Calcium-dependent Glutamate Release Research Articles

Effect of Crude Extract from the Sea Anemone Bunodeopsis globulifera on Voltage-Gated Ion Channels from Central and Peripheral Murine Nervous Systems.

Sea anemones are an important source of bioactive compounds with potential pharmacological applications. Their toxins are produced and stored in organelles called nematocysts and act on specific targets, including voltage-gated ion channels. To date, sea anemone toxins have demonstrated effects on voltage-gated sodium and potassium channels, facilitating investigations into the structure and function of these proteins. In this study, we evaluated the effect of Bunodeopsis globulifera sea anemone crude extract, and of a low molecular weight fraction, on voltage-gated sodium and calcium channels within the murine nervous system. Notably, the crude extract led to a significant reduction in total sodium current, while also triggering calcium-dependent glutamate release. Furthermore, the low molecular weight fraction, in particular, enhanced total calcium currents and current density. These findings underscore the existence of sea anemone toxins with diverse mechanisms of action beyond those previously documented.

Testing PET-[11C]ABP688 as a tool to quantify glutamate release in vivo

Abstract The excitatory neurotransmitter glutamate plays a critical role in experience-dependent neuroplasticity, including addiction-related processes. To date, however, it is not possible to measure glutamate release in the living human brain. Positron emission tomography (PET) with [11C]ABP688, a selective allosteric antagonist of metabotropic type 5 glutamate (mGlu5) receptors, could offer an effective strategy. To test this proposition, we conducted a series of studies in rats using microdialysis and [11C]ABP688 microPET imaging, and in humans using PET and magnetic resonance spectroscopy (MRS). Significant calcium-dependent glutamate release was identified in the ventral striatum of awake rats (190.5 ± 34.7%, p < 0.05; n = 7) following administration of a low dose of ethanol (EtOH; 20%, 0.5 g/kg), a pharmacological challenge readily translatable to human research. Simultaneous microdialysis and microPET studies in anesthetized rats yielded concurrent increases in glutamate release (126.9 ± 5.3%, p < 0.001; n = 11) and decreases in striatal [11C]ABP688 binding (6.8 ± 9.6%, p < 0.05). These latter two effects, however, were not significantly correlated (r = 0.25, p = 0.46). In humans, a laboratory stressor yielded significant changes in self-reported mood (ps < 0.041), sympathetic system activations (ps < 0.042), and the MRS index of striatal glutamate reuptake following excitatory neurotransmission, Glx/Cr levels (p = 0.048). These effects, however, were not accompanied by significant changes in [11C]ABP688 BPND (ps > 0.21, n = 9) or correlated with each other (ps > 0.074). Together, these studies document EtOH-induced glutamate release from neurons, EtOH-induced decreases in [11C]ABP688 binding, and stress-induced changes in glutamate turnover, yet fail to provide evidence that the PET [11C]ABP688 method can be exploited to quantify moderate changes in glutamate release. The results underscore the need for highly controlled testing conditions during PET measures of mGlu5 receptors.

NMDA-Type Glutamate Receptor Activation Promotes Vascular Remodeling and Pulmonary Arterial Hypertension.

