranasal administration of mesenchymal stem cell-derived secretome in preclinical models of morphine dependence.

Morphine is an opiate commonly used in the treatment of moderate to severe pain. However, prolonged administration can lead to physical dependence and strong withdrawal symptoms upon cessation of morphine use. These symptoms can include anxiety, irritability, increased heart rate, and muscle cramps, which strongly promote morphine use relapse. The morphine-induced increases in neuroinflammation, brain oxidative stress, and alteration of glutamate levels in the hippocampus and nucleus accumbens have been associated with morphine dependence and a higher severity of withdrawal symptoms. Due to its rich content in potent anti-inflammatory and antioxidant factors, secretome derived from human mesenchymal stem cells (hMSCs) is proposed as a preclinical therapeutic tool for the treatment of this complex neurological condition associated with neuroinflammation and brain oxidative stress. Two animal models of morphine dependence were used to evaluate the therapeutic efficacy of hMSC-derived secretome in reducing morphine withdrawal signs. In the first model, rats were implanted subcutaneously with mini-pumps which released morphine at a concentration of 10 mg/kg/day for seven days. Three days after pump implantation, animals were treated with a simultaneous intravenous and intranasal administration of hMSC-derived secretome or vehicle, and withdrawal signs were precipitated on day seven by i.p. naloxone administration. In this model, brain alterations associated with withdrawal were also analyzed before withdrawal precipitation. In the second animal model, rats voluntarily consuming morphine for three weeks were intravenously and intranasally treated with hMSC-derived secretome or vehicle, and withdrawal signs were induced by morphine deprivation. In both animal models secretome administration induced a significant reduction of withdrawal signs, as shown by a reduction in a combined withdrawal score. Secretome administration also promoted a reduction in morphine-induced neuroinflammation in the hippocampus and nucleus accumbens, while no changes were observed in extracellular glutamate levels in the nucleus accumbens. Data presented from two animal models of morphine dependence suggest that administration of secretome derived from hMSCs reduces the development of opioid withdrawal signs, which correlates with a reduction in neuroinflammation in the hippocampus and nucleus accumbens.

Mitragynine inhibits hippocampus neuroplasticity and its molecular mechanism

BackgroundMitragynine (MIT), the primary indole alkaloid of kratom (Mitragyna speciosa), has been associated with addictive and cognitive decline potentials. In acute studies, MIT decreases spatial memory and inhibits hippocampal synaptic transmission in long-term potentiation (LTP). This study investigated the impacts of 14-day MIT treatment on hippocampus synaptic transmission and its possible underlying mechanisms.MethodsUnder urethane anesthesia, field excitatory post-synaptic potentials (fEPSP) of the hippocampal CA1 region were recorded in the Sprague Dawley (SD) rats that received MIT (1, 5, and 10 mg/kg), morphine (MOR) 5 mg/kg, or vehicle (ip). The effects of the treatments on basal synaptic transmission, paired-pulse facilitation (PPF), and LTP were assessed in the CA1 region. Analysis of the brain's protein expression linked to neuroplasticity was then performed using a western blot.ResultsThe baseline synaptic transmission's amplitude was drastically decreased by MIT at 5 and 10 mg/kg doses, although the PPF ratio before TBS remained unchanged, the PPF ratio after TBS was significantly reduced by MIT (10 mg/kg). Strong and persistent inhibition of LTP was generated in the CA1 region by MIT (5 and 10 mg/kg) doses; this effect was not seen in MIT (1 mg/kg) treated rats. In contrast to MIT (1 mg/kg), MIT (5 and 10 mg/kg) significantly raised the extracellular glutamate levels. After exposure to MIT, GluR-1 receptor expression remained unaltered. However, NMDAε2 receptor expression was markedly downregulated. The expression of pCaMKII, pERK, pCREB, BDNF, synaptophysin, PSD-95, Delta fosB, and CDK-5 was significantly downregulated in response to MIT (5 and 10 mg/kg) exposure, while MOR (5 mg/kg) significantly raised synaptophysin and Delta fosB expression.ConclusionFindings from this work reveal that a smaller dose of MIT (1 mg/kg) poses no risk to hippocampal synaptic transmission. Alteration in neuroplasticity-associated proteins may be a molecular mechanism for MIT (5 and 10 mg/kg)-induced LTP disruption and cognitive impairments. Data from this work posit that MIT acted differently from MOR on neuroplasticity and its underlying mechanisms.Graphical abstract

The Key Role of Astrocytes in Amyotrophic Lateral Sclerosis and Their Commitment to Glutamate Excitotoxicity.

