Glutamate Uptake Blocker Research Articles

PhTx4, a new class of toxins from Phoneutria nigriventer spider venom, inhibits the glutamate uptake in rat brain synaptosomes

We report the characterization of a new class of glutamate uptake inhibitors isolated from Phoneutria nigriventer venom. Glutamate transport activity was assayed in rat cerebrocortical synaptosomes by using [ 3 H ]- l-glutamate. PhTx4 inhibited glutamate uptake in a dose dependent manner. The IC 50 value obtained was 2.35±0.9 μg/ml which is in the observed range reported for glutamate uptake blockers. Tx4-7, one of PhTx4 toxins, showed the strongest inhibitory activity (50.3±0.69%, n=3).

Effects of inhibiting glutamine synthetase and blocking glutamate uptake on b-wave generation in the isolated rat retina.

The purpose of the present experiments was to evaluate the contribution of the glutamate-glutamine cycle in retinal glial (Müller) cells to photoreceptor cell synaptic transmission. Dark-adapted isolated rat retinas were superfused with oxygenated bicarbonate-buffered media. Recordings were made of the b-wave of the electroretinogram as a measure of light-induced photoreceptor to ON-bipolar neuron transmission. L-methionine sulfoximine (1-10 mM) was added to superfusion media to inhibit glutamine synthetase, a Müller cell specific enzyme, by more than 99% within 5-10 min, thereby disrupting the conversion of glutamate to glutamine in the Müller cells. Threo-hydroxyaspartic acid and D-aspartate were used to block glutamate transporters. The amplitude of the b-wave was well maintained for 1-2 h provided 0.25 mM glutamate or 0.25 mM glutamine was included in the media. Without exogenous glutamate or glutamine the amplitude of the b-wave declined by about 70% within 1 h. Inhibition of glutamate transporters led to a rapid (2-5 min) reversible loss of the b-wave in the presence and absence of the amino acids. In contrast, inhibition of glutamine synthetase did not alter significantly either the amplitude of the b-wave in the presence of glutamate or glutamine or the rate of decline of the b-wave found in the absence of these amino acids. Excellent recovery of the b-wave was found when 0.25 mM glutamate was resupplied to L-methionine sulfoximine-treated retinas. The results suggest that in the isolated rat retina uptake of released glutamate into photoreceptors plays a more important role in transmitter recycling than does uptake of glutamate into Müller cells and its subsequent conversion to glutamine.

Effects of glutamate uptake blockers on stimulus-induced field potentials in rat entorhinal cortex in vitro

L-Glutamic acid (Glu) is a key excitatory transmitter in the central nervous system. Excessive amounts of Glu are highly toxic to neurons and particularly the entorhinal cortex (EC) exhibits a remarkable loss of cells in the superficial layers in acute brain injury. The accumulation of Glu is limited by a family of high-affinity Glu transporters. Using extracellular potential recordings in rat brain slices we tested whether application of the Glu uptake blockers dihydrokainate and L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-2,4-PDC) affect stimulus-induced field potentials (FPs) in superficial layer III and deep layer V of the medial EC. We found that a high concentration (400 μM) of the uptake blockers significantly reduces stimulus-induced FPs in both layers. At lower concentration (200 μM), only dihydrokainate is efficient. The data show that Glu uptake is involved in the control of extracellular Glu levels during synaptic excitation of layers III and V of the medial EC.

Characterization of [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes.

1. The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine ([3H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes. 2. [3H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg(-1) protein. Binding was not sensitive to Na+-dependent glutamate uptake blockers or Cl-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]-DCG IV binding. 3. Of the compounds observed to inhibit [3H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) inhibited [3H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nNM. 4. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]-DCG IV binding and potencies obtained for agonist activity in a GTPgamma35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-alpha-methylserine-O-phosphate (MSOP).

