Palmitic Acid Induces Oxidative Stress and Senescence in Human Brainstem Astrocytes, Downregulating Glutamate Reuptake Transporters-Implications for Obesity-Related Sympathoexcitation.

Obesity has been associated with a chronic increase in sympathetic nerve activity, which can lead to hypertension and other cardiovascular diseases. Preliminary studies from our lab found that oxidative stress and neuroinflammation in the brainstem contribute to sympathetic overactivity in high-fat-diet-induced obese mice. However, with glial cells emerging as significant contributors to various physiological processes, their role in causing these changes in obesity remains unknown. In this study, we wanted to determine the role of palmitic acid, a major form of saturated fatty acid in the high-fat diet, in regulating sympathetic outflow. Human brainstem astrocytes (HBAs) were used as a cell culture model since astrocytes are the most abundant glial cells and are more closely associated with the regulation of neurons and, hence, sympathetic nerve activity. In the current study, we hypothesized that palmitic acid-mediated oxidative stress induces senescence and downregulates glutamate reuptake transporters in HBAs. HBAs were treated with palmitic acid (25 µM for 24 h) in three separate experiments. After the treatment period, the cells were collected for gene expression and protein analysis. Our results showed that palmitic acid treatment led to a significant increase in the mRNA expression of oxidative stress markers (NQO1, SOD2, and CAT), cellular senescence markers (p21 and p53), SASP factors (TNFα, IL-6, MCP-1, and CXCL10), and a downregulation in the expression of glutamate reuptake transporters (EAAT1 and EAAT2) in the HBAs. Protein levels of Gamma H2AX, p16, and p21 were also significantly upregulated in the treatment group compared to the control. Our results showed that palmitic acid increased oxidative stress, DNA damage, cellular senescence, and SASP factors, and downregulated the expression of glutamate reuptake transporters in HBAs. These findings suggest the possibility of excitotoxicity in the neurons of the brainstem, sympathoexcitation, and increased risk for cardiovascular diseases in obesity.

Glutamate transporters are involved in direct inhibitory synaptic transmission in the vertebrate retina.

In the central nervous system of vertebrates, glutamate serves as the primary excitatory neurotransmitter. However, in the retina, glutamate released from photoreceptors causes hyperpolarization in post-synaptic ON-bipolar cells through a glutamate-gated chloride current, which seems paradoxical. Our research reveals that this current is modulated by two excitatory glutamate transporters, EAAT5b and EAAT7. In the zebrafish retina, these transporters are located at the dendritic tips of ON-bipolar cells and interact with all four types of cone photoreceptors. The absence of these transporters leads to a decrease in ON-bipolar cell responses, with eaat5b mutants being less severely affected than eaat5b/eaat7 double mutants, which also exhibit altered response kinetics. Biophysical investigations establish that EAAT7 is an active glutamate transporter with a predominant anion conductance. Our study is the first to demonstrate the direct involvement of post-synaptic glutamate transporters in inhibitory direct synaptic transmission at a central nervous system synapse.

Increased expression of SLC25A18 is associated with Alzheimer's disease and is involved in Aß42-induced mitochondrial dysfunction and apoptosis in neuronal cells.

Alzheimer's disease (AD) is currently one of the most serious public health concerns in the world. However, the best approach to treat AD has yet to be discovered, implying that we must continue to work hard to find new AD target genes. In this study, we further analysed Gene Expression Omnibus (GEO) data and discovered that the expression of the Mitochondria glutamate carrier SLC25A18 is associated with AD by screening the differentially expressed genes in different regions of the brains of Alzheimer's disease patients. To verify the expression of SLC25A18 during Alzheimer's disease development, we analysed animal models (5×FAD transgenic AD animal model, chemically induced AD animal model, natural ageing animal model), and the results showed that the expression of SLC25A18 was increased in animal models of AD. Further investigation of the different regions found that SLC25A18 expression was elevated in the EC, TeA, and CA3, and expressed in neurons. Next, We found that Aß42 treatment elevated SLC25A18 expression in Neuro 2A cells. Reducing SLC25A18 expression attenuated mitochondrial dysfunction and neuronal apoptosis caused by Aß42. Overexpression of SLC25A18 increased ATP and intracellular superoxide anions but decreased mitochondrial membrane potential. The results indicate that SLC25A18 affects mitochondrial function and neuronal apoptosis, and is related to AD, which makes it a potential target for treating brain dysfunction.