In the last two decades, there has been increasing evidence supporting non-neuronal cells as active contributors to neurodegenerative disorders. Among glial cells, astrocytes play a pivotal role in driving amyotrophic lateral sclerosis (ALS) progression, leading the scientific community to focus on the "astrocytic signature" in ALS. Here, we summarized the main pathological mechanisms characterizing astrocyte contribution to MN damage and ALS progression, such as neuroinflammation, mitochondrial dysfunction, oxidative stress, energy metabolism impairment, miRNAs and extracellular vesicles contribution, autophagy dysfunction, protein misfolding, and altered neurotrophic factor release. Since glutamate excitotoxicity is one of the most relevant ALS features, we focused on the specific contribution of ALS astrocytes in this aspect, highlighting the known or potential molecular mechanisms by which astrocytes participate in increasing the extracellular glutamate level in ALS and, conversely, undergo the toxic effect of the excessive glutamate. In this scenario, astrocytes can behave as "producers" and "targets" of the high extracellular glutamate levels, going through changes that can affect themselves and, in turn, the neuronal and non-neuronal surrounding cells, thus actively impacting the ALS course. Moreover, this review aims to point out knowledge gaps that deserve further investigation.

The antidepressant-like and glioprotective effects of the Y2 receptor antagonist SF-11 in the astroglial degeneration model of depression in rats: Involvement of glutamatergic inhibition

In this study, we explored the potential antidepressant-like properties of the brain-penetrant Y2 receptor (Y2R) antagonist SF-11 [N-(4-ethoxyphenyl)− 4-(hydroxydiphenylmethyl)− 1-piperidinecarbothioamide] in the astroglial degeneration model of depression with an emphasis on checking the possible mechanisms implicated in this antidepressant-like effect. The model of depression relies on the loss of astrocytes in the medial prefrontal cortex (mPFC) in Sprague-Dawley rats after administering the gliotoxin L-alpha-aminoadipic acid (L-AAA). SF-11 was administered intraperitoneally (i.p.) once (10 mg/kg) or for three consecutive days (10 mg/kg/day), and the effects of L-AAA and SF-11 injected alone or in combination were investigated using the forced swim test (FST), sucrose intake test (SIT), Western blotting, immunohistochemical staining, and microdialysis. SF-11 produced an antidepressant-like effect after single or three-day administration in rats subjected to astrocyte impairment, as demonstrated by the FST and SIT, respectively. Immunoblotting and immunohistochemical analyses showed that SF-11 reversed the L-AAA-induced astrocyte cell death in the mPFC, suggesting it is glioprotective. Microdialysis studies showed that SF-11 decreased extracellular glutamate (Glu) levels compared to basal value when administered alone and compared to the basal value and control group in LAAA-treated rats. The results from immunoblotting analysis indicated the involvement of Y2Rs in the astrocyte ablation model of depression and the antidepressant-like effect of SF-11. In addition, we observed the participation of the caspase-3 apoptotic pathway in the mechanism of gliotoxin action induced by L-AAA. These findings demonstrate that SF-11, a Y2R antagonist, elicited a rapid antidepressant-like response, possibly linked to its ability to inhibit glutamatergic neurotransmission.

The role of extracellular glutamate homeostasis dysregulated by astrocyte in epileptic discharges: a model evidence.

