Glutamate Transporter Activity Research Articles

Effects of Hydrocodone Overdose and Ceftriaxone on Astrocytic Glutamate Transporters and Glutamate Receptors, and Associated Signaling in Nucleus Accumbens as well as Locomotor Activity in C57/BL Mice.

Chronic opioid treatments dysregulate the glutamatergic system, inducing a hyperglutamatergic state in mesocorticolimbic brain regions. This study investigated the effects of exposure to hydrocodone overdose on locomotor activity, expression of target proteins related to the glutamatergic system, signaling kinases, and neuroinflammatory factors in the nucleus accumbens. The locomotor activity of mice was measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS). CLAMS data showed that exposure to hydrocodone overdose increased locomotion activity in mice. This study tested ceftriaxone, known to upregulate major glutamate transporter 1 (GLT-1), in mice exposed to an overdose of hydrocodone. Thus, ceftriaxone normalized hydrocodone-induced hyperlocomotion activity in mice. Furthermore, exposure to hydrocodone overdose downregulated GLT-1, cystine/glutamate antiporter (xCT), and extracellular signal-regulated kinase activity (p-ERK/ERK) expression in the nucleus accumbens. However, exposure to an overdose of hydrocodone increased metabotropic glutamate receptor 5 (mGluR5), neuronal nitric oxide synthase activity (p-nNOS/nNOS), and receptor for advanced glycation end products (RAGE) expression in the nucleus accumbens. Importantly, ceftriaxone treatment attenuated hydrocodone-induced upregulation of mGluR5, p-nNOS/nNOS, and RAGE, as well as hydrocodone-induced downregulation of GLT-1, xCT, and p-ERK/ERK expression. These data demonstrated that exposure to hydrocodone overdose can cause dysregulation of the glutamatergic system, neuroinflammation, hyperlocomotion activity, and the potential therapeutic role of ceftriaxone in attenuating these effects.

Emerging Molecular and Synaptic Targets for the Management of Chronic Pain Caused by Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) frequently experience chronic pain due to the limited effectiveness and safety profiles of current analgesics. Understanding the molecular and synaptic mechanisms underlying abnormal neuronal activation along the pain signaling pathway is essential for developing new analgesics to address SLE-induced chronic pain. Recent studies, including those conducted by our team and others using the SLE animal model (MRL/lpr lupus-prone mice), have unveiled heightened excitability in nociceptive primary sensory neurons within the dorsal root ganglia and increased glutamatergic synaptic activity in spinal dorsal horn neurons, contributing to the development of chronic pain in mice with SLE. Nociceptive primary sensory neurons in lupus animals exhibit elevated resting membrane potentials, and reduced thresholds and rheobases of action potentials. These changes coincide with the elevated production of TNFα and IL-1β, as well as increased ERK activity in the dorsal root ganglion, coupled with decreased AMPK activity in the same region. Dysregulated AMPK activity is linked to heightened excitability in nociceptive sensory neurons in lupus animals. Additionally, the increased glutamatergic synaptic activity in the spinal dorsal horn in lupus mice with chronic pain is characterized by enhanced presynaptic glutamate release and postsynaptic AMPA receptor activation, alongside the reduced activity of glial glutamate transporters. These alterations are caused by the elevated activities of IL-1β, IL-18, CSF-1, and thrombin, and reduced AMPK activities in the dorsal horn. Furthermore, the pharmacological activation of spinal GPR109A receptors in microglia in lupus mice suppresses chronic pain by inhibiting p38 MAPK activity and the production of both IL-1β and IL-18, as well as reducing glutamatergic synaptic activity in the spinal dorsal horn. These findings collectively unveil crucial signaling molecular and synaptic targets for modulating abnormal neuronal activation in both the periphery and spinal dorsal horn, offering insights into the development of analgesics for managing SLE-induced chronic pain.

