Release Of Aspartate Research Articles

Salmonella Typhimurium expansion in the inflamed murine gut is dependent on aspartate derived from ROS-mediated microbiota lysis

Inflammation boosts the availability of electron acceptors in the intestinal lumen, creating a favorable niche for pathogenic Enterobacteriaceae. However, the mechanisms linking intestinal inflammation-mediated changes in luminal metabolites and pathogen expansion remain unclear. Here, we show that mucosal inflammation induced by Salmonella enterica serovar Typhimurium (S. Tm) infection increases intestinal levels of the amino acid aspartate. S. Tm used aspartate-ammonia lyase (aspA)-dependent fumarate respiration for growth in the murine gut only during inflammation. AspA-dependent growth advantage was abolished in the gut of germ-free mice and restored in gnotobiotic mice colonized with members of the classes Bacteroidia and Clostridia. Reactive oxygen species (ROS) produced during the host response caused lysis of commensal microbes, resulting in the release of microbiota-derived aspartate that was used by S. Tm, in concert with nitrate-dependent anaerobic respiration, to outcompete commensal Enterobacteriaceae. Our findings demonstrate the role of microbiota-derived amino acids in driving respiration-dependent S. Tm expansion during colitis.

Physiological Functions of the Volume-Regulated Anion Channel VRAC/LRRC8 and the Proton-Activated Chloride Channel ASOR/TMEM206.

Volume-regulated anion channels (VRACs) and the acid-sensitive outwardly rectifying anion channel (ASOR) mediate flux of chloride and small organic anions. Although known for a long time, they were only recently identified at themolecular level. VRACs are heteromers consisting of LRRC8 proteins A to E. Combining the essential LRRC8A with different LRRC8 paralogues changes key properties of VRAC such as conductance or substrate selectivity, which is how VRACs are involved in multiple physiological functions including regulatory volume decrease, cell proliferation and migration, cell death, purinergic signalling, fat and glucose metabolism, insulin signalling, and spermiogenesis. VRACs are also involved in pathological conditions, such as the neurotoxic release of glutamate and aspartate. Certain VRACs are also permeable to larger, organic anions, including antibiotics and anti-cancer drugs, making them an interesting therapeutic target. ASOR, also named proton-activated chloride channel (PAC), is formed by TMEM206 homotrimers on the plasma membrane and on endosomal compartments where it mediates chloride flux in response to extracytosolic acidification and plays a role in the shrinking and maturation of macropinosomes. ASOR has been shown to underlie neuronal swelling which causes cell death after stroke as well as promoting the metastasis of certain cancers, making them intriguing therapeutic targets as well.

3207 – UNDERSTANDING AND TARGETING ASPARTATE METABOLISM IN THE LEUKEMIC NICHE

The bone marrow provides a specific microenvironment that sustains hematopoiesis as well as different blood cancers. Several studies have shown that non-transformed cells in the tumor microenvironment adapt and support cancer cell survival and proliferation. One mechanism by which niche cells can maintain cancer cells is by providing essential nutrients. Our prior research demonstrated aspartate release from bone marrow stromal cells and aspartate accumulation in the bone marrow. In the current study, we found that both normal hematopoietic progenitors and acute myeloid leukemia (AML) cells express aspartate transporters. AML cells take up aspartate from their microenvironment, and blocking aspartate transport reduces leukemic burden in a mouse model of AML. Current efforts aim at further exploring the therapeutic potential of targeting aspartate metabolism in AML. The bone marrow provides a specific microenvironment that sustains hematopoiesis as well as different blood cancers. Several studies have shown that non-transformed cells in the tumor microenvironment adapt and support cancer cell survival and proliferation. One mechanism by which niche cells can maintain cancer cells is by providing essential nutrients. Our prior research demonstrated aspartate release from bone marrow stromal cells and aspartate accumulation in the bone marrow. In the current study, we found that both normal hematopoietic progenitors and acute myeloid leukemia (AML) cells express aspartate transporters. AML cells take up aspartate from their microenvironment, and blocking aspartate transport reduces leukemic burden in a mouse model of AML. Current efforts aim at further exploring the therapeutic potential of targeting aspartate metabolism in AML.