In VitroBinding Characteristics of a New Selective Group II Metabotropic Glutamate Receptor Radioligand, [3H]LY354740, in Rat Brain

The in vitro binding of [3H]LY354740, the first high affinity group II-selective metabotropic glutamate (mGlu) receptor radioligand, was characterized in rat cortical, hippocampal, and thalamic membranes as well as in rat brain sections. [3H]LY354740 binding was saturable in all regions investigated. Nonspecific binding (in the presence of 10 microM DCG-IV) was approximately 8% of the total. Ionotropic glutamate receptor agonists, N-methyl-D-aspartate, (R,S)-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/kainate, a Na+-dependent glutamate uptake blocker as well as a group I-selective mGlu receptor agonist (all up to 1 mM) did not inhibit [3H]LY354740 binding to cortical membranes. However, several known metabotropic receptor ligands inhibited the binding with the following rank order of potency: LY354740 = LY341495 > (2S,2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine = (2S,1'S, 2'S)-2-(2-carboxycyclopropyl)glycine > glutamate = (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S, 2'S)-2-methyl-2-(2-carboxycyclopropyl)-glycine > quisqualate > ibotenate > L-2-amino-3-phosphonopropionic acid = (S)-alpha-methyl-4-carboxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-aspartyl-glutamate, (2S)-alpha-ethylglutamic acid, and (R, S)-alpha-methyl-4-phosphonophenylglycine inhibited [3H]LY354740 binding in a biphasic manner. Guanosine-5'-O-(3-thiotriphosphate concentration-dependently and almost completely inhibited the binding. Finally, in parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1-3 > 4-6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for presynaptic and postsynaptic mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to guanosine-5'-O-(3-thiotriphosphate show that [3H]LY354740 binding probably occurs to group II mGlu receptors in rat brain.

A Long-Lasting Calcium-Activated Nonselective Cationic Current Is Generated by Synaptic Stimulation or Exogenous Activation of Group I Metabotropic Glutamate Receptors in CA1 Pyramidal Neurons

We have shown previously that a selective metabotropic glutamate receptor (mGluR) agonist, 1S,3R-1-aminocyclo-pentane-1, 3-dicarboxylate (1S,3R-ACPD), evokes an inward current in CA1 pyramidal neurons of rat hippocampal slices in the presence of K+ channel blockers (). This current has been characterized as a Ca2+-activated nonselective cationic (CAN) current. Using whole-cell patch-clamp recordings and intracellular dialysis, we now have identified the mGluR subtype and the mechanisms underlying the CAN current (ICAN) and report for the first time the presence of a synaptic ICAN in the mammalian CNS. First, we have shown pharmacologically that activation of ICAN by 1S,3R-ACPD involves the group I mGluRs (and not the groups II and III) and a G-protein-dependent process. We also report that ICAN is modulated by the divalent cations (Mg2+, Cd2+, and Zn2+). Second, we have isolated a slow synaptic inward current evoked by a high-frequency stimulation in the presence of K+ channel blockers, ionotropic glutamate, and GABAA receptor antagonists. This current shows similar properties to the exogenously evoked ICAN: its reversal potential is close to the reversal potential of the 1S, 3R-ACPD-evoked ICAN, and it is G-protein- and Ca2+-dependent. Because the amplitude and duration of ICAN increased in the presence of a glutamate uptake blocker, we suggest that this synaptic current is generated via the activation of mGluRs. We propose that the synaptic ICAN, activated by a brief tetanic stimulation and leading to a long-lasting inward current, may be involved in neuronal plasticity and synchronized network-driven oscillations.

Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors.

The classical view of fast chemical synaptic transmission is that released neurotransmitter acts locally on postsynaptic receptors and is cleared from the synaptic cleft within a few milliseconds by diffusion and by specific reuptake mechanisms. This rapid clearance restricts the spread of neurotransmitter and, combined with the low affinities of many ionotropic receptors, ensures that synaptic transmission occurs in a point-to-point fashion. We now show, however, that when transmitter release is enhanced at hippocampal mossy fibre synapses, the concentration of glutamate increases and its clearance is delayed; this allows it to spread away from the synapse and to activate presynaptic inhibitory metabotropic glutamate receptors (mGluRs). At normal levels of glutamate release during low-frequency activity, these presynaptic receptors are not activated. When glutamate concentration is increased by higher-frequency activity or by blocking glutamate uptake, however, these receptors become activated, leading to a rapid inhibition of transmitter release. This effect may be related to the long-term depression of mossy fibre synaptic responses that has recently been shown after prolonged activation of presynaptic mGluRs (refs 2, 3). The use-dependent activation of presynaptic mGluRs that we describe here thus represents a negative feedback mechanism for controlling the strength of synaptic transmission.