Silica Nanoparticles Decrease Glutamate Uptake in Blood–Brain Barrier Components

Glutamate is the major excitatory amino acid in the vertebrate brain, playing an important role in most brain functions. It exerts its activity through plasma membrane receptors and transporters, expressed both in neurons and glia cells. Overstimulation of neuronal glutamate receptors is linked to cell death in a process known as excitotoxicity, that is prevented by the efficient removal of the neurotransmitter through glutamate transporters enriched in the glia plasma membrane and in the components of the blood–brain barrier (BBB). Silica nanoparticles (SiO2-NPs) have been widely used in biomedical applications and directed to enter the circulatory system; however, little is known about the potential adverse effects of SiO2-NPs exposure on the BBB transport systems that support the critical isolation function between the central nervous system (CNS) and the peripheral circulation. In this contribution, we investigated the plausible SiO2-NPs-mediated disruption of the glutamate transport system expressed by BBB cell components. First, we evaluated the cytotoxic effect of SiO2-NPs on human brain endothelial (HBEC) and Uppsala 87 Malignant glioma (U-87MG) cell lines. Transport kinetics were evaluated, and the exposure effect of SiO2-NPs on glutamate transport activity was determined in both cell lines. Exposure of the cells to different SiO2-NP concentrations (0.4, 4.8, 10, and 20 µg/ml) and time periods (3 and 6 h) did not affect cell viability. We found that the radio-labeled D-aspartate ([3H]-D-Asp) uptake is mostly sodium-dependent, and downregulated by its own substrate (glutamate). Furthermore, SiO2-NPs exposure on endothelial and astrocytes decreases [3H]-D-Asp uptake in a dose-dependent manner. Interestingly, a decrease in the transporter catalytic efficiency, probably linked to a diminution in the affinity of the transporter, was detected upon SiO2-NPs. These results favor the notion that exposure to SiO2-NPs could disrupt BBB function and by these means shed some light into our understanding of the deleterious effects of air pollution on the CNS.

Suppressive effect of Yokukansan on glutamate released from canine keratinocytes.

Canine atopic dermatitis (CAD) is caused by skin barrier dysfunction due to allergen exposure. Excessive glutamate release in the skin is associated with delayed skin barrier function recovery and epidermal thickening and lichenification. Treatment with Yokukansan (YKS), a traditional Japanese medicine, reduces dermatitis severity and scratching behavior in NC/Nga mice by decreasing epidermal glutamate levels. However, the association between canine keratinocytes and glutamate and the mechanism by which YKS inhibits glutamate release from keratinocytes remains unknown. We aimed to investigate glutamate release from canine progenitor epidermal keratinocytes (CPEKs) and the inhibitory effect of YKS on this release. We also explored the underlying mechanism of YKS to enable its application in CAD treatment. Glutamate produced from CPEKs in the medium at 24 hours was measured. The measurement conditions varied in terms of cell density and YKS concentration. CPEKs were treated with a glutamate receptor antagonist (MK-801), a glutamate transporter antagonist (THA), and a glutamate dehydrogenase inhibitor (epigallocatechin gallate; EGCG), and the inhibitory effect of YKS, YKS + THA, MK-801, and EGCG on this release was determined. MK-801 and glutamate dehydrogenase inhibitor were tested alone, and THA was tested in combination with YKS. Finally, glutamine incorporated into CPEKs at 24 hours was measured using radioisotope labeling. CPEKs released glutamate in a cell density-dependent manner, inhibited by YKS in a concentration-dependent manner. Moreover, YKS reduced the intracellular uptake of radioisotope-labeled glutamine in a concentration-dependent manner. No involvement of glutamate receptor antagonism or activation of glutamate transporters was found, as suggested by previous studies. In addition, EGCG could inhibit glutamate release from CPEKs. Our findings indicated that glutamate release from CPEKs could be effectively inhibited by YKS, suggesting the utility of YKS in maintaining skin barrier function during CAD. In addition, CPEKs are appropriate for analyzing the mechanism of YKS. However, we found that the mechanism of action of YKS differs from that reported in previous studies, suggesting that it may have had a similar effect to EGCG in this study. Further research is warranted to understand the exact mechanism and clinical efficacy in treating CAD.

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

AMPKα1 Deficiency in Astrocytes from a Rat Model of ALS Is Associated with an Altered Metabolic Resilience.

Alterations in the activity of the regulator of cell metabolism AMP-activated protein kinase (AMPK) have been reported in motor neurons from patients and animal models of amyotrophic lateral sclerosis (ALS). Considering the key role played by astrocytes in modulating energy metabolism in the nervous system and their compromised support towards neurons in ALS, we examined whether a putative alteration in AMPK expression/activity impacted astrocytic functions such as their metabolic plasticity and glutamate handling capacity. We found a reduced expression of AMPK mRNA in primary cultures of astrocytes derived from transgenic rats carrying an ALS-associated mutated superoxide dismutase (hSOD1G93A). The activation of AMPK after glucose deprivation was reduced in hSOD1G93A astrocytes compared to non-transgenic. This was accompanied by a lower increase in ATP levels and increased vulnerability to this insult, although the ATP production rate did not differ between the two cell types. Furthermore, soliciting the activity of glutamate transporters was found to induce similar AMPK activity in these cells. However, manipulation of AMPK activity did not influence glutamate transport. Together, these results suggest that the altered AMPK responsiveness in ALS might be context dependent and may compromise the metabolic adaptation of astrocytes in response to specific cellular stress.

