Spermine is neuroprotective against anoxia and N-methyl- -aspartate in hippocampal slices

Polyamines were implicated as either neurotoxic or neuroprotective in several models of stroke. Spermine augments the excitotoxicity mediated by the N-methyl- -aspartate (NMDA) receptor because this receptor is activated at micromolar spermine concentrations. However, at higher concentrations, spermine could be neuroprotective because it blocks the NMDA receptor and voltage-activated Ca²⁺ channels. In this work, acute hippocampal slices were exposed to 1 mM spermine and either 10 min of anoxia or 0.5 mM NMDA. The percent recovery of population spikes was the measure of neuroprotection. One millimolar spermine was robustly neuroprotective; however, 0.1 mM spermine and 1 mM putrescine were not. The neuroprotective concentration of spermine was higher than the physiological concentration of free spermine. However, during an excitotoxic episode, extracellular Ca²⁺ is decreased, enabling the inhibitory activity of lower spermine concentration. In addition, several noxious stimuli trigger the release of intracellular spermine and could raise local levels of spermine. Therefore, it is possible that spermine has a neuroprotective role in vivo.     

Spermine induces cell death in cultured human embryonic cerebral cortical neurons through N-methyl-D-aspartate receptor activation

The polyamines putrescine, spermidine, and spermine play important roles in cell proliferation, differentiation, and modulation of ion channel receptors. However, the function of increased concentrations of these compounds in brain injury and disease is unclear, in that they have been proposed as being both neuroprotective and neurotoxic. The effects of spermine and putrescine were studied in human primary cerebral cortical cultures containing both neurons and glia. No toxic effects were induced at 8 days in vitro (DIV) by either of the two polyamines at concentrations ranging from 0.3 microM to 2 mM. However, when the oxidative metabolism of spermine that generates toxic byproducts was induced by the presence of fetal calf serum, spermine caused cellular death with an LC(50) of approximately 50 microM. At 14 DIV, the coapplication of spermine 2 mM and glutamate 5 mM induced neuron cell death, but the effect of applying both components separately was null. Both spermine and glutamate were toxic to older neurons (26-42 DIV cultures), and here the coapplication of glutamate was found always to intensify the effect of spermine. Spermine showed greater toxicity than glutamate in neurons. Another effect observed is that glutamate, but not spermine, induced astrocyte swelling. Spermine toxicity was inhibited by both MK801 and ifenprodil, indicating a mechanism involving N-methyl-D-aspartate (NMDA) receptor activation. Moreover, a strong spermine modulation of the NMDA receptor was demonstrated by the inhibition of glutamate toxicity by ifenprodil. Putrescine induced minor effects also as a neurotoxic agent. In conclusion, neuronal death by spermine can be induced by its toxic byproducts as well as through NMDA receptor action. The present results confirm the potentially harmful role of the polyamines in excitotoxicity-related human disorders.     

Synapses, NMDA receptor activity and neuronal Aβ production in Alzheimer's disease

A direct relationship has been established between synaptic activity and amyloid-β secretion. Dysregulation of neuronal calcium homeostasis was shown to increase production of amyloid-β, contributing to the initiation of Alzheimer's disease. Among the different routes of Ca(2+) entry, N-methyl-d-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors, are especially involved in this process because of their ability to gate high levels of Ca(2+) influx. These receptors have been extensively studied for their crucial roles in synaptic plasticity that underlies learning and memory but also in neurotoxicity occurring during acute brain injuries and neurodegenerative diseases. For one decade, several studies provided evidence that NMDA receptor activation could have distinct consequences on neuronal fate, depending on their location. Synaptic NMDA receptor activation is neuroprotective, whereas extrasynaptic NMDA receptors trigger neuronal death and/or neurodegenerative processes. Recent data suggest that chronic activation of extrasynaptic NMDA receptors leads to a sustained neuronal amyloid-β release and could be involved in the pathogenesis of Alzheimer's disease. Thus, as for other neurological diseases, therapeutic targeting of extrasynaptic NMDA receptors could be a promising strategy. Following this concept, memantine, unlike other NMDA receptor antagonists was shown, to preferentially target the extrasynaptic NMDA receptor signaling pathways, while relatively sparing normal synaptic activity. This molecular mechanism could therefore explain why memantine is, to date, the only clinically approved NMDA receptor antagonist for the treatment of dementia.     