The mechanisms controlling physiologically stimulated changes in rat brain glucose and lactate: a microdialysis study.

1. This study is concerned with the supply of metabolic substrates for neuronal metabolism. Experiments were carried out to investigate whether mechanisms demonstrated in cultured astrocytes also occurred in vivo; these were cAMP-mediated breakdown of glycogen and glutamate uptake-stimulated release of lactate. 2. In vivo microdialysis was used in freely moving rats. Lactate and glucose in the dialysate were assayed using enzyme-based on-line assays. Drugs were given locally through the dialysis probe. Regional cerebral blood flow was measured using the hydrogen clearance method. 3. There was an increase in dialysate glucose in response to the beta-adrenoceptor agonist isoprenaline and to 8-bromo-cAMP, an analogue of cAMP, the second messenger of beta-adrenoceptor stimulation. The effect of isoprenaline was blocked by the antagonist propranolol. Isoprenaline had no effect on dialysate lactate, which was increased by the glutamate uptake blocker beta-D,L-threohydroxyaspartate (THA). 4. Physiological stimulation of neuronal activity produced an increase in both lactate and glucose. The increase in lactate was depressed in the presence of THA but was unaffected by propranolol. The increase in glucose was blocked by propranolol. Regional cerebral blood flow was increased by physiological stimulation but was unaffected by propranolol. 5. These results demonstrate that physiologically stimulated increases in glucose and lactate in the brain are mediated by different mechanisms.

Origin of aspartate-induced responses in rat cerebellar Purkinje cells.

The effects of glutamate, aspartate and N-methyl-D-aspartate (NMDA) on Purkinje cells and interneurons were investigated in cerebellar slice cultures using the whole-cell configuration of the patch-clamp technique. L-Glutamate and L-aspartate induced inward currents in Purkinje cells voltage-clamped at -60 mV. In standard external solution, the amplitude of the responses induced by the two amino-acids was a linear function of the membrane potential. L-Aspartate-induced currents were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective antagonist of non-NMDA receptors. NMDA, a selective agonist of NMDA receptors, had no effect of its own on the excitability of Purkinje cells, but was effective in blocking the responses induced by aspartate in Purkinje cells in a voltage-independent manner. In contrast, D-(-)-2-amino-5-phosphonovaleric acid (D-APV), a selective antagonist of NMDA receptors, had no effect on aspartate-induced responses. D-Aspartate also induced responses in Purkinje cells, and the amplitude of these responses was a linear function of the membrane potential. Currents induced by L- and D-aspartate were inhibited by dihydrokainate, a glutamate uptake blocker. In sodium-free external solution, glutamate still induced outward currents in Purkinje cells, whereas L- and D-aspartate no longer evoked any current. When sodium was replaced by lithium in the external medium, no change in the holding current could be detected in Purkinje cells maintained at -60 mV; moreover, in this bathing medium L-aspartate no longer evoked any current whereas glutamate-induced responses were still present. In contrast, interneurons were sensitive to both NMDA and aspartate applications, and these responses were antagonized by D-APV. In addition, aspartate still induced an outward current in sodium-free external solution. This study presents rather direct evidence in favour of L-aspartate as being a very selective NMDA receptor agonist in the cerebellum. L-Aspartate-induced currents in Purkinje cells are not due to activation of mixed NMDA/non-NMDA receptors, but are probably due to the release of L-glutamate induced by aspartate through glutamate uptake.

Serum affects the characteristics of excitatory amino acid-binding sites on Müller cells

The binding of [ 3H] l-aspartate to membranes obtained from primary cultures of chick retinal Müller cells (glia) was studied. Cells seeded in low-serum-containing medium (1%) and maintained in this condition showed an increased number of binding sites, from 1 to 5 days in vitro (DIV), when compared with controls cultured in medium containing 10% serum; these changes were not reversed by the addition of 10% serum after 48 h in vitro. Increased binding at this age was due to the expression of a low affinity binding system, competitively inhibited by the glutamate uptake blocker l-aspartate-β-hydroxamate, suggesting that high serum might inhibit the expression of uptake sites at precise maturation stages. Experiments showed the effect was due to a thermolabile serum component. The increase in binding sites is parallel in time to both an increase in aspartate uptake and the initiation of synaptogenesis in the whole retina. Our results suggest that the presence of serum at defined stages in retinal development, could result in the elevation of extracellular glutamate and the concomitant excitotoxic death of neuronal cells, due to a decreased glutamate uptake by glial cells.

