Glutamate Release Activation Research Articles

The Role of ERK1/2 Kinases in the Molecular Mechanisms of Regulation of Glutamatergic and GABAergic Neurons during the Development of Convulsive Seizures in Krushinskii–Molodkina Rats

The aim of the present work was to study the role of the ERK1/2 protein kinase cascade in regulating the activity of excitatory and inhibitory neurons in the hippocampus and temporal cortex in Krushinskii–Molodkina rats at the early stages of development of epileptiform convulsive seizures and at the ataxia stage. The content and distribution of study substances were assessed using an immunohistochemical method and western blot analysis. Levels of ERK1/2 kinase activity in the hippocampus increased significantly a few seconds after presentation of a sound signal, and this was accompanied by an increase in the content of SNARE protein, which is part of the SNAP25 complex, along with an increase in the activity of synapsin-1, which is responsible for vesicle release. Decreases in the content of the vesicular glutamate transporter (vGLUT2) on the background of increases in ERK1/2 activity and exocytosis proteins at the clonic-tonic stage of convulsive seizures constitute evidence for activation of glutamate release in the hippocampus, while decreases in ERK1/2 kinase activity and synapsin-1 in the temporal cortex, accompanied by an increase in vGLUT2, point to inhibition of glutamatergic neurotransmission. Decreased glutamate release activity in the temporal cortex thus appears to represent the origin of the decrease in the excitatory signal, which then leads to a reduction in glutamatergic system activity in the hippocampus at the ataxia stage, with termination of tonic convulsions and entry of the rats into the ataxia stage. These changes in the quantities and activities of proteins responsible for neurotransmitter release, correlating with changes in ERK1/2 activity, were most marked in glutamatergic neurons in the cortex and hippocampus. This leads to the conclusion that the kinases studied here have a predominant role in controlling glutamate release, thus taking part in the initiation of convulsive activity.

Enzyme-luminescence method: Tool for real-time monitoring of natural neurotoxins in vitro and l-glutamate release from primary cortical neurons

Novel enzyme-luminescence method is used for the rapid and sensitive in vitro detection of natural neurotoxins (e.g., shellfish and mushroom toxins) using model brain cells. Paralytic shellfish poisons gonyautoxins (e.g., GTX2,3 and GTX1,4) were detected at 1nM level by their inhibition of glutamate release from C6 glioma cells upon drug stimulation (IC50: GTX2,3=30nM and GTX1,4=8nM). Activation of glutamate release from C6 cells by ibotenic acid (a mushroom toxin) was also evaluated (EC50=10nM). The method was tested for real-time detection of glutamate release from primary rat cortical neurons. Dose-dependent effects of KCl (0–200mM) and NMDA on glutamate release from primary cortical neurons were studied. The effects of different culture conditions on K+-depolarization-induced glutamate release were also investigated. The method may be applicable to screening of drugs and toxins, and finding glutamatergic neurons in brain slices without in situ staining.

Astrocyte-Dependent Vulnerability to Excitotoxicity in Spermine Oxidase-Overexpressing Mouse.

Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca(2+)-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.

SNARE-dependent glutamate release in megakaryocytes

ObjectiveThe identification of signaling pathways involved in megakaryocytopoiesis is essential for development of novel therapeutics to treat hematological disorders. Following our previous findings that megakaryocytes express functional channel-forming N-methyl-D-aspartate-type glutamate receptors, here we aimed to determine the glutamate release capacity in undifferentiated and differentiated megakaryocytes and the role of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins that are known to be associated with vesicular exocytosis.Materials and MethodsUsing the megakaryocytic cell line MEG-01, primary megakaryocytes, and tissue sections of bone marrow, reverse transcription polymerase chain reaction, Western blot analysis, and immunolocalization were employed to detect factors required for vesicular glutamate release. Vesicle recycling was monitored by acridine orange and FM1-43 staining and glutamate release activity was assessed by an enzyme-linked fluorimetric assay. Genetically modified MEG-01 cells, with deletion or overexpression of SNARE and vesicular proteins, were also examined for glutamate release activity.ResultsWe demonstrated that megakaryocytes express numerous proteins required for vesicular glutamate release, including core SNARE proteins, vesicle-associated membrane protein, soluble N-ethyl maleimide-sensitive factor attachment protein−23, and syntaxin, as well as specific glutamate-loading vesicle proteins, VGLUT1 and VGLUT2. Moreover, active vesicle recycling and differentiation-dependent glutamate release were observed in megakaryocytes. Vesicle-associated membrane protein−deficient MEG-01 cells, which are impaired in vesicle recycling, showed a 30% decrease in released glutamate, whereas overexpression of VGLUT1 exhibited up to a 2.2-fold increase in glutamate release.ConclusionThese data show that glutamate release from megakaryocytes occurs in a SNARE-dependent, exocytotic manner and is increased during differentiation, suggesting that manipulation of glutamate signaling could influence megakaryocytopoiesis and, therefore, offer a suitable target for the treatment of thrombosis and other hematological disorders.

