Glutamate Efflux Research Articles

Analysis of the effects of polyunsaturated fatty acids on transporter expressions using a PCR array: Induction of xCT/SLC7A11 in human placental BeWo cells

ObjectivePolyunsaturated fatty acids (PUFAs), including arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are essential for adequate fetal growth. The aim of the present study was to elucidate the effects of PUFAs on the expression and function of placental transporters, which play important roles in placental functions including the supply of nutrients to the fetus, excretion of metabolites, and protection of the fetus from xenobiotics. MethodsHuman placental choriocarcinoma BeWo cells were used as a trophoblast model. PUFA-induced alteration in the gene expression of 84 transporters was investigated by a commercially available PCR array. Protein levels and the activity of transporters were assessed by western blotting and uptake experiments, respectively. The placental expression of the transporters was analyzed using pregnant Wistar rats. ResultsPUFAs (AA, EPA, and DHA) increased cystine/glutamate transporter xCT/SLC7A11, which mediates the cellular uptake of cystine coupled with the efflux of glutamate in human placental choriocarcinoma BeWo cells. These PUFAs also increased [14C]-cystine uptake in BeWo cells. PUFA-induced xCT/SLC7A11 mRNA expression was not blocked by nuclear factor-erythroid 2-related factor-2 (NRF2) knockdown. Reverse transcription (RT)-PCR analysis indicated that xCT/Slc7a11 mRNA was detected in rat placenta and the expression level at gestational day (GD) 12 was higher than that at GD 20. ConclusionThese results indicate that PUFAs promoted cystine uptake in placental cells by inducing xCT/SLC7A11 expression and NRF2 did not contribute to upregulation of xCT/SLC7A11 by PUFAs. Furthermore, xCT expression in rat placenta may change during pregnancy.

Estrone sulphate uptake by the microvillous membrane of placental syncytiotrophoblast is coupled to glutamate efflux

Organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs) are transport proteins that mediate exchange of metabolites, hormones and waste products. Directional transport by these transporters can occur when exchange is coupled to the gradients of other substrates. This study investigates whether the activity of OATP4A1 and OATP2A1 on the maternal facing microvillus membrane of the placental syncytiotrophoblast is coupled to the glutamate gradient. OAT and OATP transporter proteins were over expressed in Xenopus oocytes to study their transport characteristics. Further transport studies were performed in term human placental villous fragments. Xenopus oocytes expressing OATP4A1 mediated glutamate uptake. No glutamate transport was observed in oocytes expressing OAT1, OAT3, OAT7 or OATP2A1. In oocytes expressing OATP4A1, uptake of estrone sulphate, thyroid hormones T3 and T4 and the bile acid taurocholate stimulated glutamate efflux. In term placental villous fragments addition of estrone sulphate and taurocholate trans-stimulated glutamate efflux. Coupling of OATP4A1 to the glutamate gradient may drive placental uptake of estrone-sulphate and thyroid hormone while also facilitating uptake of potentially harmful bile acids. In contrast, if OATP2A1 is not coupled to a similar gradient, it may function more effectively as an efflux transporter, potentially mediating efflux of prostaglandins to the mother. This study provides further evidence for glutamate as an important counter-ion driving transport into the placenta.

Rapid-Acting Antidepressants.

Conventional antidepressants are thought to produce their impact on clinical symptoms by increasing the central availability of biogenic amine neurotransmitters (the monoamine hypothesis of depression). These drugs continue to be the primary medicines used in major depressive disorder. Although they have biological effects after acute dosing, full antidepressant response generally takes weeks of daily administration. Lack of rapid onset is a large limitation in antidepressant therapy (e.g., suicide, lack of medication compliance, difficulty switching medications). The present review of the literature discusses the preclinical and clinical findings on compounds that can produce immediate symptom relief. These compounds include ketamine, scopolamine, and mechanistically-related drugs. Newer additions to the list of potential rapid-acting agents include antagonists of metabotropic (mGlu) 2/3 receptors, negative allosteric modulators of α5-containing GABAA receptors, and psychedelic compounds. An additional benefit of these compounds is that they have demonstrated large effect sizes and, importantly, demonstrated efficacy in patient's refractory to other treatments. A drawback of some of these compounds, to date, is finding ways to expand the duration of clinical efficacy. In addition, for some compounds, the side-effect profile requires management. A primary mechanism by which rapid effects might be produced is through the amplification of excitatory neurotransmission through activation of AMPA receptors. The extracellular efflux of glutamate induced by these drugs has been documented and provides the hypothesized triggering mechanism for AMPA receptor amplification. The preclinical and clinical literature strongly suggests that rapid-acting antidepressants are the current focus of antidepressant drug discovery. Promising clinical findings exist for several compounds including ketamine and other NMDA receptor antagonists, scopolamine, and psilocybin. Two compounds are in late stage clinical development: GLYX-13 (Rapastinel) and eskekamine.