Pre- and postsynaptic determinants of EPSC waveform at cerebellar climbing fiber and parallel fiber to Purkinje cell synapses

Excitatory postsynaptic currents (EPSCs) at the parallel fiber and climbing fiber to Purkinje cell synapses were studied by whole-cell clamping Purkinje cells in cerebellar slices. Reducing glutamate release with adenosine or GABA decreased the amplitude of the EPSCs, with a larger suppression being produced at the parallel fiber synapse. Reducing glutamate release also speeded the decay of the EPSCs, and this effect was not a series resistance artefact since postsynaptic reduction of the current with CNQX did not speed the EPSC decay. Blocking glutamate uptake slowed the decay of the EPSCs. At the climbing fiber synapse, adenosine had little suppressive effect on the smaller EPSC evoked by the second of a pair of stimuli. Blocking desensitization of postsynaptic AMPA receptors prolonged the EPSC decay, preferentially increased the size of the second EPSC, and resulted in adenosine having a similar suppressive effect on the first and second EPSC. These data suggest that, at these synapses, the fall of glutamate concentration in the synaptic cleft overlaps with the decay of the EPSC, and that the EPSC size and duration are controlled by the amount of glutamate released, the rate of glutamate uptake, and desensitization.

NMDA and non-NMDA receptor-mediated release of [3H]GABA from granule cell dendrites of rat olfactory bulb.

We have studied the effect of glutamate and the glutamatergic agonists N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [3H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [3H]GABA release and the release evoked by a high K+ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [3H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [3H]GABA release. The presence of glycine and the absence of Mg2+ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K+ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [3H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [3H]GABA release but not the NMDA-stimulated release.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-Aminoadipic acid blocks the Na(+)-dependent glutamate transport into acutely isolated Müller glial cells from guinea pig retina.

The effect of the glial toxin alpha-aminoadipic acid (AAA) upon the Na+/glutamate cotransporter of acutely isolated guinea pig retinal glial cells was studied using the whole-cell voltage-clamp technique. Glutamate evoked an inward current in these cells at negative holding potentials dependent on the presence of extracellular Na+ and intracellular K+. A reversal potential could not be found for the current. L-trans-Pyrrolidine-2.4-dicarboxylic acid (PDC), a blocker of Na(+)-dependent glutamate uptake, diminished the glutamate current also in our cells. Application of L-AAA also generated an inward current at negative holding potentials, without a reversal potential, being suppressed if extracellular Na+ or intracellular K+ was removed. The glutamate uptake blocker, PDC (200 microM), blocked the L-AAA (1 mM) current. Thus, L-AAA proved to be transported by the Na+/glutamate transporter of Müller cells. Hence, glutamate currents were diminished by L-AAA competitively with a Km of 499 microM at a glutamate concentration of 10 microM. The Na+/glutamate uptake was less sensitive to DL- and D-AAA block. It is suggested that the blocking effect of AAA on Na(+)-dependent glutamate uptake into glial cells might be involved in the well known glia toxicity of this compound.

Intracellular pH changes produced by glutamate uptake in rat hippocampal slices.