High potassium induces taurine release by osmosensitive and osmoresistant mechanisms in the rat hippocampus in vivo

The high potassium-evoked taurine efflux in the nervous tissue has been entirely considered to be the result of the cell swelling produced by KCl influx via passive Donnan forces. However, the extracellular taurine increase evoked in the hippocampus by applying 6-100 mM KCl through microdialysis probes, which saturates at a concentration of 25 mM KCl, is not congruent with the mentioned osmosensitive release of taurine stimulated by high potassium. Therefore, we studied whether the taurine release elicited by different high KCl concentrations (25, 50, 75, or 100 mM) was blocked under hypertonic conditions (+100 mM sucrose). Taurine release stimulated by 25 mM KCl was totally osmosensitive, but that released by higher KCl concentrations became progressively osmoresistant, achieving more than the 60% of the extracellular taurine enhancement during 100 mM KCl perfusion. The osmoresistant taurine release evoked by 100 mM KCl perfusion was partially reduced by a solution without Ca(2+) and with high Mg(2+), or by D,L-2-amino-5-phosphopentanoic acid, an N-methyl-D-aspartic acid (NMDA) receptor antagonist. Moreover, the release of taurine induced by a hypoosmotic solution was reduced by the presence of either high K(+) (75 mM) or NMDA (100 microM). These results indicate that although moderately high [K(+)] evoke the osmosensitive release of taurine, higher [K(+)] inhibit it and trigger the release of taurine by an osmoresistant mechanism. This last component is partially mediated by NMDA receptors activated by the glutamate released during potassium-induced depolarization.     

AR-R15896AR blocks the early NMDA-induced loss of MAP2 in primary cortical cultures.

The low-affinity use-dependent N-methyl-D-aspartate (NMDA) receptor antagonist AR-R15896AR is neuroprotective in primary rat cortical cultures exposed to toxic concentrations of NMDA and reduces the magnitude of NMDA-triggered increases in [Ca2+]i. Here we show using fluorescence staining and measurements of microtubule-associated protein-2 (MAP2) levels, that AR-R15896AR inhibits the NMDA-induced loss of MAP2 that occurs within 2 min following NMDA exposure. Understanding the multiple, Ca(2+)-triggered intracellular events that occur following NMDA receptor stimulation is important to the development of safe and effective neuroprotective agents.     

Calcium-independent release of [3H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [3H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [3H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca2+-independent manner. Inhibition of Na+/K+-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na+ electrochemical gradients, since nigericin and veratrine did not induce release in Na+ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

Role of GluR2 expression in AMPA-induced toxicity in cultured murine cerebral cortical neurons

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R)-mediated neurotoxicity was studied in relation to subunit expression and the presence of Ca(2+)-permeable receptor channels. AMPA-mediated toxicity had two components: 1) a direct AMPA-R-mediated component, which was not due to Ca(2+) influx through voltage-gated Ca(2+) channels, reversal of the Na(+)/Ca(2+) exchanger or release of calcium from dantrolene-sensitive intracellular Ca(2+) stores, and 2) a minor, indirect component involving activation of NMDA receptor channels, because of glutamate release and removal of the Mg(2+) block of the NMDA receptor on AMPA-R stimulation. The involvement of Ca(2+) influx through AMPA-R was also examined. The number of neurons possessing Ca(2+)-permeable AMPA-R increased during culture development, concurrently with an increasing susceptibility for AMPA-induced toxicity during development. GluR2(R) levels also increased during development, and channel blockers of Ca(2+)-permeable AMPA-R lacking the GluR2(R) subunit (spermine and philanthotoxin) failed to prevent neurotoxicity or increases in [Ca(2+)](i). Thus, the direct AMPA-R-mediated toxicity may be explained by initiation of cell death by Ca(2+) fluxing through AMPA-R containing GluR2(R). The components of direct AMPA-R-mediated toxicity are proposed to be 1) toxicity mediated by GluR2(R)-lacking AMPA-R and 2) toxicity mediated by low-Ca(2+)-permeability AMPA-R containing GluR2(R).     

