Glutamate Release from Guinea-Pig Synaptosomes: Stimulation by Reuptake-Induced Depolarization

Glutamate (10-100 microM) reversibly depolarizes guinea-pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 microM N-methyl-D-aspartate (NMDA), 100 microM L-2-amino-4-phosphonobutanoate (APB), nor 100 microM quisqualate affects the Ca2+-dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists beta-N-oxalylamino-L-alanine and alpha-amino-3-hydroxy-5-methylisoxazole propionate cause a slow Ca2+-independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor-mediated neurotoxin beta-N-methylamino-L-alanine do not influence Ca2+-independent release at 100 microM. The depolarization of the plasma membrane by glutamate can be mimicked by D-aspartate, can be blocked by the transport inhibitor dihydrokainate, and is accompanied by the net uptake of acidic amino acids. L-Glutamate or D-aspartate at 100 microM increases the cytoplasmic free Ca2+ concentration. D-aspartate at 100 microM causes a Ca2+-dependent release of endogenous glutamate, superimposed on the Ca2+-independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.     

Glutamate Receptor Subtypes in Cultured Cerebellar Neurons: Modulation of Glutamate and γ-Aminobutyric Acid Release

Using cerebellar, neuron-enriched primary cultures, we have studied the glutamate receptor subtypes coupled to neurotransmitter amino acid release. Acute exposure of the cultures to micromolar concentrations of kainate and quisqualate stimulated D-[3H]aspartate release, whereas N-methyl-D-aspartate, as well as dihydrokainic acid, were ineffective. The effect of kainic acid was concentration dependent in the concentration range of 20-100 microM. Quisqualic acid was effective at lower concentrations, with maximal releasing activity at about 50 microM. Kainate and dihydrokainate (20-100 microM) inhibited the initial rate of D-[3H]aspartate uptake into cultured granule cells, whereas quisqualate and N-methyl-DL-aspartate were ineffective. D-[3H]Aspartate uptake into confluent cerebellar astrocyte cultures was not affected by kainic acid. The stimulatory effect of kainic acid on D-[3H]aspartate release was Na+ independent, and partly Ca2+ dependent; the effect of quisqualate was Na+ and Ca2+ independent. Kynurenic acid (50-200 microM) and, to a lesser extent, 2,3-cis-piperidine dicarboxylic acid (100-200 microM) antagonized the stimulatory effect of kainate but not that of quisqualate. Kainic and quisqualic acid (20-100 microM) also stimulated gamma-[3H]-aminobutyric acid release from cerebellar cultures, and kynurenic acid antagonized the effect of kainate but not that of quisqualate. In conclusion, kainic acid and quisqualic acid appear to activate two different excitatory amino acid receptor subtypes, both coupled to neurotransmitter amino acid release. Moreover, kainate inhibits D-[3H]aspartate neuronal uptake by interfering with the acidic amino acid high-affinity transport system.     

N-methyl-d-aspartate andl-aspartate activate distinct receptors in piriform cortex

The effects of ionophoretically applied N-methyl-DL-aspartate (NMDA) and aspartate on identified pyramidal neurons in rat piriform cortex were examined in isolated, submerged, and perfused brain slices. NMDA was more potent than aspartate in eliciting neuronal discharge. Perfusion of the acidic amino acid antagonists, DL-2-amino-5-phosphonovalerate (APV), 10(-6) or 10(-5) M, DL-2-amino-7-phosphonoheptanoate (APH), 10(-5) M, and gamma-D-glutamylglycine (gamma DGG), 10(-5) M, selectively blocked the response to NMDA without effect on the response to aspartate. At higher concentrations which blocked responses to both NMDA and aspartate, gamma DGG blocked kainate responses and depressed glutamate and quisqualate responses. These results suggest that in piriform neurons NMDA and aspartate act at distinct receptor sites, not a common receptor site, and that both of these sites are distinct from those that mediate responses to glutamate, quisqualate, and kainate.     