Liver-Type Glutaminase is an Essential and Druggable Metabolic Node in Defined Breast Cancer Subtypes

A key strategy targeting the metabolic changes underlying tumorigenesis involves inhibiting the glutaminase isozyme GLS to disrupt glutamine catabolism. However, identifying patient groups for this approach remains a challenge. Here, we define the roles of GLS and its less studied counterpart liver-type glutaminase (GLS2) in breast cancer. We find that GLS2 is upregulated and essential for TCA cycle anaplerosis, cell proliferation, and tumorigenesis in luminal-subtype breast cancers, which account for >70% of disease incidence, and that it mediates resistance to GLS-selective inhibitors. Although GLS is overexpressed in basal-subtype breast cancers, sensitivity to GLS inhibitors depends on glutamate efflux through the xCT antiporter. These findings establish an important role for GLS2 in tumorigenesis and suggest patient stratification approaches for glutaminase-targeted therapy.

Lucy in the sky with ketamine: Psychoactive drugs have potential for a major breakthrough in treating depression.

EMBO Reports (2018) e47118 “It felt like being in a pod […], like a little boat, going along through rooms [that had] white breathing walls.” This is how American standup comedian Neal Brennan described his experience with ketamine, a psychoactive drug also known as Special K in the club scene, in an interview with fellow comedian Joe Rogan. “I just couldn't get over the fact that this was happening in a doctor's office,” he said about taking ketamine as a medication against depression. > One may argue that treating psychologically unstable patients with an addictive drug that has psychedelic side effects is not a good idea. Ketamine was introduced commercially in the United States in 1970 as an anesthetic. At lower doses, it causes dissociations, out‐of‐body experiences, and hallucinations, which explains its career as club drug. But during the past two decades, it has also gained attention as a fast‐acting antidepressant. Its antidepressant effect was first noted by researchers at Yale University in the 1990s, and the effect was reproduced in a larger double‐blind, placebo‐controlled trial at the US National Institute of Mental Health, published in 2006. A number of studies have since shown the effectiveness of ketamine in treating patients with depression, suicidal ideation, and post‐traumatic stress disorder [1]. Researchers have called it the biggest breakthrough in depression research in half a century. Ketamine is not yet approved by regulatory agencies, but is widely used off‐label by private so‐called ketamine clinics. New medications to treat depression are urgently needed. The last significant innovation—Prozac, the first selective serotonin reuptake inhibitor (SSRI)—is now 30 years old, and SSRIs and many me‐too drugs of similar structure and function still dominate the treatment of depression. They are, however, anything but satisfactory. It takes 6–8 weeks for their effect to kick in, and …

Corynebacterium glutamicum mechanosensitive channels: towards unpuzzling "glutamate efflux" for amino acid production.

Corynebacterium glutamicum has been utilized for industrial amino acid production, especially for monosodium glutamate (MSG), the food-additive for the "UMAMI" category of taste sensation, which is one of the five human basic tastes. Glutamate export from these cells is facilitated by the opening of mechanosensitive channels in the cell membrane within the bacterial cell envelope following specific treatments, such as biotin limitation, addition of Tween 40 or penicillin. A long-unsolved puzzle still remains how and why C. glutamicum mechanosensitive channels are activated by these treatments to export glutamate. Unlike mechanosensitive channels in other bacteria, these channels are not simply osmotic safety valves that prevent these bacteria from bursting upon a hypo-osmotic shock. They also function as metabolic valves to continuously release glutamate as components of a pump-and-leak mechanism regulating the cellular turgor pressure. Recent studies have demonstrated that the opening of the mechanosensitive channel, MscCG, mainly facilitates the efflux of glutamate and not of other amino acids and that the "force-from-lipids" gating mechanism of channels also applies to the MscCG channel. The bacterial types of mechanosensitive channels are found in cell-walled organisms from bacteria to land plants, where their physiological functions have been specialized beyond their basic function in bacterial osmoregulation. In the case of the C. glutamicum MscCG channels, they have evolved to function as specialized glutamate exporters.