Thyroid hormone deficiency affects anxiety-related behaviors and expression of hippocampal glutamate transporters in male congenital hypothyroid rat offspring.

Thyroid hormones are crucial for brain development and their deficiency during fetal and postnatal periods can lead to mood and cognitive disorders. We aimed to examine the consequences of thyroid hormone deficiency on anxiety-related behaviors and protein expression of hippocampal glutamate transporters in congenital hypothyroid male offspring rats. Possible beneficial effects of treadmill exercise have also been examined. Congenital hypothyroidism was induced by adding propylthiouracil (PTU) to drinking water of pregnant Wistar rats from gestational day 6 until the end of the weaning period (postnatal day 28). Next, following 4 weeks of treadmill exercise (5 days per week), anxiety-related behaviors were examined using elevated plus maze (EPM) and light/dark box tests. Thereafter, protein expression of astrocytic (GLAST and GLT-1) and neuronal (EAAC1) glutamate transporters were measured in the hippocampus by immunoblotting. Hypothyroid rats showed decreased anxiety-like behavior, as measured by longer time spent in the open arms of the EPM and in the light area of the light/dark box, compared to control rats. Hypothyroid rats had significantly higher GLAST and GLT-1 and lower EAAC1 protein levels in the hippocampus than did the euthyroid rats. Following exercise, anxiety levels decreased in the euthyroid group while protein expression of EAAC1 increased and returned to normal levels in the hypothyroid group. Our findings indicate that thyroid hormone deficiency was associated with alterations in protein expression of glutamate transporters in the hippocampus. Up-regulation of hippocampal GLAST and GLT-1 could be at least one of the mechanisms associated with the anxiolytic effects of congenital hypothyroidism.

Structural bioinformatics studies of glutamate transporters and their AlphaFold2 predicted water-soluble QTY variants and uncovering the natural mutations of L->Q, I->T, F->Y and Q->L, T->I and Y->F.

Glutamate transporters play key roles in nervous physiology by modulating excitatory neurotransmitter levels, when malfunctioning, involving in a wide range of neurological and physiological disorders. However, integral transmembrane proteins including the glutamate transporters remain notoriously difficult to study, due to their localization within the cell membrane. Here we present the structural bioinformatics studies of glutamate transporters and their water-soluble variants generated through QTY-code, a protein design strategy based on systematic amino acid substitutions. These include 2 structures determined by X-ray crystallography, cryo-EM, and 6 predicted by AlphaFold2, and their predicted water-soluble QTY variants. In the native structures of glutamate transporters, transmembrane helices contain hydrophobic amino acids such as leucine (L), isoleucine (I), and phenylalanine (F). To design water-soluble variants, these hydrophobic amino acids are systematically replaced by hydrophilic amino acids, namely glutamine (Q), threonine (T) and tyrosine (Y). The QTY variants exhibited water-solubility, with four having identical isoelectric focusing points (pI) and the other four having very similar pI. We present the superposed structures of the native glutamate transporters and their water-soluble QTY variants. The superposed structures displayed remarkable similarity with RMSD 0.528Å-2.456Å, despite significant protein transmembrane sequence differences (41.1%->53.8%). Additionally, we examined the differences of hydrophobicity patches between the native glutamate transporters and their QTY variants. Upon closer inspection, we discovered multiple natural variations of L->Q, I->T, F->Y and Q->L, T->I, Y->F in these transporters. Some of these natural variations were benign and the remaining were reported in specific neurological disorders. We further investigated the characteristics of hydrophobic to hydrophilic substitutions in glutamate transporters, utilizing variant analysis and evolutionary profiling. Our structural bioinformatics studies not only provided insight into the differences between the hydrophobic helices and hydrophilic helices in the glutamate transporters, but they are also expected to stimulate further study of other water-soluble transmembrane proteins.