6,7-Dinitroquinoxaline-2,3-Dione Blocks the Cytotoxicity of N-Methyl-D-Aspartate and Kainate, but Not Quisqualate, in Cortical Cultures

Based on radioligand binding and electrophysiological studies, quinoxalinediones such as 6,7-dinitroquinoxaline-2,3-dione (DNQX) have been shown to be potent competitive antagonists at the quisqualate and kainate subtypes of the glutamate receptor. In this report we have examined the effects of DNQX on excitatory amino acid neurotoxicity and evoked neurotransmitter release. DNQX was found to be a potent neuroprotective agent against glutamate and N-methyl-D-aspartate (NMDA) neurotoxicity. The data suggest that this neuroprotective activity of DNQX is due to its antagonism of the coagonist activity of glycine at the NMDA receptor-channel complex. The specificity of DNQX for the glycine site associated with the NMDA receptor-channel complex was confirmed in radioligand binding and neurotransmitter release studies. DNQX also prevented kainate neurotoxicity and kainate-evoked neurotransmitter release, presumably by direct competition for the kainate receptor. DNQX, however, did not prevent quisqualate neurotoxicity, suggesting that a novel quisqualate-preferring receptor insensitive to DNQX may mediate quisqualate toxicity.     

The Glycine Site of the N-Methyl-D-Aspartate Receptor Channel: Differences Between the Binding of HA-966 and of 7-Chlorokynurenic Acid

The mechanisms of action of three different glycine-site antagonists of the N-methyl-D-aspartate (NMDA)-receptor channel were analyzed employing [3H]glycine direct binding assays, as well as functional glycine- and glutamate-induced uncompetitive blocker binding assays. The latter assays measure apparent channel opening. All three antagonists tested, viz., 7-chlorokynurenic acid (7-Cl-KYNA), kynurenic acid (KYNA), and 1-hydroxy-3-aminopyrrolidone-2 (HA-966), inhibited the binding of [3H]glycine to the NMDA receptor in a dose-dependent manner. These antagonists also inhibited the glycine-induced increase in accessibility of the uncompetitive blocker [3H]N-[1-(2-thienyl)cyclohexyl]-piperidine ([3H]TCP) to the channel. 7-Cl-KYNA and KYNA, but not HA-966, completely blocked the glutamate-induced binding of [3H]TCP, in a manner similar to the non-competitive manner in which the selective NMDA antagonist D-(-)-2-amino-5-phosphonovaleric acid (AP-5) inhibited glycine-induced [3H]TCP binding. The inhibitory effects of HA-966 and of AP-5 on glutamate-induced [3H]TCP binding were overcome when glutamate concentrations were increased. Of the three antagonists, 7-Cl-KYNA appears to be the most potent (Ki = 0.4-1.0 microM) and the most selective glycine antagonist. KYNA was found to act at both the glycine (Ki = 40-50 microM) and the glutamate sites. In contrast, HA-966 (Ki = 6-17 microM) appears to act either on a domain distinct from the glutamate and the glycine sites, but tightly associated with the latter, or at the glycine site, but according to a mechanism distinct from that of 7-Cl-KYNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Astrocyte Heterogeneity: Endogenous Amino Acid Levels and Release Evoked by Non-N-Methyl-D-Aspartate Receptor Agonists and by Potassium-Induced Swelling in Type-1 and Type-2 Astrocytes

The aim of the present study was to determine whether endogenous amino acids are released from type-1 and type-2 astrocytes following non-N-methyl-D-aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type-1 or type-2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type-1 than in type-2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type-1 than in type-2 astrocytes; that of other amino acids was similar. (c) Exposure of type-2 astrocyte cultures to 50 microM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type-1 astrocyte cultures to 50-200 microM kainate or 50 microM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type-1 or type-2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type-1 and type-2 astrocytes. Swelling and K(+)-induced release were somewhat higher in type-2 than in type-1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non-NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type-2 but not from type-1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type-1 and type-2 astrocytes. The absence of kainate- and quisqualate-evoked release in type-1 astrocytes suggests that the density of non-NMDA receptors in this cell type is very low.     

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.     