Hypothalamic PVN astrocytes fail to properly set glutamate tone in obese mice contributing to impaired glucose metabolism in this condition

The hypothalamus is key in the control of energy balance, but strategies targeting hypothalamic neurons have failed to provide viable options to treat most metabolic diseases. Conversely, the role of astrocytes in systemic metabolic control has remained largely unexplored. Here, we show that obesity promotes anatomically restricted exacerbated Ca 2+ activity in hypothalamic paraventricular nucleus (PVN) astrocytes. Bidirectional chemogenetic manipulation of astrocyte Ca 2+ activity in lean mice stimulated or inhibited the activity of neighboring parvocellular neurons. Importantly, these effects translated into bidirectional regulation of sympathetic activity, glucose metabolism, and energy balance. We found that the astrocyte-mediated effects on PVN neurons were blocked by the glutamate receptor blocker kynurenic acid, and the astrocyte glutamate transport blocker TBOA, suggesting that the astrocyte-neuron signaling and the effects on glucose metabolism involved the astrocytic control of ambient glutamate levels. Notably, we found that TBOA alone stimulated neurons in lean mice but failed to evoke a response in obese mice. This suggests that the astrocyte glutamate transporter activity is impaired in obese mice. Finally, we found that chemogenetic inhibition of astrocyte Ca 2+ activity in obese mice improved glucose metabolism in this condition. Together, our results indicate that exacerbated astrocyte Ca 2+ activity leads to impaired glutamate transporter activity, standing as a likely mechanism contributing to altered PVN neuronal activity and glucose homeostasis during this condition. Collectively, these findings highlight a yet unappreciated role for astrocytes in the direct control of systemic metabolism and suggest potential targets for anti-obesity strategy. NHLBI F32 HL158172 (MKK) NHLBI HL090948 (JES) NINDS NS094640 (JES) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cochlear glial cells mediate glutamate uptake through a sodium-independent transporter

Since glutamate is the primary excitatory neurotransmitter in the mammalian cochlea, the mechanisms for the removal of glutamate from the synaptic and extrasynaptic spaces are critical for maintaining normal function of this region. Glial cells of inner ear are crucial for regulation of synaptic transmission throughout since it closely interacts with neurons along the entire auditory pathway, however little is known about the activity and expression of glutamate transporters in the cochlea. In this study, using primary cochlear glial cells cultures obtained from newborn Balb/C mice, we determined the activity of a sodium-dependent and sodium-independent glutamate uptake mechanisms by means of High Performance Liquid Chromatography. The sodium-independent glutamate transport has a prominent contribution in cochlear glial cells which is similar to what has been demonstrated in other sensory organs, but it is not found in tissues less susceptible to continuous glutamate-mediated injuries. Our results showed that xCG− system is expressed in CGCs and is the main responsible for sodium-independent glutamate uptake. The identification and characterization of the xCG− transporter in the cochlea suggests a possible role of this transporter in the control of extracellular glutamate concentrations and regulation of redox state, that may aid in the preservation of auditory function.

Properties of REM sleep alterations with epilepsy.