A Review of Studies on the Use of New Non-Gabapentinoid Anticonvulsants and Muscle Relaxants in the Control of Acute Postoperative Pain

Postoperative pain management in any type of surgery is an important issue for patients. The pain relief contributes to decreased hospital costs, shorter hospital stay, and elevated patient consent. Correspondingly, in this review article we assessed the postoperative pain management efficacy of new non-gabapentinoid anticonvulsants and muscle relaxant drugs in pre-clinical and clinical studies. The scientific databases, including PubMed, Embase, Scopus, and Google Scholar, were searched using relevant keywords: surgery, postoperative pain, analgesia, non-gabapentinoid anticonvulsants, and muscle relaxants. New anticonvulsants reduce postoperative pain, length of stay at postoperative anesthesia care unit, and analgesic requirement after surgeries. Their mechanisms include the inhibition of glutamate release, blocking the N-type calcium channels of afferent neurons, inhibition of supraspinal 5-hydroxytryptamine-3 receptor, and prevention of cyclin-dependent kinase 5-mediated collapsin response mediator protein 2 phosphorylation. Also, muscle relaxants show the same effects by downregulation the Cacna1a, Cacna1b, and Runt-related transcription factor 1 genes of dorsal root ganglia, inhibition the release of glutamate, aspartate or substance P from the terminals of primary afferent C and Aδ fibers, expression of Neurokinin 1 receptor in the spinal dorsal horn, agonistic effect on α2-adrenoceptor, and stimulation of acetylcholine release in the spinal cord. With respect to the concerns regarding opioid abuse, muscle relaxants and non-gabapentinoid anticonvulsant drugs can be regarded as a safe option for postoperative pain control.

Neonatal Hypoglycemia and Brain Vulnerability.

Neonatal hypoglycemia is a common condition. A transient reduction in blood glucose values is part of a transitional metabolic adaptation following birth, which resolves within the first 48 to 72 h of life. In addition, several factors may interfere with glucose homeostasis, especially in case of limited metabolic stores or increased energy expenditure. Although the effect of mild transient asymptomatic hypoglycemia on brain development remains unclear, a correlation between severe and prolonged hypoglycemia and cerebral damage has been proven. A selective vulnerability of some brain regions to hypoglycemia including the second and the third superficial layers of the cerebral cortex, the dentate gyrus, the subiculum, the CA1 regions in the hippocampus, and the caudate-putamen nuclei has been observed. Several mechanisms contribute to neuronal damage during hypoglycemia. Neuronal depolarization induced by hypoglycemia leads to an elevated release of glutamate and aspartate, thus promoting excitotoxicity, and to an increased release of zinc to the extracellular space, causing the extensive activation of poly ADP-ribose polymerase-1 which promotes neuronal death. In this review we discuss the cerebral glucose homeostasis, the mechanisms of brain injury following neonatal hypoglycemia and the possible treatment strategies to reduce its occurrence.

Prevention and Healing of Calcium Signaling Mediated Neuronal Damage on successive Administration of Flavonoid Enriched Pterocarpus Marsupium Roxb in Peripheral Neuropathy Model

Background: Modern research on peripheral neuropathy circumstance utter that treatments with Vincristine (VCR) disturb the microtubular cells in sensory and motor neurons due to calcium over- load in sciatic nerve, unfortunately, VCR triggering the release of Tumor necrosis factor-α (TNFα) in central neurons causes excitotoxicity as well. Although ethnomedical information specifies that Pterocarpus marsupium Roxb (PM) is widely used for various nervous disorders, not yet justified on VCR induced peripheral neuropathy and in relation to central mechanism. Objective: This study is aimed to explore the possible central and peripheral mechanism of flavonoid enriched PM in VCR induced neuropathy model. Methods: Neuropathic pain was induced in female Wistar rats by VCR (75μg/ kg/day, i.p) for 10 days. Nociceptive thresholds were assessed by subjecting them to behavioral and biochemical estimation, proinflammatory cytokines along with morphological evaluation. Results: PM significantly increased the nociceptive threshold evident from various behavioral models in comparison to VCR group. More importantly, PM significantly reversed the VCR induced calcium elevation, glutamate and aspartate release in the brain. Discussion: It was also observed that the raised TNF-α, Interleukin-1β were controlled and interleukin- 10 was elevated in sciatic nerve after PM treatment. Evident from histology, PM markedly reversed the VCR induced axonal degeneration, Schwann cell hyperplasia, and myelin fibrosis. Conclusion: Flavonoid enriched PM both 100 & 200mg/kg post and co-administration exerted a preventive and curative effect in VCR induced neuropathic pain by controlling calcium-mediated excitotoxicity through peripheral and central mechanism.