Hippocampal mossy fiber long-term depression in Grm2/3 double knockout mice

Group II metabotropic glutamate receptors (mGluR2, encoded by Grm2, and mGluR3, encoded by Grm3) are inhibitory autoreceptors that negatively modulate the adenylate cyclase signaling cascade. Within the hippocampus, mGluR2 is believed to play a key role in the induction of long-term depression (LTD) at mossy fiber-CA3 synapses. Here, we used Grm2/3 double knockout (dko) mice to investigate to what extent group II mGluRs are necessary for mossy fiber LTD. Surprisingly, we found that these mice displayed prominent mossy fiber LTD. However, the induction of this form of LTD was sensitive to the external Ca(2+) concentration. Mossy fiber LTD in Grm2/3 dko mice was indistinguishable from that in wild-type mice at 4 mM Ca(2+) , but largely absent at 2 mM external Ca(2+) . Mossy fiber LTD in Grm2/3 dko mice was not blocked by the N-methyl-D-aspartic acid (NMDA) receptor antagonist D-AP5, confirming that the observed response did not reflect NMDA receptor-dependent LTD in contaminating associational-commissural fibers, and enabling us to use the NMDA receptor-mediated EPSC to monitor mossy fiber LTD. Using whole-cell recordings, we demonstrated that LTD of the NMDA receptor-mediated EPSC in Grm2/3 dko mice was not affected by intracellular application of BAPTA and CsF to block postsynaptic Ca(2+) and G-protein-mediated effects. This presynaptic LTD was, however, blocked by the AMPA/kainate receptor antagonist, NBQX. Thus, an activity-dependent, external Ca(2+) concentration-sensitive form of mossy fiber LTD can be induced in Grm2/3 dko mice. Two mGluR antagonists also failed to block mossy fiber LTD under 4 mM conditions in wild-type mice, strengthening the conclusion that group II mGluRs are not obligatory for mossy fiber LTD.     

Enhancement of NMDA-induced current by the putative NR2B selective antagonist ifenprodil

Ifenprodil has been widely used as an antagonist selective for NMDA receptors containing the NR2B subunit. Evidence suggests, however, that ifenprodil also increases NMDA receptor affinity. Using rat brain slices, we found that ifenprodil enhanced NMDA-induced current in both cortical and subcortical areas examined. To test whether the effect is due to an increase in NMDA receptor affinity, we compared the effect of ifenprodil on currents induced by different concentrations of NMDA. Consistent with the hypothesis, the enhancing effect (percent increase) was relatively constant at low NMDA concentrations. As NMDA concentration increased, however, the effect decreased. To test whether the effect is blocked when NMDA binding sites are saturated with NMDA, high concentrations of NMDA were applied. To partially block Ca(2+) influx and prevent cells from deteriorating, the experiments were performed in the presence of either MK801 or kynurenate, two noncompetitive antagonists. Under such conditions, ifenprodil not only failed to potentiate NMDA currents, but consistently suppressed the current. When the same concentration of NMDA was applied in the presence of the competitive antagonist CGP37849, ifenprodil regained its ability to potentiate NMDA currents. Furthermore, the higher the concentration of CGP37849 the more the NMDA current was potentiated by ifenprodil. These results, combined with previous studies, suggest that the enhancing effect is due to an increase in NMDA receptor affinity and is specific for responses induced by low NMDA concentrations. As NMDA concentration increases, the affinity-enhancing effect decreases. Consequently, the channel-suppressing effect becomes more prominent.     