1. The mean intracellular pH in area CA1 of rat hippocampal slices was monitored fluorescently after loading the cells with the dye BCECF-AM. 2. Including L-glutamate in the solution superfusing the slice led to the intracellular pH becoming more acid. This acidification had a roughly Michaelis-Menten dependence on the superfused glutamate concentration with a half-maximal dose around 200 microM: this value must overestimate the glutamate concentration at most of the cells, which will be reduced by uptake. 3. The glutamate-evoked acidification was not significantly reduced by blockers of glutamate-gated ion channels [6-cyano-7-nitroquinoxaline-2,3- dione (CNQX) and D-aminophosphonovalerate (APV)] nor by blockers of gamma-aminobutyric acid (GABA)- and glycine-gated channels (picrotoxin and strychnine), and so was not produced by H+ entry through alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptor channels nor by HCO3- exit through the chloride channels controlled by GABA or glycine. 4. The glutamate-evoked acidification was not reduced by tetrodotoxin (TTX), ruling out the possibility of it being generated by action potentials. It was also unaffected by saturation of presynaptic L-amino-4-phosphonobutanoate (AP4) receptors with AP4. 5. In the presence of blockers of glutamate-, GABA-, and glycine-gated channels, the acidification showed the pharmacology of glutamate uptake and was reduced by a glutamate uptake blocker. 6. The glutamate-evoked acidification showed an ion dependence similar to that of glutamate uptake. It was abolished by removal of extracellular sodium and was reduced by raising the extracellular potassium concentration. It was unaffected by blockers of Na+/H+ exchange (amiloride) and Na+/HCO3- cotransport [4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)] and so was not produced by the Na+ influx accompanying glutamate uptake changing the activity of these carriers. 7. These data show that the glutamate uptake carrier acidifies hippocampal cells, possibly because it transports a pH-changing anion out of the cell as in salamander glial cells. Glutamate uptake may thus contribute to activity-induced pH changes in the nervous system.

Prolonged presence of glutamate during excitatory synaptic transmission to cerebellar Purkinje cells

In the molecular layer of the cerebellar cortex, Purkinje cells and interneurons receive a common excitatory input from parallel fibers. The AMPA/kainate receptor-mediated parallel fiber excitatory postsynaptic current (EPSC) recorded in Purkinje cells decays much more slowly than that recorded in interneurons. We show that this slowness of decay does not result from dendritic filtering and that it is unlikely to reflect the deactivation kinetics of the postsynaptic receptors. Agents blocking glutamate uptake prolong the EPSC in Purkinje cells. We conclude that the slow EPSC decay results from the continued presence of transmitter glutamate. This may be due to retarded transmitter diffusion around spines or to cross-talk between neighboring active synapses.

Glutamate blocks astroglial stellation: Effect of glutamate uptake and volume changes

Neurotransmitters which increase intracellular cAMP levels can cause cultured astroglia to change from a flat, polygonal shape to a stellate morphology. Little is known about how glial stellation can be regulated by other transmitters. In the present study, we demonstrated that L-glutamate blocked isoproterenol (ISO) or dibutyryl-cAMP induced stellation in astroglia. The glutamate inhibition was concentration dependent, with its maximal effect on > 90% of cells at 500 microM. Glutamate also reversed glial stellation within a short period (< 30 min). Glutamate uptake analogues, D-glutamate and D-aspartate, rather than receptor agonists, kainate and quisqualate, mimicked the glutamate effect. Likewise, the glutamate uptake blocker, D-thero-beta-hydroxyaspartate, blocked the glutamate effect. The glutamate inhibition was not a result of inhibition of cAMP formation, since norepinephrine, which inhibited 80% of ISO-stimulated cAMP, also caused glial stellation. Increases in extracellular K+ to 50 mM also reduced glial stellation, whereas 25 mM K+ had little effect. Since 25 mM K+ caused much greater depolarization than 400 microM glutamate, it was unlikely that the effects of both glutamate and high [K+] on glial stellation were due to membrane depolarization. Hypotonic treatment (120 mOsm) enhanced, whereas hypertonic treatment (520 mOsm) prevented, the glutamate reversal of glial stellation. Thus, glial swelling appeared to be a primary mechanism for the inhibitory effect of glutamate and high [K+] on glial stellation. This mechanism could also explain the observation that glutamate inhibited stellation induced by PMA, a PKC activator. Our data suggest that glutamate released from neurons during neuronal activity or pathology can be taken up by astrocytes and alter their morphology. Changes in glial morphology may in turn affect the volume and composition of the extracellular space and, as a result, neuronal activity.