Reduced cortical serotonin 5-HT2A receptor binding and glutamate activity in high compulsive drinker rats

Serotonin2A receptors and glutamate signaling have been implicated in the pathophysiology and treatment of compulsive spectrum disorders. Schedule-Induced Polydipsia (SIP), characterized by excessive drinking under intermittent food reinforcement schedules, is a valid model for studying the compulsive phenotype in rats. We explored the expression, function, and neurochemistry of 5-HT2A receptors in the frontal cortex (FC) of rats with individual differences to compulsivity. Rats were selected for high (HD) versus low (LD) drinking on SIP. First, we measured 5-HT2A, 5-HT1A, and mGlu2/3 receptors and serotonin transporter binding in different brain regions. Second, we assessed the effect of microinfusion into the medial prefrontal cortex (mPFC) of the 5-HT2A/C receptor agonist DOI, the mGlu2/3 agonist LY379268, and the combination of DOI with the 5-HT2A receptor antagonist M100907 and the 5-HT2C receptor antagonist SB242084. Finally, we measured the serotonin and glutamate efflux in mPFC in basal condition and after DOI local application. The compulsive HD rats showed a specific reduction of 5-HT2A receptor binding in FC compared to LD rats. The highest dose of DOI reduced compulsive drinking in HD rats on SIP, whereas LY379268 did not induce any significant effect. The 5-HT2A receptor antagonist M100907 reversed the DOI induced reduction on compulsive drinking in HD rats while blocking the 5-HT2C receptor did not affect SIP. Compulsive HD rats showed increased serotonin and decreased glutamate efflux in basal conditions that were modified by the DOI application. These findings indicate that reduced 5-HT2A receptor binding and glutamate neurochemical mechanisms may underlie compulsive behavior vulnerability.

Prelimbic NMDA receptors stimulation mimics the attenuating effects of clozapine on the auditory electrophysiological rebound induced by ketamine withdrawal

Ketamine (KET) is a non-competitive N-Methyl-d-aspartate (NMDA) receptors antagonist that intensifies sensory experiences, prompts hallucinations and delusions, exacerbates previously installed psychosis and disrupts physiological evoked potentials (AEPs). Pharmacologically, KET stimulates glutamate efflux in the medial prefrontal cortex, mainly in the prelimbic (PrL) sub-region. Efferences from this region exert a top-down regulatory control of bottom-up sensory processes either directly or indirectly. In the midbrain, the central nucleus of the inferior colliculus (CIC) plays a fundamental role in the processing of auditory ascending information related to sound localization, sensorimotor gating, and preattentive event-related potentials. Auditory hallucinations elicited during a psychotic outbreak are accompanied by CIC neural activation. Thus, it is possible that NMDA-mediated glutamate neurotransmission in the PrL indirectly modulates CIC neuronal firing. The aim of the present study was to assess the effects of KET on the latency and amplitude of AEPs elicited in the CIC of rats tested during KET effects and following withdrawal from the chronic administration. Changes on emotionally induced by KET treatment were evaluated with the use of the elevated zero maze (EZM). Unlike typical neuroleptics, the atypical antipsychotic clozapine (CLZ) potently blocks the disruption of the sensorimotor gating induced by NMDA antagonists. Therefore, the effects of KET withdrawal on AEPs were challenged with a systemic injection of CLZ. In addition, we further investigated the role of NMDA receptors of the PrL on the AEPs expression recorded in the CIC through intra-PrL infusions of NMDA itself. Our results showed that the processing of sensory information in the CIC is under indirect control of PrL. These data suggest that the long-term KET treatment disrupts the collicular auditory field potentials, possibly through influencing PrL glutamate activity on intrinsic 5-HT mechanisms in the dorsal raphe and CIC.