It is usually assumed that individuals rest during sleep. However, coordinated neural activity that presumably requires high energy consumption is increased during REM sleep. Here, using freely moving male transgenic mice, the local brain environment and astrocyte activity during REM sleep were examined using the fibre photometry method with an optical fibre inserted deep into the lateral hypothalamus, a region that is linked with controlling sleep and metabolic state of the entire brain. Optical fluctuations of endogenous autofluorescence of the brain parenchyma or fluorescence of sensors for Ca2+ or pH expressed in astrocytes were examined. Using a newly devised method for analysis, changes in cytosolic Ca2+ and pH in astrocytes and changes in the local brain blood volume (BBV) were extracted. On REM sleep, astrocytic Ca2+ decreases, pH decreases (acidification) and BBV increases. Acidification was unexpected, as an increase in BBV would result in efficient carbon dioxide and/or lactate removal, which leads to alkalinization of the local brain environment. Acidification could be a result of increased glutamate transporter activity due to enhanced neuronal activity and/or aerobic metabolism in astrocytes. Notably, optical signal changes preceded the onset of the electrophysiological property signature of REM sleep by ∼20-30 s. This suggests that changes in the local brain environment have strong control over the state of neuronal cell activity. With repeated stimulation of the hippocampus, seizure response gradually develops through kindling. After a fully kindled state was obtained with multiple days of stimuli, the optical properties of REM sleep at the lateral hypothalamus were examined again. Although a negative deflection of the detected optical signal was observed during REM sleep after kindling, the estimated component changed. The decrease in Ca2+ and increase in BBV were minimal, and a large decrease in pH (acidification) emerged. This acidic shift may trigger an additional gliotransmitter release from astrocytes, which could lead to a state of hyperexcitable brain. As the properties of REM sleep change with the development of epilepsy, REM sleep analysis may serve as a biomarker of epileptogenesis severity. REM sleep analysis may also predict whether a specific REM sleep episode triggers post-sleep seizures.

Diminished motor neuron activity driven by abnormal astrocytic EAAT1 glutamate transporter activity in spinal muscular atrophy is not fully restored after lentiviral SMN delivery.

Spinal muscular atrophy (SMA) is characterized by the loss of the lower spinal motor neurons due to survival motor neuron (SMN) deficiency. The motor neuron cell autonomous and non-cell autonomous disease mechanisms driving early glutamatergic dysfunction, a therapeutically targetable phenotype prior to motor neuron cell loss, remain unclear. Using microelectrode array analysis, we demonstrate that the secretome and cell surface proteins needed for proper synaptic modulation are likely disrupted in human SMA astrocytes and lead to diminished motor neuron activity. While healthy astrocyte conditioned media did not improve SMA motor neuron activity, SMA motor neurons robustly responded to healthy astrocyte neuromodulation in direct contact cultures. This suggests an important role of astrocyte synaptic-associated plasma membrane proteins and contact-mediated cellular interactions for proper motor neuron function in SMA. Specifically, we identified a significant reduction of the glutamate Na+ dependent excitatory amino acid transporter EAAT1 within human SMA astrocytes and SMA lumbar spinal cord tissue. The selective inhibition of EAAT1 in healthy co-cultures phenocopied the diminished neural activity observed in SMA astrocyte co-cultures. Caveolin-1, an SMN-interacting protein previously associated with local translation at the plasma membrane, was abnormally elevated in human SMA astrocytes. Although lentiviral SMN delivery to SMA astrocytes partially rescued EAAT1 expression, limited activity of healthy motor neurons was still observed in SMN-transduced SMA astrocyte co-cultures. Together, these data highlight the detrimental impact of astrocyte-mediated disease mechanisms on motor neuron function in SMA and that SMN delivery may be insufficient to fully restore astrocyte function at the synapse.

Activation of Protease-Activated Receptor-1 Causes Chronic Pain in Lupus-Prone Mice Via Suppressing Spinal Glial Glutamate Transporter Function and Enhancing Glutamatergic Synaptic Activity

Systemic lupus erythematosus (SLE) is an unpredictable autoimmune disease where the body's immune system mistakenly attacks healthy tissues in many parts of the body. Chronic pain is one of the most frequently reported symptoms among SLE patients. We previously reported that MRL lupus prone (MRL/lpr) mice develop hypersensitivity to mechanical and heat stimulation. In the present study, we found that the spinal protease-activated receptor-1(PAR1) plays an important role in the genesis of chronic pain in MRL/lpr mice. Female MRL/lpr mice with chronic pain had activation of astrocytes, over-expression of thrombin and PAR1, enhanced glutamatergic synaptic activity, as well as suppressed activity of adenosine monophosphate-activated protein kinase (AMPK) and glial glutamate transport function in the spinal cord. Intrathecal injection of either the PAR1 antagonist, or AMPK activator attenuated heat hyperalgesia and mechanical allodynia in MRL/lpr mice. Furthermore, we also identified that the enhanced glutamatergic synaptic activity and suppressed activity of glial glutamate transporters in the spinal dorsal horn of MRL/lpr mice are caused by activation of the PAR1 and suppression of AMPK signaling pathways. These findings suggest that targeting the PAR1 and AMPK signaling pathways in the spinal cord may be a useful approach for treating chronic pain caused by SLE. PerspectiveOur study provides evidence suggesting activation of PAR1 and suppression of AMPK in the spinal cord induces thermal hyperalgesia and mechanical allodynia in a lupus mouse model. Targeting signaling pathways regulating the PAR1 and AMPK could potentially provide a novel approach to the management of chronic pain caused by SLE.