Neuroprotection Role of Vitamin C by Upregulating Glutamate Transporter-1 in Auditory Cortex of Noise-Induced Tinnitus Animal Model.

Vitamin C (Vc) plays a pivotal role in a series of pathological processes, such as tumors, immune diseases, and neurological disorders. However, its therapeutic potential for tinnitus management remains unclear. In this study, we find that Vc relieves tinnitus in noise-exposed rats. In the 7-day therapy groups, spontaneous firing rate (SFR) increases from 1.17 ± 0.10 Hz to 1.77 ± 0.15 Hz after noise exposure. Vc effectively reduces the elevated SFR to 0.99 ± 0.07 and 0.55 ± 0.05 Hz at different doses. The glutamate level in auditory cortex of noise-exposed rats (3.78 ± 0.42 µM) increases relative to that in the control group (1.34 ± 0.22 µM). High doses of Vc (500 mg/kg/day) effectively reduce the elevated glutamate levels (1.49 ± 0.28 µM). Mechanistic studies show that the expression of glutamate transporter 1 (GLT-1) is impaired following noise exposure and that Vc treatment effectively restores GLT-1 expression in the auditory cortex. Meanwhile, the GLT-1 inhibitor, dl-threo-beta-benzyloxyaspartic acid (dl-TBOA), invalidates the protection role of Vc. Our finding shows that Vc substantially enhances glutamate clearance by upregulating GLT-1 and consequently alleviates noise-induced tinnitus. This study provides valuable insight into a novel biological target for the development of therapeutic interventions that may prevent the onset of tinnitus.

Aryl Hydrocarbon Receptor Involvement in the Sodium-Dependent Glutamate/Aspartate Transporter Regulation in Cerebellar Bergmann Glia Cells.

Glutamate, the major excitatory neurotransmitter in the vertebrate brain, exerts its functions through the activation of specific plasma membrane receptors and transporters. Overstimulation of glutamate receptors results in neuronal cell death through a process known as excitotoxicity. A family of sodium-dependent glutamate plasma membrane transporters is responsible for the removal of glutamate from the synaptic cleft, preventing an excitotoxic insult. Glial glutamate transporters carry out more than 90% of the brain glutamate uptake activity and are responsible for glutamate recycling through the GABA/Glutamate/Glutamine shuttle. The aryl hydrocarbon receptor is a ligand-dependent transcription factor that integrates environmental clues through its ability to heterodimerize with different transcription factors. Taking into consideration the fundamental role of glial glutamate transporters in glutamatergic synapses and that these transporters are regulated at the transcriptional, translational, and localization levels in an activity-dependent fashion, in this contribution, we explored the involvement of the aryl hydrocarbon receptor, as a model of environmental integrator, in the regulation of the glial sodium-dependent glutamate/aspartate transporter. Using the model of chick cerebellar Bergmann glia cells, we report herein that the aryl hydrocarbon receptors exert a time-dependent decrease in the transporter mRNA levels and a diminution of its uptake activity. The nuclear factor kappa light chain enhancer of the activated B cell signaling pathway is involved in this regulation. Our results favor the notion of an environmentally dependent regulation of glutamate removal in glial cells and therefore strengthen the notion of the involvement of glial cells in xenobiotic neurotoxic effects.

Neuronal small extracellular vesicles carrying miR-181c-5p contribute to the pathogenesis of epilepsy by regulating the protein kinase C-δ/glutamate transporter-1 axis in astrocytes.

Information exchange between neurons and astrocytes mediated by extracellular vesicles (EVs) is known to play a key role in the pathogenesis of central nervous system diseases. A key driver of epilepsy is the dysregulation of intersynaptic excitatory neurotransmitters mediated by astrocytes. Thus, we investigated the potential association between neuronal EV microRNAs (miRNAs) and astrocyte glutamate uptake ability in epilepsy. Here, we showed that astrocytes were able to engulf epileptogenic neuronal EVs, inducing a significant increase in the glutamate concentration in the extracellular fluid of astrocytes, which was linked to a decrease in glutamate transporter-1 (GLT-1) protein expression. Using sequencing and gene ontology (GO) functional analysis, miR-181c-5p was found to be the most significantly upregulated miRNA in epileptogenic neuronal EVs and was linked to glutamate metabolism. Moreover, we found that neuronal EV-derived miR-181c-5p interacted with protein kinase C-delta (PKCδ), downregulated PKCδ and GLT-1 protein expression and increased glutamate concentrations in astrocytes both in vitro and in vivo. Our findings demonstrated that epileptogenic neuronal EVs carrying miR-181c-5p decrease the glutamate uptake ability of astrocytes, thus promoting susceptibility to epilepsy.

Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation.

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

Conditional Astrocyte Rac1KO Attenuates Hyperreflexia after Spinal Cord Injury.

Spasticity is a hyperexcitability disorder that adversely impacts functional recovery and rehabilitative efforts after spinal cord injury (SCI). The loss of evoked rate-dependent depression (RDD) of the monosynaptic H-reflex is indicative of hyperreflexia, a physiological sign of spasticity. Given the intimate relationship between astrocytes and neurons, that is, the tripartite synapse, we hypothesized that astrocytes might have a significant role in post-injury hyperreflexia and plasticity of neighboring neuronal synaptic dendritic spines. Here, we investigated the effect of selective Rac1KO in astrocytes (i.e., adult male and female mice, transgenic cre-flox system) on SCI-induced spasticity. Three weeks after a mild contusion SCI, control Rac1wt animals displayed a loss of H-reflex RDD, that is, hyperreflexia. In contrast, transgenic animals with astrocytic Rac1KO demonstrated near-normal H-reflex RDD similar to pre-injury levels. Reduced hyperreflexia in astrocytic Rac1KO animals was accompanied by a loss of thin-shaped dendritic spine density on α-motor neurons in the ventral horn. In SCI-Rac1wt animals, as expected, we observed the development of dendritic spine dysgenesis on α-motor neurons associated with spasticity. As compared with WT animals, SCI animals with astrocytic Rac1KO expressed increased levels of the glial-specific glutamate transporter, glutamate transporter-1 in the ventral spinal cord, potentially enhancing glutamate clearance from the synaptic cleft and reducing hyperreflexia in astrocytic Rac1KO animals. Taken together, our findings show for the first time that Rac1 activity in astrocytes can contribute to hyperreflexia underlying spasticity following SCI. These results reveal an opportunity to target cell-specific molecular regulators of H-reflex excitability to manage spasticity after SCI.Significance Statement Spinal cord injury leads to stretch reflex hyperexcitability, which underlies the clinical symptom of spasticity. This study shows for the first time that astrocytic Rac1 contributes to the development of hyperreflexia after SCI. Specifically, astrocytic Rac1KO reduced SCI-related H-reflex hyperexcitability, decreased dendritic spine dysgenesis on α-motor neurons, and elevated the expression of the astrocytic glutamate transporter-1 (GLT-1). Overall, this study supports a distinct role for astrocytic Rac1 signaling within the spinal reflex circuit and the development of SCI-related spasticity.

Design and Characterization of Prodrug-like Inhibitors for Preventing Glutamate Efflux through Reverse Transport.

Glutamate transporters are responsible for active transport of the major excitatory neurotransmitter glutamate across the cell membrane, regulating the extracellular glutamate concentration in the mammalian brain. Extracellular glutamate levels in the brain are usually in the submicromolar range but can increase by exocytosis, inhibition of cellular uptake, or through glutamate release by reverse transport, as well as other mechanisms, which can lead to neurodegeneration and neuronal cell death. Such conditions can be encountered upon energy deprivation during an ischemic stroke. Here, we developed acetoxymethyl (AM) ester prodrug-like derivatives of excitatory amino acid transporter (EAAT) inhibitors that permeate the cell membrane and are activated, most likely through hydrolysis by endogenous cellular esterases, to form the active EAAT inhibitor. Upon increase in external K+ concentration, the inhibitors block glutamate efflux by EAAT reverse transport. Using a novel high-affinity fluorescent prodrug-like inhibitor, dl-threo-9-anthracene-methoxy-aspartate (TAOA) AM ester, we demonstrate that the precursor rapidly accumulates inside cells. Electrophysiological methods and fluorescence assays utilizing the iGluSnFR external glutamate sensor were used to demonstrate the efficacy of AM ester-protected inhibitors in inhibiting K+-mediated glutamate release. Together, our results provide evidence for a novel method to potentially prevent glutamate release by reverse transport under pathophysiological conditions in a model cell system, as well as in human astrocytes, while leaving glutamate uptake under physiological conditions operational. This method could have wide-ranging applications in the prevention of glutamate-induced neuronal cell death.