Glutamate (Glu) is a predominant excitatory neurotransmitter that acts on glutamate receptors to transfer signals in the central nervous system. Abnormally elevated extracellular glutamate levels is closely related to the generation and transition of epileptic seizures. However, there lacks of investigation regarding the role of extracellular glutamate homeostasis dysregulated by astrocyte in neuronal epileptic discharges. According to this, we propose a novel neuron-astrocyte computational model (NAG) by incorporating extracellular Glu concentration dynamics from three aspects of regulatory mechanisms: (1) the Gluuptake through astrocyte EAAT2; (2) the binding and release Glu via activating astrocyte mGluRs; and (3) the Glu free diffusion in the extracellular space. Then the proposed model NAG is analyzed theoretically and numerically to verify the effect of extracellular Glu homeostasis dysregulated by such three regulatory mechanisms on neuronal epileptic discharges. Our results demonstrate that the neuronal epileptic discharges can be aggravated by the downregulation expression of EAAT2, the aberrant activation of mGluRs, and the elevated Glu levels in extracellular micro-environment; as well as various discharge states (including bursting, mixed-mode spiking, and tonic firing) can be transited by their combination. Furthermore, we find that such factors can also alter the bifurcation threshold for the generation and transition of epileptic discharges. The results in this paper can be helpful for researchers to understand the astrocyte role in modulating extracellular Glu homeostasis, and provide theoretical basis for future related experimental studies.

Imbalance of glutamatergic and GABAergic neurotransmission in audiogenic seizure-susceptible Leucine-rich glioma-inactivated 1 (Lgi1)-mutant rats

Leucine-rich glioma-inactivated 1 (LGI1) was identified as a causative gene of autosomal dominant lateral temporal lobe epilepsy. We previously reported that Lgi1-mutant rats carrying a missense mutation (L385R) showed audiogenic seizure-susceptibility. To explore the pathophysiological mechanisms underlying Lgi1-related epilepsy, we evaluated changes in glutamate and GABA release in Lgi1-mutant rats. Acoustic priming (AP) for audiogenic seizure-susceptibility was performed by applying intense sound stimulation (130 dB, 10 kHz, 5 min) on postnatal day 16. Extracellular glutamate and GABA levels in the hippocampus CA1 region were evaluated at 8 weeks of age, using in vivo microdialysis techniques. Under naïve conditions without AP, glutamate and GABA release evoked by high-K+ depolarization was more prominent in Lgi1-mutant than in wild-type (WT) rats. The AP treatment on day 16 significantly increased basal glutamate levels and depolarization-induced glutamate release both in Lgi1-mutant and WT rats, yielding greater depolarization-induced glutamate release in Lgi1-mutant rats. On the other hand, the AP treatment enhanced depolarization-induced GABA release only in WT rats, and not in Lgi1-mutant rats, illustrating reduced GABAergic neurotransmission in primed Lgi1-mutant rats. The present results suggest that enhanced glutamatergic and reduced GABAergic neurotransmission are involved in the audiogenic seizure-susceptibility associated with Lgi1-mutation.

An in vitro study on the stimulatory effects of extracellular glutamate on astrocytes.

In our previous research, it was found that the cerebrospinal fluid had higher levels of glutamate, astrocytes were stimulated and released pro-inflammatory factors in a subarachnoid hemorrhage model. Glutamate is a neurotransmitter produced in abundance by excitatory neurons in the central nervous system, residual glutamate can cause neurotoxicity. Recent studies indicate that most glutamate is absorbed by astrocytes, to optimize neurological functions and prevent excitotoxicity. However, it is still unclear if astrocytes could be stimulated by glutamate, and the concentration range of glutamate transportable by astrocytes. Thus, further research is necessary. This study aimed to clarify these scientific questions by stimulating primary astrocytes at different glutamate concentrations (0, 25, 50, and 100 µM) for 24h. The results showed that glutamate induced an increased response in astrocytes, the protein levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were upregulated in treatment groups with 50 and 100 µM. Additionally, the protein expression of complement component 3 (C3) significantly increased following glutamate stimulation (50 and 100 µM) for 24h. Furthermore, the supernatant of the 100 µM treatment group significantly decreased the viability of HT-22 (an immortalized mouse hippocampal neuronal cell line). In summary, our results indicate that increased extracellular glutamate levels can activate astrocytes and promote pro-inflammatory factor production. Moreover, the concentration range of glutamate transported by astrocytes is approximately less than 50-100 µM. Therefore, our study suggests that experimental antagonization of glutamate excitotoxicity is feasible.