TPA-023 attenuates subchronic phencyclidine-induced declarative and reversal learning deficits via GABAA receptor agonist mechanism: possible therapeutic target for cognitive deficit in schizophrenia

GABAergic drugs are of interest for the treatment of anxiety, depression, bipolar disorder, pain, cognitive impairment associated with schizophrenia (CIAS), and other neuropsychiatric disorders. Some evidence suggests that TPA-023, (7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b] pyridazine), a GABAA α2,3 subtype-selective GABAA partial agonist and α1/5 antagonist, and the neurosteroid, pregnenolone sulfate, a GABAA antagonist, may improve CIAS in pilot clinical trials. The goal of this study was to investigate the effect of TPA-023 in mice after acute or subchronic (sc) treatment with the N-methyl-d-aspartate receptor (NMDAR) antagonist, phencyclidine (PCP), on novel object recognition (NOR), reversal learning (RL), and locomotor activity (LMA) in rodents. Acute TPA-023 significantly reversed scPCP-induced NOR and RL deficits. Co-administration of sub-effective dose (SED) TPA-023 with SEDs of the atypical antipsychotic drug, lurasidone, significantly potentiated the effect of TPA-023 in reversing the scPCP-induced NOR deficit. Further, scTPA-023 co-administration significantly prevented scPCP-induced NOR deficit for 5 weeks. Also, administration of TPA-023 for 7 days following scPCP reversed the NOR deficit for 1 week. However, TPA-023 did not blunt acute PCP-induced hyperactivity, suggesting lack of efficacy as a treatment for psychosis. Systemic TPA-023 significantly blocked lurasidone-induced increases in cortical acetylcholine, dopamine, and glutamate without affecting increases in norepinephrine and with minimal effect on basal release of these neurotransmitters. TPA-023 significantly inhibited PCP-induced cortical and striatal dopamine, serotonin, norepinephrine, and glutamate efflux. These results suggest that TPA-023 and other GABAA agonists may be of benefit to treat CIAS.

Regional-specific effects of cerebral ischemia/reperfusion and dehydroepiandrosterone on synaptic NMDAR/PSD-95 complex in male Wistar rats

Excessive glutamate efflux and N-methyl-D-aspartate receptor (NMDAR) over-activation represent well-known hallmarks of cerebral ischemia/reperfusion (I/R) injury, still, expression of proteins involved in this aspect of I/R pathophysiology show inconsistent data. Neurosteroid dehydroepiandrosterone (DHEA) has been proposed as potent NMDAR modulator, but its influence on I/R-induced changes up to date remains questionable. Therefore, I/R-governed alteration of vesicular glutamate transporter 1 (vGluT1), synaptic NMDAR subunit composition, postsynaptic density protein 95 (PSD-95) and neuronal morphology alone or following DHEA treatment were examined. For that purpose, adult male Wistar rats were treated with a single dose of vehicle or DHEA (20 mg/kg i.p.) 4 h following sham operation or 15 min bilateral common carotid artery occlusion. Western blot was used for analyses of synaptic protein expressions in hippocampus and prefrontal cortex, while neuronal morphology was assessed using Nissl staining. Regional-specific postischemic changes were detected on protein level i.e. signs of neuronal damage in CA1 area was accompanied with hippocampal vGluT1, NR1, NR2B enhancement and PSD-95 decrement, while histological changes observed in layer III were associated with decreased NR1 subunit in prefrontal cortex. Under physiological conditions DHEA had no effect on protein and histological appearance, while in ischemic milieu it restored hippocampal PSD-95 and NR1 in prefrontal cortex to the control level. Along with intact neurons, ones characterized by morphology observed in I/R group were also present. Future studies involving NMDAR-related intracellular signaling and immunohistochemical analysis will reveal precise effects of I/R and DHEA treatment in selected brain regions.