Excessive proliferation and apoptosis resistance in pulmonary vascular cells underlie vascular remodeling in pulmonary arterial hypertension (PAH). Specific treatments for PAH exist, mostly targeting endothelial dysfunction, but high pulmonary arterial pressure still causes heart failure and death. Pulmonary vascular remodeling may be driven by metabolic reprogramming of vascular cells to increase glutaminolysis and glutamate production. The N-methyl-d-aspartate receptor (NMDAR), a major neuronal glutamate receptor, is also expressed on vascular cells, but its role in PAH is unknown. We assessed the status of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and controls through mass spectrometry imaging, Western blotting, and immunohistochemistry. We measured the glutamate release from cultured pulmonary vascular cells using enzymatic assays and analyzed NMDAR regulation/phosphorylation through Western blot experiments. The effect of NMDAR blockade on human pulmonary arterial smooth muscle cell proliferation was determined using a BrdU incorporation assay. We assessed the role of NMDARs in vascular remodeling associated to pulmonary hypertension, in both smooth muscle-specific NMDAR knockout mice exposed to chronic hypoxia and the monocrotaline rat model of pulmonary hypertension using NMDAR blockers. We report glutamate accumulation, upregulation of the NMDAR, and NMDAR engagement reflected by increases in GluN1-subunit phosphorylation in the pulmonary arteries of human patients with PAH. Kv channel inhibition and type A-selective endothelin receptor activation amplified calcium-dependent glutamate release from human pulmonary arterial smooth muscle cell, and type A-selective endothelin receptor and platelet-derived growth factor receptor activation led to NMDAR engagement, highlighting crosstalk between the glutamate-NMDAR axis and major PAH-associated pathways. The platelet-derived growth factor-BB-induced proliferation of human pulmonary arterial smooth muscle cells involved NMDAR activation and phosphorylated GluN1 subunit localization to cell-cell contacts, consistent with glutamatergic communication between proliferating human pulmonary arterial smooth muscle cells via NMDARs. Smooth-muscle NMDAR deficiency in mice attenuated the vascular remodeling triggered by chronic hypoxia, highlighting the role of vascular NMDARs in pulmonary hypertension. Pharmacological NMDAR blockade in the monocrotaline rat model of pulmonary hypertension had beneficial effects on cardiac and vascular remodeling, decreasing endothelial dysfunction, cell proliferation, and apoptosis resistance while disrupting the glutamate-NMDAR pathway in pulmonary arteries. These results reveal a dysregulation of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and identify vascular NMDARs as targets for antiremodeling treatments in PAH.

An autocrine purinergic signaling controls astrocyte-induced neuronal excitation

Astrocyte-derived gliotransmitters glutamate and ATP modulate neuronal activity. It remains unclear, however, how astrocytes control the release and coordinate the actions of these gliotransmitters. Using transgenic expression of the light-sensitive channelrhodopsin 2 (ChR2) in astrocytes, we observed that photostimulation reliably increases action potential firing of hippocampal pyramidal neurons. This excitation relies primarily on a calcium-dependent glutamate release by astrocytes that activates neuronal extra-synaptic NMDA receptors. Remarkably, our results show that ChR2-induced Ca2+ increase and subsequent glutamate release are amplified by ATP/ADP-mediated autocrine activation of P2Y1 receptors on astrocytes. Thus, neuronal excitation is promoted by a synergistic action of glutamatergic and autocrine purinergic signaling in astrocytes. This new mechanism may be particularly relevant for pathological conditions in which ATP extracellular concentration is increased and acts as a major danger signal.

Initiation of spreading depression by synaptic and network hyperactivity: Insights into trigger mechanisms of migraine aura.

Background Cortical spreading depression (SD) is thought to underlie migraine aura but mechanisms of triggering SD in the structurally normal, well-nourished cortex of migraine patients remain unknown. Synaptic and network dysfunctions appear to underlie episodic neurological disorders, including migraine. The narrative review summarizes old and recent experimental evidence for triggering SD by synaptic/network mechanisms and discusses the relevance of the data to migraine pathogenesis. Our hypothesis is that under some conditions synaptic/network hyperactivity may reliably ignite SD, and this mechanism may underlie triggering migraine aura in patients. Findings High-frequency tetanic stimulation of the cortex reliably triggers SD in synaptically connected regions; SD is a reliable cortical response to acute hyperexcitability (epileptic seizures), though chronic epilepsy prevents triggering SD; in the hyperexcitable cortex, SD may be triggered by sensory stimulation; compromised glutamatergic transmission plays the critical role in triggering SD. Conclusion SD may be triggered by dynamic network instability produced by dysfunction of calcium-dependent glutamate release. Synaptic drive from subcortical sensory processing structures (brainstem and/or thalamocortical networks) is able to evoke depolarization of hyperexcitable cortical neurons sufficient to initiate the regenerative SD process. Studying SD initiation by synaptic/network hyperexcitability may provide insights into basic mechanisms underlying SD generation in migraine brain.