A first-in-human pharmacodynamic evaluation of dual polyamine depletion for patients with high-grade gliomas.

TPS2082 Background: No drug has improved survival for patients with high-grade gliomas (HGGs) since temozolomide in 2005. Relatively little is known about the live response of in situ gliomas to therapeutic intervention. Phase 0 studies that leverage access to the live human glioma in situ, including via microdialysis to sample the live glioma microenvironment, provide an attractive and feasible solution to this problem. We performed intraoperative microdialysis to evaluate the extracellular metabolome of divergent regions in 15 HGG resections. Our data revealed a conserved metabolic signature of HGGs and a novel glioma-associated metabolite, guanidinoacetate (GAA). Pathways enriched in this tumor signature included methionine and polyamine metabolism. Although GAA is typically converted to creatine, we hypothesize that the high flux of ornithine through ornithine decarboxylase (ODC) results in excess GAA accumulation. As such, in a Phase 0 trial, we will evaluate in situ glioma responses to polyamine depletion via ODC inhibition (Difluoromethylornithine, DFMO), with or without blockade of polyamine uptake (AMXT 1501), to identify candidate extracellular biomarkers of target engagement and cytotoxicity. Methods: Patients will be recruited who are undergoing a clinically indicated subtotal resection for a known or suspected high-grade glioma (NCT05717153). At the end of the surgery, two microdialysis catheters will be implanted for post-operative microdialysis, one in residual tumor and one in brain adjacent to tumor for an internal control. Beginning on post-operative day (POD) 1, patients will be randomized to one of the following three groups, including administration of vehicle, DFMO (500 mg, PO, BID) and AMXT 1501 (600 mg, PO, BID): 1) DFMO + AMXT 1501 (POD1-5); 2) Vehicle (POD1–POD2), followed by DFMO + AMXT 1501 (POD3-POD5); 3) DFMO alone (POD1–POD2), followed by DFMO + AMXT 1501 (POD3- POD5). Throughout this time, extracellular metabolite levels of polyamines, guanidinoacetate, and glutamate will be quantified from microdialysate collected every two hours from intraoperatively implanted 100 kDA microdialysis catheters. The primary outcome of this study is to determine how polyamine depletion impacts extracellular intratumoral guanidinoacetate abundance. The secondary outcomes of this study are to (1) determine the impact of polyamine depletion on polyamine abundance and the global extracellular metabolome within live human gliomas, in situ, and (2) assess the feasibility of longitudinal microdialysis for pharmacodynamic analysis within live human gliomas in situ in the post-operative setting. Clinical trial information: NCT05717153 .

Intrahippocampal injection of a selective blocker of NMDA receptors containing the GluN2B subunit, Ro25-6981, increases glutamate neurotransmission and induces antidepressant-like effects.

Major depressive disorder (MDD) is a serious public health problem, as it is the most common psychiatric disorder worldwide. Antidepressant drugs increase adult hippocampal neurogenesis, which is required to induce some behavioral effects of antidepressants. Adult-born granule cells in the dentate gyrus (DG) and the glutamate receptors subunits 2 (GluN2B) subunit of N-methyl-D-aspartate (NMDA) ionotropic receptors play an important role in these effects. However, the precise neurochemical role of the GluN2B subunit of the NMDA receptor on adult-born GCs for antidepressant-like effects has yet to be elucidated. The present study aims to explore the contribution of the GluN2B-containing NMDA receptors in the ventral dentate gyrus (vDG) to the antidepressant drug treatment using a pharmacological approach. Thus, (αR)-(4-hydroxyphenyl)-(βS)-methyl-4-(phenylmethyl)-1-piperidinepropanol (Ro25-6981), a selective antagonist of the GluN2B subunit, was acutely administered locally into the ventral DG (vDG, 1 μg each side) following a chronic fluoxetine (18 mg/kg/day) treatment-known to increase adult hippocampal neurogenesis-in a mouse model of anxiety/depression. Responses in a neurogenesis-dependent task, the novelty suppressed feeding (NSF), and neurochemical consequences on extracellular glutamate and gamma-aminobutyric acid (GABA) levels in the vDG were measured. Here, we show a rapid-acting antidepressant-like effect of local Ro25-6981 administration in the NSF independent of fluoxetine treatment. Furthermore, we revealed a fluoxetine-independent increase in the glutamatergic transmission in the vDG. Our results suggest behavioral and neurochemical effects of GluN2B subunit independent of serotonin reuptake inhibition.