Activation of voltage-gated sodium channels by BmK NT1 augments NMDA receptor function through Src family kinase signaling pathway in primary cerebellar granule cell cultures

Voltage-gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in excitable cells and are the molecular targets of an array of neurotoxins. BmK NT1, an α-scorpion toxin obtained from the scorpion Buthus martensii Karsch (BmK), produces neurotoxicity that is associated with extracellular Ca2+ influx through Na+-Ca2+ exchangers, N-methyl-d-aspartic acid (NMDA) receptors, and L-type Ca2+ channels in cultured cerebellar granule cells (CGCs). In the present study, we demonstrated that BmK NT1 triggered concentration-dependent release of excitatory neurotransmitters, glutamate and aspartate; both effects were eliminated by VGSC blocker, tetrodotoxin. More importantly, we demonstrated that a threshold concentration of BmK NT1 that produced marginal Ca2+ influx and neuronal death augmented glutamate-induced Ca2+ elevation and neuronal death in CGCs. BmK NT1-augmented glutamate-induced Ca2+ influx and neuronal death were suppressed by tetrodotoxin and MK-801 suggesting that the augmentation was through activation of VGSCs and NMDA receptors. Consistently, BmK NT1 also enhanced NMDA-induced Ca2+ influx. Further mechanistic investigations demonstrated that BmK NT1 increased the expression level of NMDA receptors on the plasma membrane and increased the phosphorylation level of NR2B at Tyr1472. Src family kinase inhibitor, 1-tert-butyl-3-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidin-4-yl]amine (PP2), but not the inactive analogue, 4-amino-1-phenylpyrazolo[3,4-d]pyrimidine (PP3), eliminated BmK NT1-triggered NR2B phosphorylation, NMDA receptor trafficking, as well as BmK NT1-augmented NMDA Ca2+ response and neuronal death. Considered together, these data demonstrated that both presynaptic (excitatory amino acid release) and postsynaptic mechanisms (augmentation of NMDA receptor function) are critical for VGSC activation-induced neurotoxicity in primary CGC cultures.

Lamotrigine Induces Tremor among Epilepsy Patients Probably via Cerebellar Pathways.

Lamotrigine, a frequently used antiepileptic drug, inhibits voltage-gated sodium-channels. By suppressing the release of glutamate and aspartate, lamotrigine acts as a membrane stabilizer, and it is also effective in bipolar disorder and migraine. However, lamotrigine is known to induce tremor among 4-10% of patients. We examined the lamotrigine-induced tremor in 28 epilepsy patients (age: 38.06 ± 13.56 years; 24 females and 4 males) receiving lamotrigine monotherapy and compared the data to 30 age- and sex-matched controls (age: 33.06 ± 10.71 years; 25 females and 5 males). Tremor was visually assessed by clinical tremor rating scales. Quantitative characteristics (intensity, center frequency and frequency dispersion) which are regularly used to differentiate various tremor syndromes were measured by validated, sensitive biaxial accelerometry in resting, postural and intentional positions. Regularity of repetitive finger and hand movements and reaction time were also determined. Data were statistically analyzed. Clinical tremor rating scales detected pathological tremor in three patients (10%), while accelerometry revealed tremor in seven patients (25%). Center frequency of patients with pathological tremor was similar to controls, but the frequency dispersion was significantly lower and tremor intensity was significantly higher in both postural and intentional positions. Rhythmic movements and reaction time were normal. Our results show that objective measurements detect pathological intention tremor in 25% of epilepsy patients receiving lamotrigine monotherapy. Quantitative characteristics suggest the involvement of the cerebellum in the pathomechanism of lamotrigine-induced tremor. Determining the parameters of drug-induced tremor syndromes might help to understand the complex action of tremor generator networks.

Folic Acid Protects Against Glutamate-Induced Excitotoxicity in Hippocampal Slices Through a Mechanism that Implicates Inhibition of GSK-3β and iNOS.