Role of astrocytes in neuronal energetic production and its relationship with neurodegeneration

Event Abstract Back to Event Role of astrocytes in neuronal energetic production and its relationship with neurodegeneration Felipe Vargas-Restrepo1, Angelica M. Sabogal-Guaqueta1 and Gloria Patricia Cardona Gomez1* 1 University of Antioquia, Group of Neuroscience of Antioquia, Colombia Astrocytes are specialized glial cells that make important contributions to neuronal support and the blood-brain barrier. This barrier limits the passage of certain fatty acids and lipids to the Central Nervous System (CNS), which the majority of lipids found in this tissue must be synthesized right there. Astrocytes are involved in the conversion and production of fatty acids in the CNS as well as production of lipid messengers that apparently are involved in the activity of neurons and neuroprotective or neurodegenerative processes. For this reason, we review the role of astrocytes in energy production and its relationship to neuronal lipid changes in neuroprotective and neurodegenerative processes. Figure 1 Acknowledgements We want to thank to the Colciencias project # 111565740581. References Amruthesh S.C., Boerschel M.F., McKinney J.S., Willoughby K.A., Ellis E.F. (1993) Metabolism of arachidonic acid to epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and prostaglandins in cultured rat hippocampal astrocytes. J Neurochem. 61, 150-159. Bezzi P., Carmignoto G., Pasti L., Vesce S., Rossi D., Rizzini B.L., et al. (1998) Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature. 391, 281-285. Moore S.A., Yoder E., Murphy S., Dutton G.R., Spector A.A. (1991) Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6). Journal of Neurochemistry. 56, 518-524. Posada-Duque R.A., Barreto G.E., Cardona-Gomez G.P. (2014) Protection after stroke: cellular effectors of neurovascular unit integrity. Front Cell Neurosci. 8, 231. doi: 10.3389/fncel.2014.00231. Staub F., Winkler A., Peters J., Goerke U., Kempski O., Baethmann A. (1995) Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes. Neurochem Res. 20, 1449-1456. Keywords: Fatty Acids, Astrocytes, neurodegeneration, Neuroprotection, energy production Conference: Latin-American School on glial cells in the diseased brain (IBRO), Bogotá, Colombia, 13 Jul - 17 Jul, 2015. Presentation Type: Poster Presentation Topic: Traumatic and Cerebrovascular diseases Citation: Vargas-Restrepo F, Sabogal-Guaqueta AM and Cardona Gomez G (2015). Role of astrocytes in neuronal energetic production and its relationship with neurodegeneration. Conference Abstract: Latin-American School on glial cells in the diseased brain (IBRO). doi: 10.3389/conf.fncel.2015.35.00006 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Mar 2015; Published Online: 16 May 2015. * Correspondence: Prof. Gloria Patricia Cardona Gomez, University of Antioquia, Group of Neuroscience of Antioquia, Medellin, Antioquia, 05001000, Colombia, patricia.cardona@neurociencias.udea.edu.co Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Felipe Vargas-Restrepo Angelica M Sabogal-Guaqueta Gloria Patricia Cardona Gomez Google Felipe Vargas-Restrepo Angelica M Sabogal-Guaqueta Gloria Patricia Cardona Gomez Google Scholar Felipe Vargas-Restrepo Angelica M Sabogal-Guaqueta Gloria Patricia Cardona Gomez PubMed Felipe Vargas-Restrepo Angelica M Sabogal-Guaqueta Gloria Patricia Cardona Gomez Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

An Unconventional Glutamatergic Circuit in the Retina Formed by vGluT3 Amacrine Cells