Novel regimen through combination of memantine and tea polyphenol for neuroprotection against brain excitotoxicity

NMDA receptors are abundant, ubiquitously distributed throughout the brain, fundamental to excitatory neurotransmission, and critical for normal CNS function. However, excessive glutamate overstimulates NMDA receptors, leading to increased intracellular calcium and excitotoxicity. Mitochondrial dysfunction associated with loss of Ca(2+)homeostasis and enhanced cellular oxidative stress has long been recognized to play a major role in cell damage associated with excitotoxicity. In this experiment, we attempted to explore whether treatment with memantine (an NMDA receptor antagonist) and tea polyphenol (an antioxidant and anti-inflammatory agent), either alone or in combination, is effective in neuroprotection in a mouse excitotoxic injury model. Memantine (10 mg/kg/day), tea polyphenol (60 mg/kg/day), or a combination (memantine 5 mg/kg/day plus tea polyphenol 30 mg/kg/day) was administered by oral gavage for 2 consecutive days before causing excitotoxic injury. Mice received a 0.3-microL NMDA [335 mM (pH 7.2)] injection into the left striatum. Locomotor activity was assessed 24 hr before and after excitotoxic injury. Brain synaptosomes were harvested 24 hr after excitotoxic injury for assessment of Na(+), K(+)-ATPase and Mg(2+)-ATPase activity, reactive oxygen species production, mitochondrial membrane potential (Delta Psi m), mitochondrial reductase activity (MTT test), and Ca(2+)concentration. The results showed that treatment with memantine could significantly rescue mitochondrial function by attenuating the decreased mitochondrial membrane potential (Delta Psi m) and mitochondrial reductase activity in mouse excitotoxic injury. Treatment with tea polyphenol could significantly decrease the increased production of synaptosomal reactive oxygen species (ROS) and thus reduced the deteriorative ROS-sensitive Na(+), K(+)-ATPase and Mg(2+)-ATPase activity. However, neither memantine nor tea polyphenol alone could significantly improve the impaired locomotor activity unless treatment was combined. Combined treatment with memantine and tea polyphenol could significantly protect mice against excitotoxic injury by reducing the increased synaptosomal ROS production, attenuating the decreased Na(+), K(+)-ATPase and Mg(2+)-ATPase activity, the mitochondrial membrane potential (Delta Psi m), the mitochondrial reductase activity, and the increased synaptosomal Ca(2+)concentration. In addition, the impairment in locomotor activity was also significantly improved. Therefore, the combined treatment of memantine and tea polyphenol is more effective in neuroprotection than either memantine or tea polyphenol alone in mouse excitotoxic injury. These findings provide useful information about the potential application of memantine and tea polyphenols in preventing clinical excitotoxic injury such as brain trauma, brain ischemia, epilepsy, and Alzheimer's disease.     

Carnosine protects a primary cerebellar cell culture from acute NMDA toxicity

Activation of extrasynaptic NMDA receptors by high glutamate concentrations is one of the key pathogenic factors following a stroke. For this reason, the search for efficient neuroprotective agents that could reduce glutamate toxicity is a pressing need. Ca²⁺ overload in response to glutamate leads to activation of signaling cascades in the cell and the development of oxidative stress, which ultimately leads to apoptosis. Using a model system of acute excitotoxicity caused by 50 μM NMDA, which was used as a specific NMDA receptor activator, we demonstrated that during 2 hours of incubation the viability of the primary neuronal culture decreased by 30–50%. To demonstrate that the observed effect is associated not only with the Ca²⁺ influx into the cytoplasm through the activated NMDA receptors, we decreased the Ca²⁺ concentration in the medium. The lowered Ca2+ concentration, as well as its complete absence, did not affect NMDA toxicity. We tested carnosine, a naturally occurring dipeptide and promising antioxidant, as a neuroprotective agent. The addition of 2 mM carnosine prevented the decrease in cell viability caused by a 2-hour incubation with 50 μM NMDA, while it showed no effect on the viability of the cell culture in the control. Based on the results, we consider the further study of carnosine, its complexes, and analogues as neuroprotectors in cerebral ischemia promising.     