Nucleus accumbens GLT-1a overexpression reduces glutamate efflux during reinstatement of cocaine-seeking but is not sufficient to attenuate reinstatement

Cocaine use disorder is a chronically relapsing disease without FDA-approved treatments. Using a rodent model of cocaine relapse, we and others have previously demonstrated that the beta-lactam antibiotic ceftriaxone attenuates cue- and cocaine-primed reinstatement of cocaine-seeking. Ceftriaxone restores cocaine-induced deficits in both system xc- and GLT-1 expression and function in the nucleus accumbens core (NAc). We recently demonstrated that restoration of GLT-1 expression in the NAc is necessary for ceftriaxone to attenuate reinstatement of cocaine-seeking. Here we used an adeno-associated virus (AAV) to overexpress GLT-1a in the NAc to investigate whether such restoration is sufficient to attenuate cue- and cocaine-primed reinstatement. Rats self-administered cocaine for two weeks and received injections of either AAV-GFAP-GLT-1a or AAV-GFAP-eGFP in the NAc following the last day of self-administration. Rats then underwent three weeks of extinction training (during which time transduction and expression occurred) before undergoing a cue- or cocaine-primed reinstatement test. Microdialysis for the quantification of glutamate efflux in the NAc was conducted during the cocaine-primed test. Rats that received AAV-GFAP-GLT-1a reinstated cue-primed cocaine-seeking in a similar manner as rats that received the control AAV-GFAP-eGFP. Upregulation of GLT-1a attenuated glutamate efflux during a cocaine-primed reinstatement test, but was not sufficient to attenuate reinstatement. We confirmed that GLT-1a upregulation resulted in functional upregulation of glutamate transport and expression, without affecting sodium-independent glutamate uptake, indicating system xc-was not altered. These results indicate that upregulation of NAc GLT-1 transporters alone is not sufficient to prevent the reinstatement of cocaine-seeking and implicate additional mechanisms in regulating glutamate efflux.

Bidirectional variation in glutamate efflux in the medial prefrontal cortex induced by selective positive and negative allosteric mGluR5 modulators.

Dysregulation of prefrontal cortical glutamatergic signalling via NMDA receptor hypofunction has been implicated in cognitive dysfunction and impaired inhibitory control in such neuropsychiatric disorders as schizophrenia, attention‐deficit hyperactivity disorder and drug addiction. Although NMDA receptors functionally interact with metabotropic glutamate receptor 5 (mGluR5), the consequence of this interaction for glutamate release in the prefrontal cortex (PFC) remains unknown. We therefore investigated the effects of positive and negative allosteric mGluR5 modulation on changes in extracellular glutamate efflux in the medial PFC (mPFC) induced by systemic administration of the non‐competitive NMDA receptor antagonist dizocilpine (or MK801) in rats. Extracellular glutamate efflux was measured following systemic administration of the positive allosteric mGluR5 modulator [S‐(4‐Fluoro‐phenyl)‐{3‐[3‐(4‐fluoro‐phenyl)‐[1,2,4]‐oxadiazol‐5‐yl]‐piperidin‐1‐yl}‐methanone] (ADX47273; 100 mg/kg, p.o.) and negative allosteric mGluR5 modulator [2‐chloro‐4‐{[1‐(4‐fluorophenyl)‐2,5‐dimethyl‐1H‐imidazol‐4‐yl]ethynyl}pyridine] (RO4917523; 0.3 mg/kg, p.o.), using a wireless glutamate biosensor in awake, freely moving rats. The effect of MK801 (0.03–0.06 mg/kg, s.c.) on mPFC glutamate efflux was also investigated in addition to the effects of MK801 (0.03 mg/kg, s.c.) following ADX47273 (100 mg/kg, p.o.) pre‐treatment. ADX47273 produced a sustained increase in glutamate efflux and increased the effect of NMDA receptor antagonism on glutamate efflux in the mPFC. In contrast, negative allosteric mGluR5 modulation with RO4917523 decreased glutamate efflux in the mPFC. These findings indicate that positive and negative allosteric mGluR5 modulators produce long lasting and opposing actions on extracellular glutamate efflux in the mPFC. Positive and negative allosteric modulators of mGluR5 may therefore be viable therapeutic agents to correct abnormalities in glutamatergic signalling present in a range of neuropsychiatric disorders.

Glutamate Excitotoxicity Linked to Spermine Oxidase Overexpression.