Dendrimer-enabled targeted delivery attenuates glutamate excitotoxicity and improves motor function in a rabbit model of cerebral palsy.

Glutamate carboxypeptidase II (GCPII), localized on the surface of astrocytes and activated microglia, regulates extracellular glutamate concentration in the central nervous system (CNS). We have previously shown that GCPII is upregulated in activated microglia in the presence of inflammation. Inhibition of GCPII activity could reduce glutamate excitotoxicity, which may decrease inflammation and promote a 'normal' microglial phenotype. 2-(3-Mercaptopropyl) pentanedioic acid (2-MPPA) is the first GCPII inhibitor that underwent clinical trials. Unfortunately, immunological toxicities have hindered 2-MPPA clinical translation. Targeted delivery of 2-MPPA specifically to activated microglia and astrocytes that over-express GCPII has the potential to mitigate glutamate excitotoxicity and attenuate neuroinflammation. In this study, we demonstrate that 2-MPPA when conjugated to generation-4, hydroxyl-terminated polyamidoamine (PAMAM) dendrimers (D-2MPPA) localize specifically in activated microglia and astrocytes only in newborn rabbits with cerebral palsy (CP), not in controls. D-2MPPA treatment led to higher 2-MPPA levels in the injured brain regions compared to 2-MPPA treatment, and the extent of D-2MPPA uptake correlated with the injury severity. D-2MPPA was more efficacious than 2-MPPA in decreasing extracellular glutamate level in ex vivo brain slices of CP kits, and in increasing transforming growth factor beta 1 (TGF-β1) level in primary mixed glial cell cultures. A single systemic intravenous dose of D-2MPPA on postnatal day 1 (PND1) decreased microglial activation and resulted in a change in microglial morphology to a more ramified form along with amelioration of motor deficits by PND5. These results indicate that targeted dendrimer-based delivery specifically to activated microglia and astrocytes can improve the efficacy of 2-MPPA by attenuating glutamate excitotoxicity and microglial activation.

Administration of the glutamate-modulating drug, riluzole, after stress prevents its delayed effects on the amygdala in male rats.

Extracellular glutamate levels are elevated across brain regions immediately after stress. Despite sharing common features in their genesis, the patterns of stress-induced plasticity that eventually take shape are strikingly different between these brain areas. While stress causes structural and functional deficits in the hippocampus, it has the opposite effect on the amygdala. Riluzole, an FDA-approved drug known to modulate glutamate release and facilitate glutamate clearance, prevents stress-induced deficits in the hippocampus. But whether the same drug is also effective in countering the opposite effects of stress in the amygdala remains unexplored. We addressed this question by using a rat model wherein even a single 2-h acute immobilization stress causes a delayed expression of anxiety-like behavior, 10 days later, alongside stronger excitatory synaptic connectivity in the basolateral amygdala (BLA). This temporal profile-several days separating the acute stressor and its delayed impact-allowed us to test if these effects can be prevented by administering riluzole in drinking water after acute stress. Poststress riluzole not only prevented the delayed increase in anxiety-like behavior on the elevated plus maze but also blocked the increase in spine density on BLA neurons 10 days later. Further, stress-induced increase in the frequency of miniature excitatory postsynaptic currents recorded in BLA slices, 10 days later, was also blocked by the same poststress riluzole administration. Together, these findings underscore the importance of therapeutic strategies, aimed at glutamate uptake and modulation, in correcting the delayed behavioral, physiological, and morphological effects of stress on the amygdala.