The effects of potassium-induced depolarization, glutamate receptor antagonists and N-methyl-D-aspartate on neuronal survival in cultured neocortex explants

The effects of elevating the potassium concentration of the growth medium of neocortical explants was studied. Under control conditions, 10 mM potassium resulted in ca 20% decrease in the number of surviving neurons. The same potassium concentration, however, was clearly neurotrophic in tetrodotoxin-grown cultures: tetrodotoxin-induced neuronal death was significantly reduced. Both effects could be mimicked by the addition of 10 microM N-methyl-D-aspartate (NMDA); lower concentrations were without effect; higher concentrations were neurotoxic under both control and tetrodotoxin conditions. The neurotoxic, as well as the neurotrophic effects of 10 mM potassium appear to be mediated through depolarization-induced glutamate release since they could be influenced by the application of glutamate receptor antagonists. The addition of the NMDA receptor antagonist D-2-amino-7-phosphonoheptanoate (APH) blocked the trophic effect of 10 mM potassium in tetrodotoxin-grown cultures, resulting in low survival. On the other hand, the addition of the non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) resulted in neuronal survival similar to control cultures, indicating that it blocked the toxic effects of glutamate, leaving the trophic effects on the NMDA receptor untouched. Under control (non-TTX) conditions, neither DNQX nor APH showed significant effects on 10 mM potassium-induced cell death, indicating that stimulation of the non-NMDA, as well as the NMDA receptors is neurotoxic. This differential effect of NMDA receptor stimulation on neuronal survival is discussed with respect to the maturational and/or functional state of the neurons in the culture.     

Polyamines regulate glycine interaction with the N-methyl-D-aspartate receptor

[3H]Glycine binding studies have been performed to further characterize polyamine interactions with the rat brain N-methyl-D-aspartate (NMDA) receptor. Strychnine-insensitive [3H]glycine binding to washed cortical membranes was enhanced by spermine, spermidine, and hirudonin. Spermine stimulation of binding was additive with that produced by the NMDA receptor agonist L-glutamate. A high concentration of the L-glutamate antagonist 2-amino-5-phosphonovaleric acid reduced, but did not eliminate, spermine effects. Saturation experiments indicated that L-glutamate and spermine enhancement of binding was due to an increase in [3H]glycine binding affinity. Kinetic studies showed that optimal concentrations of spermine and L-glutamate reduced [3H]glycine association and dissociation rates by approximately fivefold and 30-fold, respectively. In competition experiments, the presence of L-glutamate and spermine had differential effects on the affinities of compounds that act as either agonists or antagonists at the glycine site of the NMDA receptor. The affinities of the agonists glycine, D-serine, and D-alanine, were increased about fivefold, while antagonist (HA-966, 7-chlorokynurenic acid) inhibitory potencies were unchanged. These data support our previous results showing that the NMDA receptor possesses a novel polyamine recognition site and demonstrate that these compounds directly modulate glycine's interactions with the receptor complex.     

Spermine depresses NMDA, K/AMPA and GABAA-mediated synaptic transmission in the rat hippocampal slice preparation

The effects of spermine, an endogenous polyamine, were examined in area CA1 of the rat hippocampal slice preparation. Spermine, at low millimolar concentrations, rapidly and potently depressed NMDA and K/AMPA-mediated population EPSPs, and GABA-mediated monosynaptic population IPSPs. These effects contrast with its well-known potentiation of NMDA currents at lower concentrations. Our results raise the possibility that the large intracellular stores of spermine that are released after various neural insults could act as an endogenous neuroprotective mechanism by limiting excessive calcium entry.     

Effects of inorganic potassium channel blockers on calcium requirement of transmission in a sympathetic ganglion.