Folic acid (folate) is a vitamin of the B-complex group crucial for neurological function. Considering that excitotoxicity and cell death induced by glutamate are involved in many disorders, the potential protective effect of folic acid on glutamate-induced cell damage in rat hippocampal slices and the possible intracellular signaling pathway involved in such effect were investigated. The treatment of hippocampal slices with folic acid (100μM) significantly abrogated glutamate (1mM)-induced reduction of cell viability measured by MTT reduction assay and inhibited glutamate-induced D-[3H]-aspartate release. To investigate the putative intracellular signaling pathways implicated in the protective effect of folic acid, we used a PI3K inhibitor, LY294002, which abolished the protective effects of folic acid against glutamate-induced cell damage and D-[3H] aspartate release. Moreover, hippocampal slices incubated with folic acid alone for 30min presented increased phosphorylation of GSK-3β at Ser9, indicating an inhibition of the activity of this enzyme. Furthermore, folic acid in the presence of glutamate insult in hippocampal slices maintained for an additional period of 6h in fresh culture medium without glutamate and/or folic acid induced phosphorylation of GSK-3β and β-catenin expression. In addition, glutamate-treated hippocampal slices showed increased iNOS expression that was reversed by folic acid. In conclusion, the results of this study show that the protective effect of folic acid against glutamate-induced excitotoxicity may involve the modulation of PI3K/GSK-3β/β-catenin pathway and iNOS inhibition.

Influence of Volatile Anesthesia on the Release of Glutamate and other Amino Acids in the Nucleus Accumbens in a Rat Model of Alcohol Withdrawal: A Pilot Study.

BackgroundAlcohol withdrawal syndrome is a potentially life-threatening condition, which can occur when patients with alcohol use disorders undergo general anesthesia. Excitatory amino acids, such as glutamate, act as neurotransmitters and are known to play a key role in alcohol withdrawal syndrome. To understand this process better, we investigated the influence of isoflurane, sevoflurane, and desflurane anesthesia on the profile of excitatory and inhibitory amino acids in the nucleus accumbens (NAcc) of alcohol-withdrawn rats (AWR).MethodsEighty Wistar rats were randomized into two groups of 40, pair-fed with alcoholic or non-alcoholic nutrition. Nutrition was withdrawn and microdialysis was performed to measure the activity of amino acids in the NAcc. The onset time of the withdrawal syndrome was first determined in an experiment with 20 rats. Sixty rats then received isoflurane, sevoflurane, or desflurane anesthesia for three hours during the withdrawal period, followed by one hour of elimination. Amino acid concentrations were measured using chromatography and results were compared to baseline levels measured prior to induction of anesthesia.ResultsGlutamate release increased in the alcohol group at five hours after the last alcohol intake (p = 0.002). After 140 min, desflurane anesthesia led to a lower release of glutamate (p < 0.001) and aspartate (p = 0.0007) in AWR compared to controls. GABA release under and after desflurane anesthesia was also significantly lower in AWR than controls (p = 0.023). Over the course of isoflurane anesthesia, arginine release decreased in AWR compared to controls (p < 0.001), and aspartate release increased after induction relative to controls (p20min = 0.015 and p40min = 0.006). However, amino acid levels did not differ between the groups as a result of sevoflurane anesthesia.ConclusionsEach of three volatile anesthetics we studied showed different effects on excitatory and inhibitory amino acid concentrations. Under desflurane anesthesia, both glutamate and aspartate showed a tendency to be lower in AWR than controls over the whole timecourse. The inhibitory amino acid arginine increased in AWR compared to controls, whereas GABA levels decreased. However, there were no significant differences in amino acid concentrations under or after sevoflurane anesthesia. Under isoflurane, aspartate release increased in AWR following induction, and from 40 min to 140 min arginine release in controls was elevated. The precise mechanisms through which each of the volatile anesthetics affected amino acid concentrations are still unclear and further experimental research is required to draw reliable conclusions.

Metabolic response of glioblastoma cells associated with glucose withdrawal and pyruvate substitution as revealed by GC-MS.