Excitotoxic stress has been associated with several different neurological disorders, and it is one of the main causes of neuronal degeneration and death. To identify new potential proteins that could represent key factors in excitotoxic stress and to study the relationship between polyamine catabolism and excitotoxic damage, a novel transgenic mouse line overexpressing spermine oxidase enzyme in the neocortex (Dach-SMOX) has been engineered. These transgenic mice are more susceptible to excitotoxic injury and display a higher oxidative stress, highlighted by 8-Oxo-2'-deoxyguanosine increase and activation of defense mechanisms, as demonstrated by the increase of nuclear factor erythroid 2-related factor 2 (Nrf-2) in the nucleus. In Dach-SMOX astrocytes and neurons, an alteration of the phosphorylated and non-phosphorylated subunits of glutamate receptors increases the kainic acid response in these mice. Moreover, a decrease in excitatory amino acid transporters and an increase in the system xc- transporter, a Nrf-2 target, was observed. Sulfasalazine, a system xc- transporter inhibitor, was shown to revert the increased susceptibility of Dach-SMOX mice treated with kainic acid. We demonstrated that astrocytes play a crucial role in this process: neuronal spermine oxidase overexpression resulted in an alteration of glutamate excitability, in glutamate uptake and efflux in astrocytes involved in the synapse. Considering the involvement of oxidative stress in many neurodegenerative diseases, Dach-SMOX transgenic mouse can be considered as a suitable in vivo genetic model to study the involvement of spermine oxidase in excitotoxicity, which can be considered as a possible therapeutic target.

Increased acetylcholine and glutamate efflux in the prefrontal cortex following intranasal orexin-A (hypocretin-1).

Orexin/hypocretin neurons of the lateral hypothalamus and perifornical area are integrators of physiological function. Previous work from our laboratory and others has shown the importance of orexin transmission in cognition. Age-related reductions in markers of orexin function further suggest that this neuropeptide may be a useful target for the treatment of age-related cognitive dysfunction. Intranasal administration of orexin-A (OxA) has shown promise as a therapeutic option for cognitive dysfunction. However, the neurochemical mechanisms of intranasal OxA administration are not fully understood. Here, we use immunohistochemistry and invivo microdialysis to define the effects of acute intranasal OxA administration on: (i) activation of neuronal populations in the cortex, basal forebrain, and brainstem and (ii) acetylcholine (ACh) and glutamate efflux in the prefrontal cortex (PFC) of Fischer 344/Brown Norway F1 rats. Acute intranasal administration of OxA significantly increased c-Fos expression, a marker for neuronal activation, in the PFC and in subpopulations of basal forebrain cholinergic neurons. Subsequently, we investigated the effects of acute intranasal OxA on neurotransmitter efflux in the PFC and found that intranasal OxA significantly increased both ACh and glutamate efflux in this region. These findings were independent from any changes in c-Fos expression in orexin neurons, suggesting that these effects are not resultant from direct activation of orexin neurons. In total, these data indicate that intranasal OxA may enhance cognition through activation of distinct neuronal populations in the cortex and basal forebrain and through increased neurotransmission of ACh and glutamate in the PFC.

Metabolic correction by pyruvate halts acquired epilepsy in multiple rodent models

Metabolic intervention strategy of epilepsy treatment has been gaining broader attention due to accumulated evidence that hypometabolism, manifested in humans as reduced brain glucose consumption, is a principal factor in acquired epilepsy. Therefore, targeting deficient energy metabolism may be an effective approach for treating epilepsy. To confront this pathology we utilized pyruvate, which besides being an anaplerotic mitochondrial fuel possesses a unique set of neuroprotective properties as it: (i) is a potent reactive oxygen species scavenger; (ii) abates overactivation of Poly [ADP-ribose] polymerase 1 (PARP-1); (iii) facilitates glutamate efflux from the brain; (iv) augments brain glycogen stores; (v) is anti-inflammatory; (vi) prevents neuronal hyperexcitability; and (vii) normalizes the cytosolic redox state. In vivo, chronic oral pyruvate administration completely abolished established epileptic phenotypes in three accepted and fundamentally different rodent acquired epilepsy models. Our study reports metabolic correction by pyruvate as a potentially highly effective treatment of acquired epilepsies.

Arctic ground squirrel hippocampus tolerates oxygen glucose deprivation independent of hibernation season even when not hibernating and after ATP depletion, acidosis, and glutamate efflux.