Excitatory amino acids inhibit stimulated phosphoinositide hydrolysis in the rat prefrontal cortex

In rat prefrontal cortical slices, the excitatory amino acids N-methyl-D-aspartate (NMDA), ibotenate, L-aspartate, quisqualate, kainate and L-glutamate inhibit carbachol-induced phosphoinositide hydrolysis as measured by the accumulation of [3H]inositol-1-phosphate ([3H]IP1). NMDA dose-dependently inhibited the carbachol response (IC50 = 14.4 microM), and this inhibition was blocked by the NMDA receptor antagonist D,L-aminophosphonovaleric acid. Lowering medium Na+ concentration to 10 mM or exposing slices to pertussis toxin alleviated the inhibitory effect of NMDA on carbachol-induced [3H]IP1 formation. Serotonin-induced stimulation of [3H]IP1 was also inhibited by NMDA; in contrast, stimulation by norepinephrine, epinephrine or dopamine was unaffected. The results suggest that excitatory amino acids, besides their traditional role as stimulatory substances, can also act to inhibit the production of 2nd messengers activated by certain neurotransmitters in the brain.     

Autoreceptor Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area

Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and gamma-aminobutyrate evoked by 50 mM K+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(+/- )-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 microM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K(+)-evoked release of aspartate. Conversely, 6-cyano-7-nitro-quinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.     

Excitatory amino acid receptor agonists stimulate membrane inositol phospholipid hydrolysis and increase cytoplasmic free Ca2+ in primary cultures of retinal neurons

A variety of neurotransmitters are believed to elicit effects through receptor-stimulated inositol phospholipid metabolism. It appears that most major types of retinal neurons receive a direct glutamatergic input. The aim of the present studies was to characterize excitatory amino acid (EAA) receptor-mediated breakdown of inositol phospholipids and changes in Ca2+ homeostasis in primary avian retinal cell cultures. Cell monolayers, prepared from 8-day-old chick embryo neural retina, were labelled with [3H]inositol for 48 h, and used after 7 days in vitro. Kainic acid stimulated the accumulation of inositol phosphates in a time- and dose-dependent manner (ED50 = 30 microM). The EAA receptor agonists glutamate, N-methyl-D-aspartate (NMDA), ibotenate and quisqualate were all active, with the rank order: glutamate greater than kainate greater than NMDA much greater than ibotenate approximately quisqualate. External Ca2+ was required for these effects. Agonist actions were inhibited by type-specific antagonists, and also Mg2+ in the case of glutamate and NMDA. Glutamate, NMDA and kainate also elevated cytosolic free Ca2+ in individual retinal cells loaded with the Ca2(+)-sensitive dye Fura-2, as assessed by digital fluorescence ratio imaging microscopy. The agonist-induced increases in [Ca2+]i were largely dependent on extracellular Ca2+, independent of membrane depolarization and were blocked by Mg2+ for glutamate and NMDA. These results demonstrate that vertebrate retinal cells possess EAA receptors coupled to intracellular signal transduction pathways.     

Quisqualic Acid Modulates Kainate Responses in Cultured Cerebellar Granule Cells

The activation of kainic acid and quisqualic acid receptors in cultured cerebellar granule cells stimulated the release of preaccumulated D-[3H]aspartate. The effect of kainate could be distinguished from that of quisqualate by its sensitivity to the antagonists kynurenic acid and 2,3-cis-piperidine dicarboxylic acid. At a concentration of kainic acid (50 microM) close to its half-maximal releasing effect, simultaneous addition of quisqualic acid (10-50 microM) resulted in a significant dose-dependent inhibition of the kainate-induced component of D-[3H]aspartate release, which was monitored by the progressive decrease in sensitivity of the evoked release to kynurenic acid. In contrast, when kainic acid was used at a subeffective concentration (10 microM), addition of low doses of quisqualate (2-5 microM) resulted in a synergistic effect on D-[3H]aspartate release. Under these conditions, the effect of the two agonists was sensitive to kynurenic acid. Kainic acid (50-100 microM) also caused a dose-dependent, kynurenic acid-sensitive accumulation of cyclic GMP (cGMP) in granule cell cultures. Quisqualic acid was, by itself, ineffective and prevented, in a dose-dependent manner, the kainate-induced cGMP formation (IC50 = 5 microM). Finally, the guanylate cyclase activator sodium nitroprusside greatly enhanced cGMP formation but had no effect on D-[3H]aspartate release. Together, these results demonstrate the existence of complex interactions between quisqualic and kainic acids and indicate that the effects of the two glutamate agonists on D-[3H]aspartate release and on cGMP accumulation are independent.     