Possible role of glutamatergic neurotransmission in regulating ethanol-evoked brain ascorbate release

It was found that systemic application of ethanol induced brain ascorbate (AA) release. In order to study the mechanism of ethanol-evoked AA release, the role of brain glutamatergic neurotransmission was investigated using in vivo voltammetry in the striatum of freely moving rats. Pretreatment with l- trans-pyrrolidine-2,4-dicarboxylate (PDC, 10 nmol, i.c.v.), a glutamate (Glu) uptake blocker, potentiated ethanol (1 g/kg, intraperitoneal injection, i.p.)-evoked release of brain AA. N- methyl- d-aspartate (NMDA, 1 nmol, i.c.v.) produced a fast transient increase in extracellular AA, whereas α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA, 1 nmol, i.c.v.) produced a decrease in extracellular AA (75.8 ± 3% of control). Kainate (KA, 1 nmol, i.c.v.) produced an initial decrease (48.7 ± 11.7% of control) then an increase (250 ± 68.5% of control) in extracellular AA. These results suggest that systemic administration of ethanol may affect the release or uptake of brain glutamatergic neurotransmitters which appear to regulate brain AA release. The NMDA, but not the non-NMDA, type of Glu receptor may be responsible for this regulation.

Glucose can fuel glutamate uptake in ischemic brain.

Astrocytes in culture can maintain glutamate uptake during hypoxia if glucose is available. To determine whether this capacity is shared by brain in situ, extracellular glutamate levels were measured in ischemic brain under conditions of continued glucose delivery. Microdialysis probes were placed bilaterally in caudate nuclei of rats and perfused with artificial cerebrospinal fluid (CSF) containing either 30 or 0 mM glucose. Global cerebral ischemia was induced by cardiac arrest. Dialysate collected from probes not perfused with glucose showed a 50-fold increase in glutamate levels over the 60 min following cardiac arrest. Addition of glucose to the perfusate reduced the glutamate rise to < 20% of the levels attained in the glucose-free probes. The glucose effect was negated by the addition of 0.5 mM of the glutamate uptake blocker threo-beta-hydroxyaspartate to the artificial CSF. These results show that oxygen is not required to maintain efficient uptake of extracellular glutamate in brain and suggest that elevations in extracellular glutamate levels during ischemia result from metabolic perturbations other than hypoxia.

The uptake inhibitor L-trans-PDC enhances responses to glutamate but fails to alter the kinetics of excitatory synaptic currents in the hippocampus.

1. We have used patch-clamp recording techniques to study the physiological properties of a recently described glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), in the CA1 region of the guinea pig hippocampus. 2. L-trans-PDC markedly potentiated the action of exogenously applied glutamate and raised the ambient extracellular levels of glutamate in hippocampal slices. Despite these actions, L-trans-PDC did not affect the time course of either the N-methyl-D-aspartate (NMDA) or non-NMDA receptor-mediated synaptic currents evoked by the stimulation of a large number of neighboring synapses. 3. These findings are consistent with models of fast synaptic transmission in which transmitter is rapidly cleared from the synaptic cleft by diffusion. However, in marked contrast to fast gamma-aminobutyric acid A (GABAA) synapses in the hippocampus, uptake does not appear to play a role in regulating the "spill-over" of transmitter from neighboring, co-activated glutamatergic synapses. Therefore, either diffusion alone can effectively limit the temporal and spatial domain of synaptically released glutamate, or alternatively, L-trans-PDC like other currently available blockers is not sufficiently potent to reveal a role for transmitter uptake at glutamatergic synapses.

The triggering of spreading depression in the chicken retina: a pharmacological study

Spreading depression (S.D.) can be reproducibly evoked in the retinas of 3- to 6-day-old chickens by K + ions. N-methyl- d-aspartate (NMDA), kainate (KA), and quisqualate (QA). Specific NMDA antagonist inhibit S.D. evoked by all the above agents. The very selective non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) blocks QA- and KA-evoked S.D. but not NMDA- or K +-evoked S.D. These findings indicate that NMDA receptor activation is the vital event in the triggering of S.D. in this tissue, and that QA and KA trigger S.D. indirectly via excitation of NMDA receptors. Tetrodotoxin, cadnium chloride, conotoxin, baclofen and adenosine agonists are all ineffective in blocking K +-, NMDA-, QA-or KA-evoked S.D. l- trans-Pyrolidine-2,4-dicar☐ylic acid, a glutamate uptake blocker, does inhibit QA-evoked S.D. It is therefore argued that a presynaptic release of vesicular glutamate ‘neurotransmitter stores’ is not the mechanism of action of QA and KA. A mechanism involving a reversal of the glutamate uptake carrier is suggested.