Cerebral ischemia/reperfusion (I/R) triggers a cascade of uncontrolled cellular processes that perturb cell homeostasis. The arctic ground squirrel (AGS), a seasonal hibernator resists brain damage following cerebral I/R caused by cardiac arrest and resuscitation. However, it remains unclear if tolerance to I/R injury in AGS depends on the hibernation season. Moreover, it is also not clear if events such as depletion of ATP, acidosis, and glutamate efflux that are associated with anoxic depolarization are attenuated in AGS. Here, we employ a novel microperfusion technique to test the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is independent of the hibernation season and persists even after glutamate efflux. Acute hippocampal slices were harvested from summer euthermic AGS, hibernating AGS, and interbout euthermic AGS. Slices were subjected to oxygen glucose deprivation (OGD), an invitro model of I/R injury to determine cell death marked by lactate dehydrogenase (LDH) release. ATP was assayed using ENLITEN ATP assay. Glutamate and aspartate efflux was measured using capillary electrophoresis. For acidosis, slices were subjected to pH 6.4 or ischemic shift solution (ISS). Acute hippocampal slices from rats were used as a positive control, susceptible to I/R injury. Our results indicate that when tissue temperature is maintained at 36°C, hibernation season has no influence on OGD-induced cell death in AGS hippocampal slices. Our data also show that tolerance to OGD in AGS hippocampal slices occurs despite loss of ATP and glutamate release, and persists during conditions that mimic acidosis and ionic shifts, characteristic of cerebral I/R. Read the Editorial Comment for this article on page 10.

Upregulation of orexin/hypocretin expression in aged rats: Effects on feeding latency and neurotransmission in the insular cortex

Aging is associated with changes in numerous homeostatic functions, such as food intake, that are thought to be mediated by the hypothalamus. Orexin/hypocretin neurons of the hypothalamus regulate several physiological functions, including feeding, sleep and wakefulness. Evidence from both clinical and animal studies supports the notion that aging is associated with loss or dysregulation of the orexin system. Here, we used virus-mediated gene transfer to manipulate expression of orexin peptides in young and aged rats and examined behavioral and neurochemical correlates of food intake in these animals. Aged rats showed slower feeding latencies when presented with palatable food compared to young control rats, and these deficits were ameliorated by upregulation of orexin expression. Similarly, young animals treated with a virus designed to decrease preproorexin expression showed longer feeding latencies reminiscent of aged control rats. Feeding was also associated with increased acetylcholine, glutamate and GABA efflux in insular cortex of young control animals. Orexin upregulation did not restore deficits in feeding-elicited release of these neurotransmitters in aged rats, but did enhance basal neurotransmitter levels which may have contributed to the behavioral correlates of these genetic manipulations. These studies demonstrate that age-related deficits in behavioral and neurochemical measures of feeding are likely to be mediated, in part, by the orexin system. Because these same neurotransmitter systems have been shown to underlie orexin effects on cognition, treatments which increase orexin function may have potential for improving both physiological and cognitive manifestations of certain age-related disorders.

HIF-1α triggers long-lasting glutamate excitotoxicity via system xc- in cerebral ischaemia-reperfusion.

Hypoxia-inducible factor 1α (HIF-1α) controls many genes involved in physiological and pathological processes. However, its roles in glutamatergic transmission and excitotoxicity are unclear. Here, we proposed that HIF-1α might contribute to glutamate-mediated excitotoxicity during cerebral ischaemia-reperfusion (CIR) and investigated its molecular mechanism. We showed that an HIF-1α conditional knockout mouse displayed an inhibition in CIR-induced elevation of extracellular glutamate and N-methyl-d-aspartate receptor (NMDAR) activation. By gene screening for glutamate transporters in cortical cells, we found that HIF-1α mainly regulates the cystine-glutamate transporter (system xc- ) subunit xCT by directly binding to its promoter; xCT and its function are up-regulated in the ischaemic brains of rodents and humans, and the effects lasted for several days. Genetic deletion of xCT in cortical cells of mice inhibits either oxygen glucose deprivation/reoxygenation (OGDR) or CIR-mediated glutamate excitotoxicity in vitro and in vivo. Pharmaceutical inhibition of system xc- by a clinically approved anti-cancer drug, sorafenib, improves infarct volume and functional outcome in rodents with CIR and its therapeutic window is at least 3 days. Taken together, these findings reveal that HIF-1α plays a role in CIR-induced glutamate excitotoxicity via the long-lasting activation of system xc- -dependent glutamate outflow and suggest that system xc- is a promising therapeutic target with an extended therapeutic window in stroke. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel mode of operation of SLC22A11: Membrane insertion of estrone sulfate versus translocation of uric acid and glutamate