Sulfasalazine, a potent cystine-glutamate transporter inhibitor, enhances osteogenic differentiation of canine adipose-derived stem cells.

Cystine-glutamate transporter (xCT) is a plasma membrane transporter that imports cystine and indirectly contributes to the oxidative stress resistance associated with increased intracellular glutathione levels. Canine adipose-derived stem cells (CADSCs) include an xCT-positive subpopulation and show relatively low expression of osteogenic markers during in vitro osteogenic differentiation. Sulfasalazine (SSZ), a drug used to treat rheumatoid arthritis, suppresses xCT expression in cancer cells. In this study, we found that the SSZ treatment at 100 µM significantly suppressed xCT mRNA expression in CADSCs but did not significantly affect cell proliferation under the same conditions. Additionally, this treatment decreased the intracellular glutathione concentration. During in vitro osteogenic differentiation, the SSZ treatment at 50 µM and 100 µM significantly increased alizarin red staining and its quantification, as well as the concentration-dependent osteogenic differentiation markers (BMP1 and SPP) mRNA expression. Our results suggested that SSZ enhances the osteogenic differentiation potential of CADSCs and can potentially exhibit a superior therapeutic profile in canine bone regenerative medicine.

Neuronal MML-1/MXL-2 regulates systemic aging via glutamate transporter and cell nonautonomous autophagic and peroxidase activity.

Accumulating evidence has demonstrated the presence of intertissue-communication regulating systemic aging, but the underlying molecular network has not been fully explored. We and others previously showed that two basic helix-loop-helix transcription factors, MML-1 and HLH-30, are required for lifespan extension in several longevity paradigms, including germlineless Caenorhabditis elegans. However, it is unknown what tissues these factors target to promote longevity. Here, using tissue-specific knockdown experiments, we found that MML-1 and its heterodimer partners MXL-2 and HLH-30 act primarily in neurons to extend longevity in germlineless animals. Interestingly, however, the downstream cascades of MML-1 in neurons were distinct from those of HLH-30. Neuronal RNA interference (RNAi)-based transcriptome analysis revealed that the glutamate transporter GLT-5 is a downstream target of MML-1 but not HLH-30. Furthermore, the MML-1-GTL-5 axis in neurons is critical to prevent an age-dependent collapse of proteostasis and increased oxidative stress through autophagy and peroxidase MLT-7, respectively, in long-lived animals. Collectively, our study revealed that systemic aging is regulated by a molecular network involving neuronal MML-1 function in both neural and peripheral tissues.

Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity.

Human induced pluripotent stem cell (hiPSC)-derived neural cells have started to be used in safety/toxicity tests at the preclinical stage of drug development. As previously reported, hiPSC-derived neurons exhibit greater tolerance to excitotoxicity than those of primary cultures of rodent neurons; however, the underlying mechanisms remain unknown. We here investigated the functions of L-glutamate (L-Glu) transporters, the most important machinery to maintain low extracellular L-Glu concentrations, in hiPSC-derived neural cells. We also clarified the contribution of respective L-Glu transporter subtypes. At 63 days in vitro (DIV), we detected neuronal circuit functions in hiPSC-derived neural cells by a microelectrode array system (MEA). At 63 DIV, exposure to 100 µM L-Glu for 24 h did not affect the viability of neural cells. 100 µM L-Glu in the medium decreased to almost 0 µM in 60 min. Pharmacological inhibition of excitatory amino acid transporter 1 (EAAT1) and EAAT2 suppressed almost 100% of L-Glu decrease. In the presence of this inhibitor, 100 µM L-Glu dramatically decreased cell viability. These results suggest that in hiPSC-derived neural cells, EAAT1 and EAAT2 are the predominant L-Glu transporters, and their uptake potentials are the reasons for the tolerance of hiPSC-derived neurons to excitotoxicity.