In the vertebrate retina, glutamate is traditionally thought to be released only by photoreceptors and bipolar cells to transmit visual signals radially along parallel ON and OFF channels. Lateral interactions in the inner retina are mediated by amacrine cells, which are thought to be inhibitory neurons. Here, we report calcium-dependent glutamate release from vGluT3-expressing amacrine cells (GACs) in the mouse retina. GACs provide an excitatory glutamatergic input to ON-OFF and ON direction-selective ganglion cells (DSGCs) and a subpopulation of W3 ganglion cells, but not to starburst amacrine cells. GACs receive excitatory inputs from both ON and OFF channels, generate ON-OFF light responses with a medium-center, wide-surround receptive field structure, and directly regulate ganglion cell activity. The results reveal a functional glutamatergic circuit that mediates noncanonical excitatory interactions in the retina and probably plays a role in generating ON-OFF responses, crossover excitation, and lateral excitation.

Crotoxin from Crotalus durissus terrificus snake venom induces the release of glutamate from cerebrocortical synaptosomes via N and P/Q calcium channels

Crotoxin (Crtx), the main toxin in the venom of Crotalus durissus terrificus snake, is a heterodimer with a basic subunit, CB, and an acidic subunit, CA. CB is a phospholipase A2 that depends on CA to specifically bind to the cell membrane. This toxin acts in the central nervous system (CNS) causing chronic seizure effects and other cytotoxic effects. Here, we report its action on glutamate release in rat cerebral cortex synaptosomes. Aiming at a better understanding of the mechanism of action of Crtx, calcium channel blockers were used and internalization studies were performed in cerebellar granule neurons. Our results show that Crtx induces calcium-dependent glutamate release via N and P/Q calcium channels. In addition, the CB subunit of Crtx is shown to be internalized. This internalization does not depend on the presence of CA subunit neither on the PLA2 activity of CB. A correlation between CB internalization and glutamate release remains to be established.

M‐type potassium channels modulate Schaffer collateral–CA1 glutamatergic synaptic transmission

Previous studies have suggested that muscarinic receptor activation modulates glutamatergic transmission. M-type potassium channels mediate the effects of muscarinic activation in the hippocampus, and it has been proposed that they modulate glutamatergic synaptic transmission. We tested whether M1 muscarinic receptor activation enhances glutamatergic synaptic transmission via the inhibition of the M-type potassium channels that are present in Schaffer collateral axons and terminals. Miniature excitatory postsynaptic currents (mEPSCs) were recorded from CA1 pyramidal neurons. The M1 receptor agonist, NcN-A-343, increased the frequency of mEPSCs, but did not alter their amplitude. The M-channel blocker XE991 and its analogue linopirdine also increased the frequency of mEPSCs. Flupirtine, which opens M-channels, had the opposite effect. XE991 did not enhance mEPSCs frequency in a calcium-free external medium. Blocking P/Q- and N-type calcium channels abolished the effect of XE991 on mEPSCs. These data suggested that the inhibition of M-channels increases presynaptic calcium-dependent glutamate release in CA1 pyramidal neurons. The effects of these agents on the membrane potentials of presynaptic CA3 pyramidal neurons were studied using current clamp recordings; activation of M1 receptors and blocking M-channels depolarized neurons and increased burst firing. The input resistance of CA3 neurons was increased by the application of McN-A-343 and XE991; these effects were consistent with the closure of M-channels. Muscarinic activation inhibits M-channels in CA3 pyramidal neurons and its efferents – Schaffer collateral, which causes the depolarization, activates voltage-gated calcium channels, and ultimately elevates the intracellular calcium concentration to increase the release of glutamate on CA1 pyramidal neurons.