Blockade of A1 receptors by caffeine induces c-fos, zif-268 and ARC expression in the striatum through different interactions with the dopamine system.

Altogether, these results demonstrate that caffeine- and DPCPX-mediated IEG inductions involved different mechanisms in striatonigral and striatopallidal neurons through blockade of A1 receptors. IEG inductions result from a stimulation of dopamine release in striatonigral neurons and from activation of glutamate release and probably also acetylcholine release in striatopallidal neurons. Acute blockade of A1 receptors induces high striatal expression of transcription factors IEG from leucine zipper (c-fos) and zinc finger (zif-268) families and this expression could reflect the behavioral depression induced by high dose of caffeine. These transcription factors could also be involved in the plastic changes resulting from a chronic treatment with caffeine such as the variations in several receptors and G protein sububits16 which may be involved in the development of tolerance to caffeine. Moreover, A2A receptor-deficient mice showed unexpected alterations in striatal gene expression or motor behavior probably related to adaptative changes in which A1 receptors might be involved.17

Caffeine-mediated induction of c-fos, zif-268 and arc expression through A1 receptors in the striatum: different interactions with the dopaminergic system.

Adenosine and the adenosine receptor antagonist, caffeine, modulate locomotor activity and striatal neuropeptide expression through interactions with the dopaminergic system by mechanisms which remain partially undetermined. We addressed this question by using quantitative immunocytochemistry and in situ hybridization, combined with retrograde tracing of striatal neurons, to characterize the mechanism(s) leading to the striatal increase in the immediate early genes (IEG), c-fos, zif-268 and arc, following a single injection of caffeine or the A1 antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Caffeine and DPCPX induced c-fos, zif-268 and arc expression, both at mRNA and protein levels, in large proportions of striatonigral and striatopallidal neurons. The involvement of dopamine systems was evaluated by manipulations of the dopaminergic transmission. Quinpirole, a D2 agonist, almost completely blocked the caffeine-induced IEG increase in both striatopallidal and striatonigral neurons. Conversely, the lesion of the nigrostriatal pathway and the D1 antagonist SCH23390 abolished the caffeine effects in striatonigral neurons but had no or slight effect, respectively, on its action in striatopallidal neurons. These observations demonstrate that caffeine- and DPCPX-mediated IEG inductions involved different mechanisms in striatonigral and striatopallidal neurons through blockade of A1 receptors. Immediate early gene inductions result from a stimulation of dopamine release in striatonigral neurons and from activation of glutamate release and probably also acetylcholine release in striatopallidal neurons. These results also support the idea that, besides A2A receptors, adenosine acting at the A1 receptor plays pivotal functions in the basal ganglia physiology and that blockade of these receptors by specific or nonspecific antagonists, DPCPX and caffeine, may influence a broad range of neuronal functions in the striatum.

Nicotine-stimulated release of [3H]norepinephrine from fetal rat locus coeruleus cells in culture.

Acute nicotine administration stimulated [3H]norepinephrine ([3H]NE) release from cultured fetal locus coeruleus (LC) cells. The effect was concentration dependent, with an EC50 of 0.9 microM, and was abolished by removal of calcium from, or addition of tetrodotoxin (500 nM) to, the assay buffer. Other nicotinic receptor agonists stimulated [3H]NE release, with the rank order of potency being (+)-epibatidine > (-)-nicotine > 1,1-dimethyl-4-phenylpiperazinium (DMPP). Whereas (-)-nicotine and (+/-)-epibatidine exhibited equal maximal responses, DMPP was a partial agonist and (-)-cytisine had no agonist activity. Nicotine-stimulated release of [3H]NE was blocked by nicotinic receptor antagonists, with an order of potency of mecamylamine > lobeline > cytisine > methyllycaconitine > dihydro-beta-erythroidine. The pharmacological profile of this nicotinic receptor is largely consistent with that described previously for an alpha4beta2 subunit combination, although discrepancies in the efficacies of agonists were observed. No additivity in NMDA- and nicotine-stimulated [3H]NE release was observed, suggesting a common signal transduction mechanism. However, the pharmacological characteristics of MK-801 blockade of nicotine-induced responses were not consistent with those of an NMDA receptor. We therefore conclude that nicotine directly releases [3H]NE from LC cells and does not act indirectly via activation of glutamate release.