Proteomic Analysis of the Amygdala Reveals Dynamic Changes in Glutamate Transporter-1 During Progression of Complete Freund's Adjuvant-Induced Pain Aversion.

Pain sufferer usually show an aversion to the environment associated with pain, identified as pain aversion. The amygdala, an almond-shaped limbic structure in the medial temporal lobe, exerts a critical effect on emotion and pain formation. However, studies on inflammatory pain-induced aversion are still relatively limited, and the available evidence is not enough to clarify its inherent mechanisms. Proteomics is a high-throughput, comprehensive, and objective study method that compares the similarities and differences of protein expression under different conditions to screen potential targets. The current study aimed to identify potential pivotal proteins in the amygdala of rats after complete Freund's adjuvant (CFA)-induced pain aversion via proteomics analysis. Immunohistochemistry was performed to confirm the expression of glutamate transporter-1 (GLT-1) in the amygdala during different periods of pain aversion. Thirteen proteins were found to be different between the day 2 and day 15 groups. Among the 13 differentially expressed proteins, Q8R64 denotes GLT-1, which utilises synaptic glutamate to remain optimal extracellular glutamic levels, thereby preventing accumulation in the synaptic cleft and consequent excitotoxicity. The variation in GLT-1 expression was correlated with the variation tendency of pain aversion, which implies a potential link between the modulation of pain aversion and the excitability of glutamatergic neurons. This study demonstrated that exposure to inflammatory pain results in aversion induced from pain, leading to extensive biological changes in the amygdala.

ß_lactam antibiotics against drug addiction: A novel therapeutic option.

Drug addiction as a problem for the health of the individual and the society is the result of a complex process in which there is an interaction between brain nuclei and neurotransmitters (such as glutamate). ß-lactam antibiotics, due to their enhancing properties on the glutamate transporter glutamate transporter-1, can affect and counteract the addictive mechanisms of drugs through the regulation of extracellular glutamate. Since glutamate is a key neurotransmitter in the development of drug addiction, it seems that ß-lactams can be considered as a promising treatment for addiction. However, more research in this field is necessary to identify other mechanisms involved in their effectiveness. This article is a review of the studies conducted on the effect of ß-lactam administration in preventing the development of drug addiction, as well as their possible cellular and molecular mechanisms. This review suggests the clinical use of ß-lactam antibiotics that have weak antimicrobial properties (such as clavulanic acid) in the treatment of drug dependence.

Depressed glutamate transporter 1 expression in a mouse model of Dravet syndrome.

Dravet syndrome (DS) is a monogenic, often refractory, epilepsy resultant from SCN1A haploinsufficiency in humans. A novel therapeutic target in DS that can be engaged in isolation or as adjunctive therapy is highly desirable. Here, we demonstrate reduced expression of the rodent glutamate transporter type 1 (GLT-1) in a DS mouse model, and in wild type mouse strains where Scn1a haploinsufficiency is most likely to cause epilepsy, indicating that GLT-1 depression may play a role in DS seizures. As GLT-1 can be upregulated by common and safe FDA-approved medications, this strategy may be an attractive, viable, and novel avenue for DS treatment.

Glutamate-aspartate transporter dysfunction enhances aminoglycoside-induced cochlear hair cell death via NMDA receptor activation.

Glutamate is a crucial neurotransmitter for hearing transduction in the cochlea, but excess glutamate is detrimental to the survival of cochlear sensory cells. Glutamate-aspartate transporter (GLAST) is the major transporter for glutamate removal; however, its role in aminoglycoside-induced hair cell loss is not well studied. In the present study, we first investigated the localization and expression of GLAST over the course of development of the mouse cochlea, and we found that inhibition of GLAST increased hair cell death. However, when the glutamate receptor NMDAR was inhibited by D-AP5, hair cell death was no longer increased by the GLAST inhibitor. Our results indicate that GLAST inhibition aggravates damage to cochlear hair cells, which may occur via NMDAR, and this suggests new clinical strategies for ameliorating the ototoxicity associated with the dysfunction of glutamate metabolism.