The effects of the alkali metal ions cesium (Cs+) and rubidium (Rb+) and alkaline earth metal ions barium (Ba2+) and strontium (Sr2+) on ganglionic transmission in various calcium (Ca2+) concentrations were investigated in rat isolated superior cervical ganglia. Cesium (1 and 2 mM) moderately supported transmission in low Ca2+ and potentiated the compound action potential (CAP) at high Ca2+ concentration. Higher concentrations of Cs+ caused depression of CAP especially at lower Ca2+ concentration. Cesium (1-4 mM) induced large spontaneous rhythmic spikes or burst of spikes. At 4 mM, Rb+ potentiated CAP at all Ca2+ concentrations. As with Cs+, higher concentrations of Rb+ inhibited CAP except at large Ca2+ concentrations. Barium (1-6 mM) effectively enhanced transmission at all Ca2+ concentrations. Strontium enhanced transmission only at very low Ca2+ concentrations. No enhancement was seen with Sr2+ in the presence of normal or higher Ca2+. The results indicate that Cs+ and Rb+ may antagonize Ca2+ effects at release sites while allowing more Ca2+ influx into the nerve terminal as a result of K+ channel blockade. Barium and to a lesser extent Sr2+ may substitute for Ca2+ in the process of transmitter release in the superior cervical ganglia of rats.     

Effects of calcium on the discharge pattern of primary and secondary endings of isolated cat muscle spindles recorded under a ramp-and-hold stretch

The impulse activity of muscle spindles isolated from the cat tenuissimus muscle was investigated under varying concentrations of external calcium (Ca(2+)). The outer capsule of the muscle spindle represents an effective diffusion barrier for Ca(2+) ions since activity changes were strong and rapid only if the capsule was partly removed from the sensory region of the receptor. The impulse activity of both primary and secondary muscle spindle endings was lowered by an increase in the external Ca(2+) concentration from 1.8 mM (normal Ringer's solution) to 2.7 mM and raised by a decrease in the Ca(2+) concentration from 1.8 to 0.9 mM. Primary endings were generally more strongly affected than secondary endings. With primary endings the firing rate changed by 23-52% when the external Ca(2+) concentration was altered by 0.9 mM. With secondary endings the discharge frequency changed by 15-24%. The afferent discharge patterns were obtained under repetitive ramp-and-hold stretches and were analyzed with regard to influences of external Ca(2+) ions on the static and dynamic components of the endings' responses. The stretch sensitivity and the adaptive response of both types of ending increased in the low Ca(2+) solution and decreased in the high Ca(2+) solution, but a specific effect on a single component of the responses to stretch was not observed. These findings indicate an overall change in excitability when the external Ca(2+) concentration was varied. The mechanical properties of the receptor were probably not affected since changes in the Ca(2+) concentration did not elicit a contraction or relaxation of the intrafusal muscle fibers. On the one hand, the observed effects can be explained according to the surface potential theory by an indirect influence of extracellular Ca(2+) ions on ion channels of the sensory nerve terminals, with Ca(2+) ions binding to negative charged sites at the endings' outer membrane. On the other hand, the results are consistent with the supposition that Ca(2+) ions act directly on ion channels of the sensory membrane of muscle spindle endings.     

N-methyl-D-aspartate receptor-mediated mitochondrial Ca(2+) overload in acute excitotoxic motor neuron death: a mechanism distinct from chronic neurotoxicity after Ca(2+) influx

Mitochondrial uptake of Ca(2+) has recently been found to play an important role in glutamate-induced neurotoxicity (GNT) as well as in the activation of Ca(2+)-dependent molecules, such as calmodulin and neuronal nitric oxide synthase (nNOS), in the cytoplasm. Prolonged exposure to glutamate injures motor neurons predominantly through the activation of Ca(2+)/calmodulin-nNOS, as previously reported, and is, in part, associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we investigated how mitochondrial uptake of Ca(2+) is involved in GNT in spinal motor neurons. Acute excitotoxicity induced by exposure to 0.5 mM glutamate for 5 min was found in both motor and nonmotor neurons in cultured spinal cords from rat embryos and was dependent on extracellular Ca(2+) and on N-methyl-D-aspartate (NMDA) receptor activation. Mitochondrial uncouplers markedly blocked acute excitotoxicity, and membrane-permeable superoxide dismutase mimics attenuated acute excitotoxicity induced by glutamate and NMDA but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate. Fluorimetric analysis showed that mitochondrial Ca(2+) was elevated promptly with subsequent accumulation of reactive oxygen species (ROS) in the mitochondria. An NMDA receptor antagonist and a mitochondrial uncoupler eliminated the increase in fluorescence of mitochondrial Ca(2+) and ROS indicators. These data indicate that acute excitotoxicity in spinal neurons is mediated by mitochondrial Ca(2+) overload and ROS generation through the activation of NMDA receptors. This mechanism is different from that of chronic GNT.     