Role of M-Type Potassium Channels Glutamatergic Synaptic Transmission and Status Epilepticus (IN5-1.003)

Objective: Whether M-currents modulate glutamatergic synaptic transmission and whether the drugs that open these channels could terminate seizures induced by muscarinic stimulation. Background Muscarinic activation by pilocarpine and anologs result in seizures presumably by the inhibition of M-currents. Muscarinic stimulation modulates glutamatergic synaptic transmission but the mechanisms remain uncertain. Design/Methods: Whole-cell patch clamp recordings from CA3 and CA1 hippocampal pyramidal neurons and video EEG recoprdings from rats. Results: In voltage clamp recordings, XE-991 an M channel blocker increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), but had no effect on their amplitude. XE-991 and its anolog linopordine also increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) but had no effect on their amplitude. M1 receptor agonist NcN-A-343 also increased the frequency of mEPSCs. The M-channel opener flupirtine had the effect opposite of XE-991. The XE-991 enhancement of mEPSCs frequency was not observed in calcium free external ACSF. When P/Q- or N-type calcium channel was blocked, the effect of XE-991 on mEPSCs was partially prevented. These data suggest that M-channel blockage increases presynaptic calcium dependent glutamate release in CA1 neurons. Current clamp recordings performed in CA3 neurons revealed that XE-991 depolarized membrane potential and increased burst firing. McN-A-343 had similar effect on CA3 neurons as XE-991. We then tested whether M-channel opener flupirtine could terminate status epilepticus (SE). Administration of flupirtine (50 mg/kg) and diazepam (10 mg/kg) individually 10 minutes after continuous EEG activity of first grade seizure was observed did not terminate SE; however, co-administration both drugs significantly reduced the length of SE, with 80% (4/5) rats returning to normal EEG within 90 minutes of treatment. Conclusions: Cholinergic activation causes depolarization of presynaptic CA3 neurons via M-channels, which activates calcium channels to increase glutamate release. Cholinergic inhibition of M-channel contributes to seizure generation.Drugs targeting M currents can terminate status epilepticus. Supported by: Congessionaly Directed Medical Research Program of the Department of Defense. NIH. Disclosure: Dr. Kapur has received (royalty or license fee or contractual rights) payments from a patent. Dr. Sun has nothing to disclose.

A calcium-dependent glutamate release induced by metabotropic glutamate receptors I/II promotes GABA efflux from amacrine cells via a transporter-mediated process

Glutamate and GABA are, respectively, the major excitatory and inhibitory neurotransmitters in the retina, participating in the two pathways through which the retina processes light information. It has already been shown that glutamate induces GABA release from amacrine cells through a transporter-mediated mechanism, and that this process is mediated by ionotropic glutamate receptors. It is well established that glutamate can also activate metabotropic glutamate receptors, which are widely distributed in the retina, and can be detected in amacrine cell bodies and synaptic contacts. Thus, we decided to investigate the role of the activation of groups I and II metabotropic glutamate receptors in GABA release from amacrine cells in the chicken retina. Group I/II agonist trans-ACPD promoted a 40% decrease in the number of GABA-positive cells in relation to the control, effect that was prevented by antagonists of both groups. Also, the trans-ACPD effect was blocked by GAT-1 inhibitor or by antagonists of ionotropic glutamate receptors. Trans-ACPD induced release of GABA was abolished when the experiment was conducted in absence of calcium ions. Under the superfusing conditions used, trans-ACPD promoted an increase in endogenous glutamate release that was prevented when calcium was omitted from the bathing medium. The results suggest that mGluRI/II regulate the release of glutamate, likely from bipolar cells, that in turn activates GABA release from amacrine cells via a transporter mediated process.

Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research

There are no pharmacological treatments to rescue botulinum neurotoxin (BoNT)-mediated paralysis of neuromuscular signaling. In part, this failure can be attributed to the lack of a cell culture model system that is neuron-based, allowing detailed elucidation of the mechanisms underlying BoNT pathogenesis, yet still compatible with modern cellular and molecular approaches. We have developed a method to derive highly enriched, glutamatergic neurons from suspension-cultured murine embryonic stem (ES) cells. Hypothesizing that ES cell-derived neurons (ESNs) might comprise a novel platform to investigate the neurotoxicology of BoNTs, we evaluated the susceptibility of ESNs to BoNT/A and BoNT/E using molecular and functional assays. ESNs express neuron-specific proteins, develop synapses and release glutamate in a calcium-dependent manner under depolarizing conditions. They express the BoNT substrate SNARE proteins SNAP25, VAMP2 and syntaxin, and treatment with BoNT/A and BoNT/E holotoxin results in proteolysis of SNAP25 within 24 h with EC50s of 0.81 and 68.6 pM, respectively. Intoxication with BoNT/A results in the functional inhibition of potassium-induced, calcium-dependent glutamate release. ESNs remain viable and susceptible to intoxication for up to 90 days after plating, enabling longitudinal screens exploring toxin-specific mechanisms underlying persistence of synaptic blockade. The evidence suggests that derived neurons are a novel, biologically relevant model system that combines the verisimilitude of primary neurons with the genetic tractability and scalable expansion of a continuous cell line, and thus should significantly accelerate BoNT research and drug discovery while dramatically decreasing animal use.

The Effect of Spider Toxin PhTx3-4, ω-Conotoxins MVIIA and MVIIC on Glutamate Uptake and on Capsaicin-Induced Glutamate Release and [Ca2+]i in Spinal cord Synaptosomes

In spinal cord synaptosomes, the spider toxin PhTx3-4 inhibited capsaicin-stimulated release of glutamate in both calcium-dependent and -independent manners. In contrast, the conus toxins, ω-conotoxin MVIIA and xconotoxin MVIIC, only inhibited calcium-dependent glutamate release. PhTx3-4, but not ω-conotoxin MVIIA or xconotoxin MVIIC, is able to inhibit the uptake of glutamate by synaptosomes, and this inhibition in turn leads to a decrease in the Ca(2+)-independent release of glutamate. No other polypeptide toxin so far described has this effect. PhTx3-4 and ω-conotoxins MVIIC and MVIIA are blockers of voltage-dependent calcium channels, and they significantly inhibited the capsaicin-induced rise of intracellular calcium [Ca(2+)](i) in spinal cord synaptosomes, which likely reflects calcium entry through voltage-gated calcium channels. The inhibition of the calcium-independent glutamate release by PhTx3-4 suggests a potential use of the toxin to block abnormal glutamate release in pathological conditions such as pain.

Nitric oxide/Reactive oxygen species signaling pathway in astrocytes: Regulation of calcium-dependent glutamate release

Nitric oxide/Reactive oxygen species signaling pathway in astrocytes: Regulation of calcium-dependent glutamate release

Cytokine-induced enhancement of calcium-dependent glutamate release from astrocytes mediated by nitric oxide

Cytokines are produced in the central nervous system (CNS) and exhibit various effects on neurons, microglia, and astrocytes. Astrocytes can release chemical transmitters, including glutamate, in a calcium-dependent manner, which may mediate communication between neurons and astrocytes. To date, no studies have been conducted on the effects of cytokines on calcium-dependent glutamate release from astrocytes. Here, we studied the effects of cytokines on calcium-dependent glutamate release. Cytokines enhanced glutamate release and induced the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO). The inhibition of iNOS eliminated the cytokine-induced enhancement of glutamate release, and treatment with a NO donor, even in the absence of cytokines, increased glutamate release. Thus, cytokines enhance glutamate release, and this enhancement is mediated by NO.