The new 2,3-benzodiazepine derivative EGIS-8332 inhibits AMPA/kainate ion channels and cell death

We observed in vitro neuroprotective and AMPA/kainate receptor antagonist effects of the new 2,3-benzodiazepine derivative EGIS-8332 (R,S-1-(4-aminophenyl)-7,8-methylenedioxy-4-cyano-4-methyl-3-N-acetyl-5H-3,4-dihydro-2,3-benzodiazepine) using the lactate dehydrogenase (LDH) release assay and patch clamp recordings on primary cultures of rat embryonic telencephalon neurons exposed to AMPA/kainate receptor agonists. EGIS-8332 potently decreased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate induced LDH release (IC(50)=5.2+/-0.4 and 7.4+/-1.3 microM, respectively) from the cells. Whole-cell patch clamp studies carried out on the ionotropic glutamate receptors N-methyl D-aspartate (NMDA), as well as AMPA (and kainate) in cultured telencephalon neurons verified that EGIS-8332 blocked steady state responses to AMPA and kainate (IC(50)=1.7+/-0.4 and 6.2+/-1.6 microM, respectively), but hardly influenced currents evoked by NMDA. EGIS-8332 also inhibited kainate-evoked response in CHO cells expressing the flop variant of GluR1 receptor and, in cerebellar Purkinje cells at similar efficiency. The stereoselectivity of the inhibitory site is established by the clearly dissimilar inhibitory potency of the enantiomer components of EGIS-8332 differing in the configuration of methyl and cyano substituents on carbon C(4): the R(-) enantiomer was found to be the efficient species. This finding suggests that the inhibitory interaction between the channel protein and drug is promoted by presence of the C(4) methyl group. The inhibition of the AMPA/kainate ion channels by EGIS-8332 is non-competitive, not use dependent, and depends neither on the closed/open state of the channel, nor the membrane potential. These findings suggest an allosteric mechanism for the inhibition. These in vitro observations suggest that the compound might be useful in the treatments of certain acute and chronic neurological syndromes initiated by derangements of ionotropic glutamate receptor function.     

Glutamate Inhibits Adenylate Cyclase Activity in Dispersed Rat Hippocampal Cells Directly via an N-Methyl-d-Aspartate-Like Metabotropic Receptor

Three major subtypes of glutamate receptors that are coupled to cation channels--N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors--are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin-stimulated cyclic AMP (cAMP) accumulation; half-maximal effects were obtained with 5.6 +/- 2.2 and 6.4 +/- 2.3 microM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3-diaminopropionate and 2-amino-5-phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 microM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 microM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP-dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective blockade of NMDA-activated channel currents may be implicated in learning deficits caused by lead.

The effect of Pb2+ on glutamate receptor activity in rat hippocampal neurons was investigated with a view of explaining the cognitive and learning deficits produced by this heavy metal. Pb2+ (2.5-50 microM) selectively inhibited N-methyl-D-aspartate (NMDA)-induced whole-cell and single-channel currents in a concentration-dependent but voltage-independent manner, without significantly altering currents induced by either quisqualate or kainate. The frequency of NMDA-induced channel activation was decreased by Pb2+. Neither glycine (10-100 microM), nor Ca2+ (10 mM) reversed the effect of Pb2+. Pb2+ also inhibited the [3H]MK-801 binding to rat hippocampal membranes in vitro. The elucidation of the actions of Pb2+ on the NMDA receptor ion channel complex provides important insights into the clinical and toxic effects of this cation.     

Modulation of Dendritic Release of Dopamine by N-Methyl-D-Aspartate Receptors in Rat Substantia Nigra

A superfusion system was used to study the effects of excitatory amino acids (EAA) on release of [3H]dopamine ([3H]DA) previously taken up by rat substantia nigra (SN) slices. The EAA tested (20-250 microM), with the exception of quisqualate and kainate, markedly evoked [3H]DA release from nigral slices when Mg2+ ions were omitted from the superfusion medium. The EAA receptor agonists exhibited the following relative potency in stimulating [3H]DA release: L-glutamate (L-Glu) greater than N-methyl-D-aspartate (NMDA) greater than NM(D,L)A greater than D-Glu much greater than quisqualate = kainate. D-2-Amino-5-phosphonovalerate (100-200 microM), an antagonist for NMDA receptors, substantially reduced [3H]DA release evoked by L-Glu or NMDA. In contrast, L-Glu diethyl ester (100-200 microM) produced a lesser blocking effect on [3H]DA release evoked by the EAA. Further experiments showed that the NMDA-mediated release of [3H]DA was totally suppressed by the omission of Ca2+ or by the addition of tetrodotoxin (0.1 microM) to the superfusion medium. In addition, strychnine, an antagonist for glycine (Gly) receptors, significantly decreased NMDA (100 microM)-evoked as well as glycine (100 microM)-evoked release of [3H]DA from nigral slices. The results shown support the idea that activation of NMDA subtype receptors in SN may trigger a Ca2+-dependent release of DA from dendrites of nigro-striatal DA-containing neurons. Furthermore, a transsynaptic mechanism that may partially involve Gly-containing interneurons is proposed to account for some of the events mediating NMDA receptor activation and DA release in SN.     