Astrocytic aryl hydrocarbon receptor mediates chronic kidney disease-associated mental disorders involving GLT1 hypofunction and neuronal activity enhancement in the mouse brain.

Chronic kidney disease (CKD)-associated mental disorders have been attributed to the excessive accumulation of hemodialysis-resistant indoxyl-3-sulfate (I3S) in the brain. I3S not only induces oxidative stress but is also a potent endogenous agonist of the aryl hydrocarbon receptor (AhR). Here, we investigated the role of AhR in CKD-induced brain disorders using a 5/6 nephrectomy-induced CKD mouse model, which showed increased I3S concentration in both blood and brain, anxiety and impaired novelty recognition, and AhR activation in the anterior cortex. GFAP+ reactive astrocytes were increased accompanied with the reduction of glutamate transporter 1 (GLT1) on perineuronal astrocytic processes (PAPs) in the anterior cingulate cortex (ACC) in CKD mice, and these alterations were attenuated in both neural lineage-specific and astrocyte-specific Ahr conditional knockout mice (nAhrCKO and aAhrCKO). By using chronic I3S treatment in primary astrocytes and glia-neuron (GN) mix cultures to mimic the CKD brain microenvironment, we also found significant reduction of GLT1 expression and activity in an AhR-dependent manner. Chronic I3S treatment induced AhR-dependent pro-oxidant Nox1 and AhR-independent anti-oxidant HO-1 expressions. Notably, AhR mediates chronic I3S-induced neuronal activity enhancement and synaptotoxicity in GN mix, not neuron-enriched cortical culture. In CKD mice, neuronal activity enhancement was observed in ACC and hippocampal CA1, and these responses were abrogated by both nAhrCKO and aAhrCKO. Finally, intranasal AhR antagonist CH-223191 administration significantly ameliorated the GLT1/PAPs reduction, increase in c-Fos+ neurons, and memory impairment in the CKD mice. Thus, astrocytic AhR plays a crucial role in the CKD-induced disturbance of neuron-astrocyte interaction and mental disorders.

The role of glial glutamate transporter in the baseline synaptic response and short-term synaptic plasticity of CA1 area of the hippocampus in male Wistar rat

Background. Glial cells release different gliotransmitters and response to neurotransmitters released from neurons. These cells especially astrocytes, having different transporters, play an important role in synaptic space homeostasis and synaptic plasticity. In this study, the role of hippocampal glial glutamate transporter (EAAT2) in baseline synaptic response and short term synaptic plasticity were investigated. Methods. In this experimental study, ceftriaxone, EAAT2 activator (0.5mmol/0.5μl), was microinjected intrahippcampally for activation of hippocampal glial glutamate transporter in male wistar rats. Baseline synaptic response and short term synaptic plasticity were evaluated by field potential recording. fEPSP was recorded from CA1 following Schaffer collaterals stimulation. After Input/Output curve construction, short term synaptic plasticity was induced by paired pulse stimulations. Results. Activation of EAAT2 by ceftriaxone microinjection in CA1 did not have any effect on baseline synaptic response (P> 0.05, Two Way ANOVA). There was no significant difference in Paired Pulse Index at 20, 80, and 200 ms inter-pulse interval between ceftriaxone treated and control group (P> 0.05, Two Way ANOVA). Conclusion. The results suggest that hippocampal glial glutamate transporter activation does not have effect on baseline synaptic response and short term synaptic plasticity in CA1 area of the hippocampus. Practical implications. Considering the role of glial cells in regulating the excitability of the nervous system as well as synaptic plasticity, correcting these features of the nervous system by manipulating glial cells can help the treatment or prevention of neurological diseases. In this study, the role of glial cells in the homeostasis of the glutamate in the synaptic space of the hippocampus was evaluated, through the stimulation of its uptake, on the basic synaptic activity and short-term synaptic plasticity.

Repeated sevoflurane exposures inhibit neurogenesis by inducing the upregulation of glutamate transporter 1 in astrocytes.