Cerebral Malaria and Cytotoxic Lesions of the Corpus Callosum.

To the Editors: Malaria is a common cause of morbidity and mortality worldwide. According to the WHO, 229 million cases and 409,000 deaths were reported worldwide in 2020.1 Children account for a high burden of malaria-related mortality and are at a higher risk of developing cerebral malaria (CM).1 A 12-year-old, previously healthy female from Guinea-Bissau was admitted 1 day after arriving in Portugal with a 2-day history of headache, generalized myalgia, and high-grade fever. Physical examination was unremarkable, except for pallor, icteric sclerae, and nontender hepatomegaly (4 cm). The patient was hemodynamically stable (BP, 101/52 mmHg; HR, 111 bpm), but shortly after admission, vomiting, lethargy, and impaired consciousness were observed (Glasgow coma score 11). She presented with anemia (hemoglobin, 10.0 g/dL; normal reference [NR]: 12.0–16.0), thrombocytopenia (78 × 109/L; NR: 150–450) and significant elevation of inflammatory markers (C-reactive protein, 301.0 mg/L; NR, <5; and procalcitonin, >100 ng/mL; NR, <0.1). No electrolyte disturbances, hypoglycemia, or acidosis. Plasmodium falciparum blood smear was positive, and severe malaria was diagnosed due to high parasitemia (28.5%/erythrocytes–11.872.000 parasites/µL) and central nervous system (CNS) involvement other infections were excluded. The cerebrospinal fluid revealed pleocytosis (15 cells), generalized slowing on the electroencephalogram, and a lesion in the splenium of the corpus callosum with T2 hypersignal and restricted diffusion (Fig. 1), compatible with a cytotoxic lesion of the corpus callosum (CLOCCs) on brain magnetic resonance imaging (MRI). CM was diagnosed based on severely impaired consciousness, CLOCCs, and no other identifiable causes of coma.FIGURE 1.: MRI axial images T2 weighted (A), DWI (B) and ADC map (C): oval lesion in the splenium of the corpus callosum (arrow) with T2 hypersignal and diffusion restriction with DWI hypersignal and ADC map hyposignal, corresponding to cytotoxic edema.The patient was treated with parenteral quinine and clindamycin, due to the immediate unavailability of artesunate, plus ceftriaxone. She became afebrile with progressive improvement in neurological status after 48 h and complete parasite clearance on day 3. Artemether-lumefantrine was initiated on day 5, and the patient was discharged on day 8. CM is defined as a disturbance of consciousness in a patient with Plasmodium parasitemia and no other identifiable cause.2 Despite the reported low mortality rate of pediatric imported malaria in Europe (<1%), CM entails a risk of life-threatening acute CNS effects with diffuse encephalopathy and often rapidly progressive coma, as observed in our patient.3 Distinctive brain MRI features involving both white and gray matter structures have been reported in a large cohort of Malawian children with CM. Increased volume, T2 hyperintensity, and Diffusion-weighted imaging (DWI) abnormalities affecting the cortical area (61.7%), subcortical and periventricular white matter (71.7%), basal ganglia (84.2%) and the corpus callosum (49.2%) were the most frequent features.4 CLOCCS was a common MRI finding in CM, as suggested by DWI/Apparent diffusion coefficient mismatch with DWI increased signal and Apparent diffusion coefficient low signal,4 as observed in our patient (Fig. 1). The corpus callosum, mostly the splenium, is a region vulnerable to inflammatory cytokinopathy, driven by malaria, seizures or other causes.5 The higher density of cytokines and glutamate receptors in this area leads to high extracellular glutamate levels, inducing cytotoxic edema upon infection, which is frequently reversible.5 CM in children is the most severe complication of P. falciparum infection and needs a high suspicion index in nonendemic countries. Altered MRI and CLOCCs, although unspecific, are associated with CM.