BackgroundTumor cells are highly dependent on glucose even in the presence of oxygen. This concept called the Warburg effect is a hallmark of cancer and strategies are considered to therapeutically exploit the phenomenon such as ketogenic diets. The success of such strategies is dependent on a profound understanding of tumor cell metabolism. With new techniques it is now possible to thoroughly analyze the metabolic responses to the withdrawal of substrates and their substitution by others. In the present study we used gas chromatography coupled to mass spectrometry (GC-MS) to analyze how glioblastoma brain tumor cells respond metabolically when glucose is withdrawn and substituted by pyruvate.MethodsGlioblastoma brain tumor cells were cultivated in medium with high (25 mM), medium (11 mM) or low (5.5 mM) glucose concentration or with pyruvate (5 mM). After 24 h GC-MS metabolite profiling was performed.ResultsThe abundances of most metabolites were dependent on the supply of glucose in tendency but not in a linear manner indicating saturation at high glucose. Noteworthy, a high level of sorbitol production and release was observed at high concentrations of glucose and high release of alanine, aspartate and citrate were observed when glucose was substituted by pyruvate. Intermediates of the TCA cycle were present under all nutritional conditions and evidence was found that cells may perform gluconeogenesis from pyruvate.ConclusionsOur experiments reveal a high plasticity of glioblastoma cells to changes in nutritional supply which has to be taken into account in clinical trials in which specific diets are considered for therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-016-0131-9) contains supplementary material, which is available to authorized users.

Oligodendrocytes Do Not Export NAA-Derived Aspartate In Vitro

Oligodendroglial cells are known to de-acetylate the N-acetylaspartate (NAA) synthesized and released by neurons and use it for lipid synthesis. However, the role of NAA regarding their intermediary metabolism remains poorly understood. Two hypotheses were proposed regarding the fate of aspartate after being released by de-acetylation: (1) aspartate is metabolized in the mitochondria of oligodendrocyte lineage cells; (2) aspartate is released to the medium. We report here that aspartoacylase mRNA expression increases when primary rat oligodendrocyte progenitor cells (OPCs) differentiate into mature cells in culture. Moreover, characterising metabolic functions of acetyl coenzyme A and aspartate from NAA catabolism in mature oligodendrocyte cultures after 5 days using isotope-labelled glucose after 5-days of differentiation we found evidence of extensive NAA metabolism. Incubation with [1,6-13C]glucose followed by gas chromatography–mass spectrometry and high performance liquid chromatography analyses of cell extracts and media in the presence and absence of NAA established that the acetate moiety produced by hydrolysis of NAA does not enter mitochondrial metabolism in the form of acetyl coenzyme A. We also resolved the controversy concerning the possible release of aspartate to the medium: aspartate is not released to the medium by oligodendrocytes in amounts detectable by our methods. Therefore we propose that: aspartate released from NAA joins the cytosolic aspartate pool rapidly and takes part in the malate–aspartate shuttle, which transports reducing equivalents from glycolysis into the mitochondria for ATP production and enters the tricarboxylic acid cycle at a slow rate.

Neuroprotection Promoted by Guanosine Depends on Glutamine Synthetase and Glutamate Transporters Activity in Hippocampal Slices Subjected to Oxygen/Glucose Deprivation.

Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by D-[(3)H]aspartate release) that was prevented in the presence of GUO (100µM). The glutamate transporter blockers, DL-TBOA (10µM), DHK (100µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250µM, Xc(-) system inhibitor) decreased OGD-induced D-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protectedhippocampal slices from cellular damage by modulation of glutamate transporters,however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing D-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity.

Dangerous Liaisons between Beta-Amyloid and Cholinergic Neurotransmission

The review examines the multifaceted interactions between cholinergic transmission and beta-amyloid suggesting a continuum in the action of the peptide that at low concentrations (picomolar-low nanomolar) may directly stimulate nicotinic cholinergic receptor while desensitizing them at increasing concentrations (high nanomolar-low micromolar). In addition high beta-amyloid concentrations may reduce the synaptic release of several neurotransmitters, including glutamate, aspartate, GABA, glycine and dopamine, when the release is elicited through cholinergic stimulation but not following depolarization. The effect of beta-amyloid has been observed both in vitro and in vivo in at least three different brain areas (nucleus accumbens, striatum, hippocampus) suggesting that the peptide may exert some general effects even if not all the brain areas have been evaluated. In turn the activation of cholinergic receptors may affect the amyloid precursor protein processing diverting the metabolism toward non-amyloidogenic products. These actions, dissociated from those described in the case of high beta-amyloid concentrations leading to neurotoxic oligomers, may participate to cause dysfunctions in the neurotransmitter activity, in turn leading, at least from a theoretical point of view, to early neuropsychiatric disturbances in the disease. Complexively these observations underscore novel relationships between two main players in Alzheimer's disease pathogenesis that are beta-amyloid and cholinergic transmission. Also emerges the inherent difficulty of targeting beta-amyloid in a context in which the peptide exerts several actions beyond neurotoxicity.