Effects of Cd 2+ on AMPA receptor-mediated synaptic transmission in rat hippocampal CA1 area

Cadmium (Cd 2+) is a common pollutant that causes a wide variety of toxic effects on the central nervous system. However, the mechanism of Cd 2+ neurotoxicity remains to be elucidated. In the present study, we examined the effects of Cd 2+ on AMPA receptor-mediated synaptic transmission and short-term synaptic plasticity in hippocampal CA1 area, using whole-cell patch clamp technique. Cd 2+ significantly inhibited the peak amplitude of evoked EPSCs (eEPSCs) in a concentration-dependent manner and enhanced the short-term synaptic plasticity including paired-pulse facilitation and frequency facilitation. Cd 2+ also decreased the frequency and amplitude of spontaneous EPSCs (sEPSCs) but had no effect on those of miniature EPSCs (mEPSCs). These effects of Cd 2+ may involve a presynaptic mechanism of blockade of action potential-sensitive, calcium-dependent release of glutamate. In addition, Cd 2+ prolonged the decay time of both sEPSCs and mEPSCs, which suggested a postsynaptic action site of Cd 2+. This study demonstrates that Cd 2+ impairs the Schaffer collateral-commissural-CA1 glutamatergic synaptic transmission and short-term plasticity in rat hippocampal slices, which may be a possible contributing mechanism for the Cd 2+-induced neurotoxic effects.

Glial cell‐derived glutamate mediates autocrine cell volume regulation in the retina: activation by VEGF

Astroglial cells are a source for gliotransmitters such as glutamate and ATP. We demonstrate here that gliotransmitters have autocrine functions in the regulation of cellular volume. Hypoosmotic stress in the presence of inflammatory mediators or oxidative stress, and during blockade or down-regulation of potassium channels, induces swelling of retinal glial cells. Vascular endothelial growth factor inhibits the osmotic swelling of glial cells in retinal slices or isolated cells. This effect was mediated by a kinase domain region/flk-1 receptor-evoked calcium dependent release of glutamate from glial cells, and subsequent stimulation of glial group I/II metabotropic glutamate receptors. Activation of kinase domain region/flk-1 or glutamate receptors evoked an autocrine swelling-inhibitory purinergic signaling cascade that was calcium-independent. This cascade involved the release of ATP and adenosine, and the activation of purinergic P2Y(1) and adenosine A1 receptors, resulting in the opening of potassium and chloride channels and inhibition of cellular swelling. The glutamatergic-purinergic regulation of the glial cell volume may be functionally important in the homeostasis of the extracellular space volume during intense neuronal activation which is associated with a swelling of neuronal cell structures in the retina. However, glial cell-derived glutamate may also contribute to the swelling of activated neurons since metabolic poisoning of glial cells by iodoacetate inhibits the neuronal cell swelling mediated by activation of ionotropic glutamate receptors.

P033. Nitric oxide mediates cytokine-induced enhancement of calcium-dependent glutamate release from astrocytes

P033. Nitric oxide mediates cytokine-induced enhancement of calcium-dependent glutamate release from astrocytes

Acute Insult of Ammonia Leads to Calcium-dependent Glutamate Release from Cultured Astrocytes, an Effect of pH

Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-beta-benzyloxyaspartate. NH4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH4+/NH3 were mediated by an increase in pH(i), we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 mM) induced a similar transient increase in both pH(i) and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE.

Synaptic proteins and SNARE complexes are localized in lipid rafts from rat brain synaptosomes

The biochemical characterization of the SNARE proteins present in lipid microdomains, also known as “lipid rafts,” has been addressed in earlier studies, with conflicting data from different laboratories. In this study, we use rat brain synaptosomes as a model with which to examine the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM), also known as ‘lipid rafts.’ By means of buoyancy analysis in sucrose gradients of Triton X-100-solubilized synaptosomes, we identified a pool of SNARE proteins (SNAP 25, syntaxin 1, and synaptobrevin2/VAMP2) significantly associated with DRM. Furthermore, Munc18, synaptophysin, and high amounts of the isoforms I and II of synaptotagmin were also found in DRM. In addition, SDS-resistant and temperature-dependent SNARE complexes were also detected in DRM. Treatment of synaptosomes with methyl-β-cyclodextrin resulted in persistence of the proteins present in the DRM isolated using Triton X-100, whilst strongly impairing calcium-dependent glutamate release. The results from the present work show that lipid microdomains are sites where SNARE proteins and complexes are actually present, as well as important elements in the control of regulated exocytosis.