Sevoflurane is a widely used general anaesthetic in paediatric patients. Although repeated sevoflurane exposure is known to cause neurodevelopmental disorders in children, the mechanism of this neurotoxicity remains largely unknown. Herein, we investigated the role of glutamate transporter 1 (GLT1) in sevoflurane-induced decreased neurogenesis. Neonatal rat pups (postnatal Day 7, PN7) were exposed to 3% sevoflurane for 2 h for three consecutive days. Neuron loss and decreased neurogenesis have been observed in the neonatal rat brain, along with decreased number of astrocytes. Apoptotic astrocytes were observed after repeated sevoflurane exposure in vitro, resulting in decreased levels of brain-derived neurotrophic factor (BDNF). Calcium overload was observed in astrocytes after repeated sevoflurane exposure, in addition to upregulation of GLT1. Inhibition of GLT1 activity ameliorates repeated sevoflurane exposure-induced cognitive deficits in adult rats. Mechanically, the upregulation of GLT1 was caused by the activation of mRNA translation. RNA-sequencing analysis further confirmed that translation-related genes were activated by repeated sevoflurane exposure. These results indicate that cognitive deficits caused by repeated sevoflurane exposure during PN7-9 are triggered decreased neurogenesis. The proposed underlying mechanism involves upregulation of apoptosis in astrocytes induced by GLT1; therefore, we propose GLT1 as a potential pharmacological target for brain injury in paediatric practice.

Local brain environment changes associated with epileptogenesis.

Plastic change of the neuronal system has traditionally been assumed to be governed primarily by the long-term potentiation/depression mechanisms of synaptic transmission. However, a rather simple shift in the ambient ion, transmitter and metabolite concentrations could have a pivotal role in generating plasticity upon the physiological process of learning and memory. Local brain environment and metabolic changes could also be the cause and consequences of the pathogenesis leading to epilepsy. Governing of the local brain environment is the primal function of astrocytes. The metabolic state of the entire brain is strongly linked to the activity of the lateral hypothalamus. In this study, plastic change of astrocyte reactions in the lateral hypothalamus was examined using epileptogenesis as an extreme form of plasticity. Fluorescent sensors for calcium or pH expressed in astrocytes were examined for up to one week by in vivo fibre photometry in freely moving transgenic male mice. Optical fluctuations on a timescale of seconds is difficult to assess because these signals are heavily influenced by local brain blood volume changes and pH changes. Using a newly devised method for the analysis of the optical signals, changes in Ca2+ and pH in astrocytes and changes in local brain blood volume associated with hippocampal-stimulated epileptic seizures were extracted. Following a transient alkaline shift in the astrocyte triggered by neuronal hyperactivity, a prominent acidic shift appeared in response to intensified seizure which developed with kindling. The acidic shift was unexpected as transient increase in local brain blood volume was observed in response to intensified seizures, which should lead to efficient extrusion of the acidic CO2. The acidic shift could be a result of glutamate transporter activity and/or due to the increased metabolic load of astrocytes leading to increased CO2 and lactate production. This acidic shift may trigger additional gliotransmitter release from astrocytes leading to the exacerbation of epilepsy. As all cellular enzymic reactions are influenced by Ca2+ and pH, changes in these parameters could also have an impact on the neuronal circuit activity. Thus, controlling the astrocyte pH and/or Ca2+ could be a new therapeutic target for treatment of epilepsy or prevention of undesired plasticity associated with epileptogenesis.

Glutamate transporter activator LDN‐212320 attenuates CFA‐induced cognitive deficits and anxiety‐like behavior in mice

The astroglial glutamate transporter‐1 (GLT‐1) in the hippocampus and anterior cingulate cortex (ACC) is critically involved in acute and chronic nociceptive pain. We have previously shown that 3‐[[(2‐Methylphenyl) methyl] thio]‐6‐(2‐pyridinyl)‐pyridazine (LDN‐212320), a GLT‐1 activator, in the hippocampus attenuates acute and chronic nociceptive pain. The cellular and molecular mechanisms of GLT‐1 modulation in the hippocampus and ACC in chronic pain‐induced cognitive deficits and anxiety‐like behaviors are unknown. Here, we have examined the effects of LDN‐212320 in complete Freund’s adjuvant (CFA)‐induced cognitive deficits and anxiety‐like behaviors in mice. Furthermore, we have measured CFA‐induced impaired spatial, working, and recognition memory using Y‐maze and object‐place recognition test. In addition, we have determined chronic pain‐induced anxiety‐like behaviors using elevated plus maze and marble burying test. We have also evaluated the effects of LDN‐212320 on cAMP response element‐binding protein (pCREB) expression in the hippocampus and ACC during CFA‐induced cognitive deficits and anxiety‐like behaviors using Western blot analysis and immunofluorescence assay. Pretreatment of LDN‐212320 (20 mg/kg) significantly attenuated CFA‐induced impaired spatial, working, and recognition memory. The LDN‐212320 (20 mg/kg) significantly reduced CFA‐induced anxiety‐like behaviors. Additionally, LDN‐212320 (20 mg/kg) significantly reversed CFA‐induced decreased‐pCREB expression in the hippocampus and ACC. Overall, these results suggest that the LDN‐212320 prevents CFA‐induced impaired cognitive behaviors and neuronal deficits via GLT‐1 modulation in the hippocampus and ACC. Therefore, LDN‐212320 may have therapeutic utility for the prevention and treatment of chronic pain‐associated with cognitive impairments and anxiety‐like behaviors.