Neuroprotective effects of the MAO-B inhibitor, PF9601N, in an in vivo model of excitotoxicity.

PF9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] is an inhibitor of monoamine oxidase B (MAO-B), which has shown to possess neuroprotective properties in several in vitro and in vivo models of Parkinson's disease (PD). As there is evidence that excitotoxicity may be implicated in the pathophysiology of several neurodegenerative diseases, the aim of the present work was to investigate the effects of PF9601N in an acute in vivo model of excitotoxicity induced by the local administration of kainic acid during striatal microdialysis in adult rats. The basal and evoked release of neurotransmitters was monitored by HPLC analysis of microdialysate samples and tissue damage was evaluated histologically "ex vivo." PF9601N (40 mg/kg, single i.p. administration) reduced the kainate-evoked release of glutamate and aspartate and increased taurine release, but it had no effect on the release of dopamine, DOPAC, and HVA. PF9601N pretreatment also resulted in a significant reduction in the kainate-induced astrocytosis, microgliosis, and apoptosis. The results suggest PF9601N to be a good candidate for the treatment of neurodegenerative diseases mediated by excitotoxicity.

Thrombin-Facilitated Efflux of d-[3H]-Aspartate from Cultured Astrocytes and Neurons Under Hyponatremia and Chemical Ischemia

Thrombin effect increasing swelling-induced glutamate efflux was examined in cultured cortical astrocytes, cerebellar granule neurons (CGN), hippocampal and cortical neurons. Hypotonic glutamate efflux (monitored by D-[(3)H]aspartate) from cortical astrocytes was increased by thrombin (5 U/mL) to reach 16% of the cell pool in 5 min. Thrombin had lower effects in CGN, and marginal effects in hippocampal and cortical neurons. These differences were related to the magnitude of thrombin-evoked cytosolic calcium rise. The protease-activated receptor 1 is expressed in astrocytes and neurons. In astrocytes exposed to chemical ischemia (sodium iodoacetate plus sodium azide) D-[(3)H]aspartate release showed a first phase (20-40 min) of initial low efflux which progressively increases; and a second phase (40-60 min) of larger efflux coincident with cell swelling. Efflux at the first phase was 52% inhibited by the glutamate transporter blocker DL-threo-β-benzyloxyaspartic-acid (TBOA) and 11% by the volume-sensitive pathway blocker phloretin. At the second phase, efflux was reduced 52 and 38% respectively, by these blockers. In CGN D-[(3)H]aspartate efflux increased sharply and then decreased. This efflux was 32% reduced by calcium omission, 46% by TBOA and 32% by 4-[(2-butyl-6,7dichloro-2-cyclopentyl-2,3-dihydro-1oxo-1H-inden-5-yl)oxy] butanoic-acid. Thrombin enhanced this release by 32%. Ischemic treatment increased astrocyte mortality from 4% in controls to 39 and 61% in ischemia and ischemia plus thrombin, respectively. Cell death was prevented by phloretin. CGN viability was unaffected by the treatment. These results suggest that coincidence of swelling and thrombin during ischemia elevates extracellular glutamate prominently from astrocyte efflux, which may endanger neurons in vivo.

Influence of parafascicular thalamic input on neuronal activity within the nucleus accumbens is mediated by nitric oxide — An in vivo study

AimsThalamostriatal fibers are involved in cognitive tasks such as acquisition, learning, processing of sensory events, and behavioral flexibility and might play a role in Parkinson's disease. The aim of the present study was the in vivo electrochemical characterization of the projection from the lateral aspect of the parafascicular thalamus (Pfl) to the dorsolateral aspect of the nucleus accumbens (dNAc). Since nitric oxide (NO) plays a crucial role in striatal synaptic transmission, its implication in Pfl-evoked signaling within the dNAc was investigated. Main methodsThe Pfl was electrically stimulated utilizing paired pulses and extracellular potentials were recorded within the dNAc. Simultaneously, the dNAc was superfused using the push–pull superfusion technique for local application of compounds and for assessing the influence of NO on release of glutamate, aspartate and GABA. Key findingsStimulation of the Pfl evoked a negative-going component at 9–14ms followed by a positive-going component at 39–48ms. The early response was current-dependent and diminished by superfusion of the dNAc with tetrodotoxin, kynurenic acid or NG-nitro-l-arginine methyl ester (L-NAME), while 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA/NO) increased this evoked potential. Transmitter release was inhibited by L-NAME and facilitated by PAPA/NO. SignificanceThis study describes for the first time in vivo extracellular electrical responses of the dNAc on stimulation of the Pfl. Synaptic transmission within the dNAc on stimulation of the Pfl seems to be facilitated by NO.