EAAT5 glutamate transporter rapidly binds glutamate with micromolar affinity in mouse rods.

Light responses of rod photoreceptor cells in the retina are encoded by changes in synaptic glutamate release that is in turn shaped by reuptake involving EAAT5 plasma membrane glutamate transporters. Heterologously expressed EAAT5 activates too slowly upon glutamate binding to support significant uptake. We tested EAAT5 activation in mouse rods in vivo by stimulating glutamate transporter anion currents (IA(glu)) with UV flash photolysis of MNI-glutamate, varying flash intensity to vary glutamate levels. Responses to uncaging rose rapidly with time constants of 2-3 ms, similar to IA(glu) events arising from spontaneous release. Spontaneous release events and IA(glu) evoked by weak flashes also declined with similar time constants of 40-50 ms. Stronger flashes evoked responses that decayed more slowly. Time constants were twofold faster at 35°C, suggesting that they reflect transporter kinetics, not diffusion. Selective EAAT1 and EAAT2 inhibitors had no significant effect, suggesting IA(glu) in rods arises solely from EAAT5. We calibrated glutamate levels attained during flash photolysis by expressing a fluorescent glutamate sensor iGluSnFr in cultured epithelial cells. We compared fluorescence at different glutamate concentrations to fluorescence evoked by photolytic uncaging of MNI-glutamate. The relationship between flash intensity and glutamate yielded EC50 values for EAAT5 amplitude, decay time, and rise time of ∼10 µM. Micromolar affinity and rapid activation of EAAT5 in rods show it can rapidly bind synaptic glutamate. However, we also found that EAAT5 currents are saturated by the synchronous release of only a few vesicles, suggesting limited capacity and a role for glial uptake at higher release rates.

Combinatory Actions of Co-transmitters in Dopaminergic Systems Modulate Drosophila Olfactory Memories.

Dopamine neurons (DANs) are extensively studied in the context of associative learning, in both vertebrates and invertebrates. In the acquisition of male and female Drosophila olfactory memory, the PAM cluster of DANs provides the reward signal, and the PPL1 cluster of DANs sends the punishment signal to the Kenyon cells (KCs) of mushroom bodies, the center for memory formation. However, thermo-genetical activation of the PPL1 DANs after memory acquisition impaired aversive memory, and that of the PAM DANs impaired appetitive memory. We demonstrate that the knockdown of glutamate decarboxylase, which catalyzes glutamate conversion to GABA in PAM DANs, potentiated the appetitive memory. In addition, the knockdown of glutamate transporter in PPL1 DANs potentiated aversive memory, suggesting that GABA and glutamate co-transmitters act in an inhibitory manner in olfactory memory formation. We also found that, in γKCs, the Rdl receptor for GABA and the mGluR DmGluRA mediate the inhibition. Although multiple-spaced training is required to form long-term aversive memory, a single cycle of training was sufficient to develop long-term memory when the glutamate transporter was knocked down, in even a single subset of PPL1 DANs. Our results suggest that the mGluR signaling pathway may set a threshold for memory acquisition to allow the organisms' behaviors to adapt to changing physiological conditions and environments.SIGNIFICANCE STATEMENT In the acquisition of olfactory memory in Drosophila, the PAM cluster of dopamine neurons (DANs) mediates the reward signal, while the PPL1 cluster of DANs conveys the punishment signal to the Kenyon cells of the mushroom bodies, which serve as the center for memory formation. We found that GABA co-transmitters in the PAM DANs and glutamate co-transmitters in the PPL1 DANs inhibit olfactory memory formation. Our findings demonstrate that long-term memory acquisition, which typically necessitates multiple-spaced training sessions to establish aversive memory, can be triggered with a single training cycle in cases where the glutamate co-transmission is inhibited, even within a single subset of PPL1 DANs, suggesting that the glutamate co-transmission may modulate the threshold for memory acquisition.