Activation of Glutamate Transport Increases Arteriole Diameter in v ivo: Implications for Neurovascular Coupling.

In order to meet the energetic demands of cell-to-cell signaling, increases in local neuronal signaling are matched by a coordinated increase in local blood flow, termed neurovascular coupling. Multiple different signals from neurons, astrocytes, and pericytes contribute to this control of blood flow. Previously, several groups demonstrated that inhibition/ablation of glutamate transporters attenuates the neurovascular response. However, it was not determined if glutamate transporter activation was sufficient to increase blood flow. Here, we used multiphoton imaging to monitor the diameter of fluorescently labeled cortical arterioles in anesthetized C57/B6J mice. We delivered vehicle, glutamate transporter substrates, or a combination of a glutamate transporter substrate with various pharmacologic agents via a glass micropipette while simultaneously visualizing changes in arteriole diameter. We developed a novel image analysis method to automate the measurement of arteriole diameter in these time-lapse analyses. Using this workflow, we first conducted pilot experiments in which we focally applied L-glutamate, D-aspartate, or L-threo-hydroxyaspartate (L-THA) and measured arteriole responses as proof of concept. We subsequently applied the selective glutamate transport substrate L-THA (applied at concentrations that do not activate glutamate receptors). We found that L-THA evoked a significantly larger dilation than that observed with focal saline application. This response was blocked by co-application of the potent glutamate transport inhibitor, L-(2S,3S)-3-[3-[4-(trifluoromethyl)-benzoylamino]benzyloxy]-aspartate (TFB-TBOA). Conversely, we were unable to demonstrate a reduction of this effect through co-application of a cocktail of glutamate and GABA receptor antagonists. These studies provide the first direct evidence that activation of glutamate transport is sufficient to increase arteriole diameter. We explored potential downstream mechanisms mediating this transporter-mediated dilation by using a Ca2+ chelator or inhibitors of reversed-mode Na+/Ca2+ exchange, nitric oxide synthetase, or cyclo-oxygenase. The estimated effects and confidence intervals suggested some form of inhibition for a number of these inhibitors. Limitations to our study design prevented definitive conclusions with respect to these downstream inhibitors; these limitations are discussed along with possible next steps. Understanding the mechanisms that control blood flow are important because changes in blood flow/energy supply are implicated in several neurodegenerative disorders and are used as a surrogate measure of neuronal activity in widely used techniques such as functional magnetic resonance imaging (fMRI).

A Selective Histamine H4 Receptor Antagonist, JNJ7777120, Role on glutamate Transporter Activity in Chronic Depression

Glutamate release and reuptake play a key role in the pathophysiology of depression. glutamatergic nerves in the hippocampus region are modulated by histaminergic afferents. Excessive accumulation of glutamate in the synaptic area causes degeneration of neuron cells. The H4 receptor is defined as the main immune system histamine receptor with a pro-inflammatory role. To understand the role of this receptor, the drug JNJ7777120 was used to reveal the chronic depression-glutamate relationship. We have important findings showing that the H4 antagonist increases the glutamate transporters’ instantaneous activity. In our experiment, it has been shown that blocking the H4 receptor leads to increased neuron cell viability and improvement in behavioral ability due to glutamate. Therefore, JNJ can be used to prevent neurotoxicity, inhibit membrane phospholipase activation and free radical formation, and minimize membrane disruption. In line with our findings, results have been obtained that indicate that JNJ will contribute to the effective prevention and treatment of depression.

Abnormal glutamatergic neurotransmission and cognitive impairment in male APPNL-F/NL-F mice.