The nitric oxide donor SNAP-induced amino acid neurotransmitter release in cortical neurons. Effects of blockers of voltage-dependent sodium and calcium channels.

BackgroundThe discovery that nitric oxide (NO) functions as a signalling molecule in the nervous system has radically changed the concept of neuronal communication. NO induces the release of amino acid neurotransmitters but the underlying mechanisms remain to be elucidated.FindingsThe aim of this work was to study the effect of NO on amino acid neurotransmitter release (Asp, Glu, Gly and GABA) in cortical neurons as well as the mechanism underlying the release of these neurotransmitters. Cortical neurons were stimulated with SNAP, a NO donor, and the release of different amino acid neurotransmitters was measured by HPLC. The involvement of voltage dependent Na+ and Ca2+ channels as well as cGMP in its mechanism of action was evaluated.ConclusionsOur results indicate that NO induces release of aspartate, glutamate, glycine and GABA in cortical neurons and that this release is inhibited by ODQ, an inhibitor of soluble guanylate cyclase. Thus, the NO effect on amino acid neurotransmission could be mediated by cGMP formation in cortical neurons. Our data also demonstrate that the Na+ and Ca2+ voltage- dependent calcium channels are involved in the NO effects on cortical neurons.

Piracetam ameliorated oxygen and glucose deprivation-induced injury in rat cortical neurons via inhibition of oxidative stress, excitatory amino acids release and P53/Bax.

Our previous work has demonstrated that piracetam inhibited the decrease in amino acid content induced by chronic hypoperfusion, ameliorated the dysfunction of learning and memory in a hypoperfusion rat model, down-regulated P53, and BAX protein, facilitated the synaptic plasticity, and may be helpful in the treatment of vascular dementia. To explore the precise mechanism, the present study further evaluated effects of piracetam on Oxygen and glucose deprivation (OGD)-induced neuronal damage in rat primary cortical cells. The addition of piracetam to the cultured cells 12 h before OGD for 4 h significantly reduced neuronal damage as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release experiments. Piracetam also lowered the levels of malondialdehyde, nitrogen monoxidum, and xanthine oxidase which was increased in the OGD cells, and enhanced the activities of superoxide dismutase and glutathione peroxidase, which were decreased in the OGD cells. We also demonstrated that piracetam could decrease glutamate and aspartate release when cortical cells were subjected to OGD. Furthermore, Western blot study demonstrated that piracetam attenuated the increased expression of P53 and BAX protein in OGD cells. These observations demonstrated that piracetam reduced OGD-induced neuronal damage by inhibiting the oxidative stress and decreasing excitatory amino acids release and lowering P53/Bax protein expression in OGD cells.

Chapter 33 - Neurologic damage in hypoglycemia

Hypoglycemia occurs in diabetic patients as a consequence of treatment with hypoglycemic agents, in insulinoma patients as a result of excessive insulin production, and in infants as a result of abnormal regulation of metabolism. Profound hypoglycemia can cause structural and functional disturbances in both the central (CNS) and the peripheral nervous system (PNS). The brain is damaged by a short and severe episode of hypoglycemia, whereas PNS pathology appears after a mild and prolonged episode. In the CNS, damaged mitochondria, elevated intracellular Ca2(+) level, released cytochrome c to the cytosol, extensive production of superoxide, increased caspase-3 activity, release of aspartate and glutamate from presynaptic terminals, and altered biosynthetic machinery can lead to neuronal cell death in the brain. Considering the PNS, chronic hypoglycemia is associated with delayed motor and sensory conduction velocities in peripheral nerves. With respect to pathology, hypoglycemic neuropathy in the PNS is characterized by Wallerian-like axonal degeneration that starts at the nerve terminal and progresses to a more proximal part of the axon, and motor axons to the muscles may be more severely damaged than sensory axons. Since excitatory neurotransmitters primarily involve the neuron in the CNS, this "dying back" pattern of axonal damage in the PNS may involve mechanisms other than excitotoxicity.