N-methyl-D-aspartate autoreceptors respond to low and high agonist concentrations by facilitating, respectively, exocytosis and carrier-mediated release of glutamate in rat hippocampus

Presynaptic NMDA autoreceptors regulating glutamate release have rarely been investigated. High-micromolar N-methyl-D-aspartate (NMDA) was reported to elicit glutamate release from hippocampal synaptosomes in a Ca(2+)-independent manner by reversal of excitatory amino acid transporters. The aim of this work was to characterize excitatory amino acid release evoked by low-micromolar NMDA from glutamatergic axon terminals. Purified rat hippocampal synaptosomes were prelabelled with [(3)H]D-aspartate ([(3)H]D-ASP) and exposed in superfusion to varying concentrations of NMDA in the presence of 1 microM glycine. The release of [(3)H]D-ASP and also that of endogenous glutamate provoked by 10 microM NMDA were external Ca(2+) dependent and sensitive to the NMDA channel blocker MK-801 but insensitive to the glutamate transporter inhibitor DL-TBOA, which, on the contrary, prevented the Ca(2+)-independent release evoked by 100 microM NMDA. The NMDA (10 microM) response was blocked by 1 nM Zn(2+) and 1 microM ifenprodil, compatible with the involvement of a NR1/NR2A/NR2B assembly, although the presence of two separate receptor populations, i.e., NR1/NR2A and NR1/NR2B, cannot be excluded. This response was strongly antagonized by submicromolar (0.01-1 microM) concentrations of kynurenic acid and was mimicked by quinolinic acid (1-100 microM) plus 1 microM glycine. Finally, the HIV-1 protein gp120 potently mimicked the NMDA co-agonists glycine and D-serine, being significantly effective at 30 pM. In conclusion, glutamatergic nerve terminals possess NMDA autoreceptors mediating different types of release when activated by different agonist concentrations: low-micromolar glutamate would potentiate glutamate exocytosis, whereas higher glutamate concentrations would also provoke carrier-mediated release.     

NMDA receptor-mediated synaptic excitation selectively inhibited by ethanol in hippocampal slice from adult rat

The effect of ethanol (EtOH) on synaptic transmission mediated by N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors was investigated in slices from adult rat hippocampus. Synaptic responses were elicited by stimulation of stratum radiatum and were recorded in CA1 stratum radiatum or stratum pyramidale. Population EPSPs (pEPSPs) mediated by NMDA receptor activation were isolated by application of a solution containing the kainate/quisqualate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione and either low (0.1 mM) Mg2+ or 100 microM bicuculline. Increasing concentrations of EtOH produced increasing inhibition of NMDA receptor-mediated pEPSPs with EtOH concentrations between 1 and 50 mM. At a concentration of 50 mM, EtOH inhibited NMDA receptor-mediated pEPSPS by 43%; the inhibition by 100 mM EtOH was not significantly different from that produced by 50 mM. Methanol and 1-butanol also inhibited the NMDA receptor-mediated pEPSPs; the potency of the alcohols for inhibition of NMDA receptor-mediated pEPSPs was 1-butanol greater than ethanol greater than methanol. pEPSPs mediated by non-NMDA glutamate receptors were isolated by the application of the NMDA receptor antagonist d,1-2-amino-5-phosphonovaleric acid in the presence of 1.5 mM Mg2+. These pEPSPs were not significantly affected by 50 mM EtOH, whereas 100 mM EtOH reduced the amplitude of these pEPSPs by 9%. The observations indicate that synaptic excitation mediated by NMDA receptors in tissue from adult rat is inhibited by intoxicating concentrations of EtOH. The data are consistent with the hypothesis that EtOH-induced inhibition of EPSPs mediated NMDA receptors may contribute to the intoxicating effects of EtOH.