It is well established that glutamatergic neurotransmission plays an essential role in learning and memory. In Alzheimer's disease (AD), previous studies indicate that glutamate dynamics shift with disease progression, inciting cognitive impairment and further perpetuating disease pathology. Here, we build upon previous work by utilizing a novel knock-in mouse model of AD (APPNL-F/NL-F ) to characterize hippocampal glutamate signaling and cognition during late stage AD. At 18-24 months old, male APPNL-F/NL-F and genetic background control (C57BL/6) mice underwent cognitive assessment through a Morris water maze (MWM) task. Following MWM, basal and 70 mM KCl stimulus-evoked glutamate release was measured in the CA1 region of the hippocampus using a glutamate-selective microelectrode in isoflurane anesthetized mice. Briefly, microelectrodes were coated with L-glutamate oxidase for the detection of glutamate at the electrode surface. A layer of 1,3-phenylenediamine was applied to prevent detection of electroactive compounds at the recording potential (+0.7 volts). A micropipette (diameter = ∼20 µm) was attached 50-100 µm from the recording surface for local application of KCl). Calibration and recordings are taken using the FAST-16 recording software. Preliminary MWM data indicates no significant differences in spatial learning performance (corrected integrated path length and cumulative distance from the platform) between C57BL/6 and APPNL-F/NL-F mice. We observed a trend of decreased platform entries during the probe trial, supporting long-term spatial memory impairment in APPNL-F/NL-F mice. Preliminary in vivo CA1 data support a trend of decreased basal and stimulus-evoked glutamate levels in APPNL-F/NL-F mice compared to control mice. Preliminary findings support impaired long-term spatial memory and hypoactive glutamatergic neurotransmission in aged male APPNL-F/NL-F mice compared to age-matched C57BL/6 mice. Lack of glutamate activity in late-stage AD may underlie cognitive decline in APPNL-F/NL-F mice. Our laboratory previously observed similar findings in a transgenic AD model. However, unlike APPNL-F/NL-F mice, those mice also displayed increased basal glutamate levels. This divergence could be attributed to possible differences in glutamate transporter expression or activity with disease progression. Together, these data support loss of glutamate excitation with amyloid accumulation. Studies are ongoing.

MC-100093, a Novel β-Lactam Glutamate Transporter-1 Enhancer Devoid of Antimicrobial Properties, Attenuates Cocaine Relapse in Rats.

Cocaine use disorder currently lacks Food and Drug Administration-approved treatments. In rodents, the glutamate transporter-1 (GLT-1) is downregulated in the nucleus accumbens after cocaine self-administration, and increasing the expression and function of GLT-1 reduces the reinstatement of cocaine seeking. The β-lactam antibiotic ceftriaxone upregulates GLT-1 and attenuates cue- and cocaine-induced cocaine seeking without affecting motivation for natural rewards. Although ceftriaxone shows promise for treating cocaine use disorder, it possesses characteristics that limit successful translation from bench to bedside, including poor brain penetration, a lack of oral bioavailability, and a risk of bacterial resistance when used chronically. Thus, we aimed to develop novel molecules that retained the GLT-1-enhancing effects of ceftriaxone but displayed superior drug-like properties. Here, we describe a new monocyclic β-lactam, MC-100093, as a potent upregulator of GLT-1 that is orally bioavailable and devoid of antimicrobial properties. MC-100093 was synthesized and tested in vitro and in vivo to determine physiochemical, pharmacokinetic, and pharmacodynamic properties. Next, adult male rats underwent cocaine self-administration and extinction training. During extinction training, rats received one of four doses of MC-100093 for 6-8 days prior to a single cue-primed reinstatement test. Separate cohorts of rats were used to assess nucleus accumbens GLT-1 expression and MC-100093 effects on sucrose self-administration. We found that 50 mg/kg MC-100093 attenuated cue-primed reinstatement of cocaine seeking while upregulating GLT-1 expression in the nucleus accumbens core. This dose did not produce sedation, nor did it decrease sucrose consumption or body weight. Thus, MC-100093 represents a potential treatment to reduce cocaine relapse. SIGNIFICANCE STATEMENT: Increasing GLT-1 activity reliably reduces drug-seeking across classes of drugs; however, existing GLT1-enhancers have side effects and lack oral bioavailability. To address this issue, novel GLT-1 enhancers were synthesized, and the compound with the most favorable pharmacokinetic and pharmacodynamic properties, MC-100093, was selected for further testing. MC-100093 attenuated cued cocaine seeking without reducing food seeking or locomotion and upregulated GLT-1 expression in the nucleus accumbens.