Estrone sulfate alias estrone-3-sulfate (E3S) is considerably larger and much more hydrophobic than typical substrates of SLC22 transporters. It is puzzling that many otherwise unrelated transporters have been reported to transport E3S. Here we scrutinized the mechanism of transport of E3S by SLC22A11 (alias OAT4), by direct comparison with uric acid (UA), an important physiological substrate. Heterologous expression of SLC22A11 in human 293 cells gave rise to a huge unidirectional efflux of glutamate (Glu) and aspartate, as determined by LC–MS/MS. The uptake of E3S was 20-fold faster than the uptake of UA. Yet, the outward transport of Glu was inhibited by extracellular E3S, but not by UA. The release of E3S after preloading was trans-stimulated by extracellular dehydroepiandrosterone sulfate (DHEAS), but neither by UA nor 6-carboxyfluorescein (6CF). The equilibrium accumulation of E3S was enhanced 3-fold by replacement of chloride with gluconate, but the opposite effect was observed for UA. These results establish that SLC22A11 provides entirely different transport mechanisms for E3S and UA. Therefore, E3S must not be used as a substitute for UA to assay the function of SLC22A11. In equilibrium accumulation experiments, the transporter-mediated uptake was a linear function of the concentration of UA and 6CF. By contrast, in the same concentration range the graph for E3S was hyperbolic. This suggests that SLC22A11 inserts E3S into a small volume with limited capacity, the plasma membrane. Our data support the notion that the reverse process, extraction from the membrane, is also catalyzed by the carrier.

The Role of Primary Motor Cortex (M1) Glutamate and GABA Signaling in l-DOPA-Induced Dyskinesia in Parkinsonian Rats.

Long-term treatment of Parkinson's disease with l-DOPA almost always leads to the development of involuntary movements termed l-DOPA-induced dyskinesia. Whereas hyperdopaminergic signaling in the basal ganglia is thought to cause dyskinesia, alterations in primary motor cortex (M1) activity are also prominent during dyskinesia, suggesting that the cortex may represent a therapeutic target. The present study used the rat unilateral 6-hydroxydopamine lesion model of Parkinson's disease to characterize in vivo changes in GABA and glutamate neurotransmission within M1 and determine their contribution to behavioral output. 6-Hydroxydopamine lesion led to parkinsonian motor impairment that was partially reversed by l-DOPA. Among sham-lesioned rats, l-DOPA did not change glutamate or GABA efflux. Likewise, 6-hydroxydopamine lesion did not impact GABA or glutamate among rats chronically treated with saline. However, we observed an interaction of lesion and treatment whereby, among lesioned rats, l-DOPA given acutely (1 d) or chronically (14-16 d) reduced glutamate efflux and enhanced GABA efflux. Site-specific microinjections into M1 demonstrated that l-DOPA-induced dyskinesia was reduced by M1 infusion of a D1 antagonist, an AMPA antagonist, or a GABAA agonist. Overall, the present study demonstrates that l-DOPA-induced dyskinesia is associated with increased M1 inhibition and that exogenously enhancing M1 inhibition may attenuate dyskinesia, findings that are in agreement with functional imaging and transcranial magnetic stimulation studies in human Parkinson's disease patients. Together, our study suggests that increasing M1 inhibitory tone is an endogenous compensatory response designed to limit dyskinesia severity and that potentiating this response is a viable therapeutic strategy. Most Parkinson's disease patients will receive l-DOPA and eventually develop hyperkinetic involuntary movements termed dyskinesia. Such symptoms can be as debilitating as the disease itself. Although dyskinesia is associated with dynamic changes in primary motor cortex physiology, to date, there are no published studies investigating in vivo neurotransmitter release in M1 during dyskinesia. In parkinsonian rats, l-DOPA administration reduced M1 glutamate efflux and enhanced GABA efflux, coincident with the emergence of dyskinetic behaviors. Dyskinesia could be reduced by local M1 modulation of D1, AMPA, and GABAA receptors, providing preclinical support for the notion that exogenously blunting M1 signaling (pharmacologically or with cortical stimulation) is a therapeutic approach to the treatment of debilitating dyskinesias.