Effects of excitatory amino acids on inositol phosphate accumulation in slices of the cerebral cortex of young and aged rats

The effects of glutamate, NMDA and quisqualate on carbachol-and norepinephrine-elicited formation of inositol phosphate (IP) were evaluated in slices prepared from the cerebral cortex of 3-and 24-month Sprague-Dawley rats. Glutamate, NMDA, and quisqualate antagonized the IP response to carbachol in a concentration-dependent fashion. This antagonism was more pronounced in aged than in young rats, both for glutamate (IC5O 0.114 and 0.210 mM) and NMDA (IC5O 0.0029 and 0.127 mM), but not for quisqualate. Glutamate (but not NMDA) also antagonized in a concentration-dependent fashion the IP response to norepinephrine, IC50s were 0.061 and 0.126 mM for aged and young rats, respectively; quisqualate had an inhibitory effect only at 1 mM concentration in the two age-groups, while in aged rats some stimulatory effect was present at 0.1 mM concentration. Glutamate, NMDA and quisqualate (1 mM) did not affect basal IP accumulation in either young or aged rats; quisqualate, however, at 0.1 mM concentration had some stimulatory effect, more pronounced in aged rats. This effect was probably responsible for the biphasic effect of quisqualate in this age-group. The most important finding consists of the demonstration of an age-related increase in the inhibitory effects of NMDA on carbachol-induced IP accumulation. This implies an altered modulation of cholinergic post-receptor mechanisms by glutamatergic mechanisms.     

Excitotoxicity, reflex responses, and evoked changes in extracellular potassium in the frog spinal cord

1. The effects of the excitatory amino acid agonists kainate (KA), quisqualate (QUIS), and N-methyl-D-aspartate (NMDA) were studied in vitro on the hemisected frog spinal cord. 2. Prolonged (1.0 hr) application of excitatory amino acid agonists (KA, 50 or 300 microM; QUIS, 30 microM; NMDA, 300 microM) significantly reduced the ventral root potentials (VRPs) and [K+]0 evoked by a dorsal root tetanus (10 sec, 25 Hz), by brief (10 sec) applications of the same agonists (KA, 30 microM; QUIS, 30 microM; NMDA, 300 microM), and by GABA (10 sec, 1.0 mM). 3. The effect was essentially irreversible and persisted despite 2-4 hr of washing. 4. Excitatory amino acid antagonists (APV, 30 microM and kynurenate, 2 mM) blocked the neurotoxic effects of the excitatory agonists NMDA and KA respectively, an observation which indicates the observed effects of the agonists require the activation of specific excitatory receptors. 5. TTX did not alter the neurotoxic effects of KA suggesting that interneuronal firing does not contribute to the observed changes. 6. Addition of high K+ did not duplicate the effect of prolonged excitatory amino acid agonist exposure, an indication that elevation of K+ does not cause the decreased responses. 7. Light microscopy did not provide any evidence of gross tissue damage. 8. The parallel reduction of postsynaptic responses and delta [K+]0 support the idea that elevation of extracellular [K+] by afferent stimuli results from interneuronal activity.     

Neuroprotective Effects of NMDA Receptor Glycine Recognition Site Antagonism: Dependence on Glycine Concentration

Abstract: High-affinity NMDA receptor glycine recognition site antagonists protect brain tissue from ischemic damage. The neuroprotective effect of 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021), a selective NMDA receptor antagonist with nanomolar affinity for the glycine binding site, was examined in rat cortical mixed neuronal/glial cultures. ACEA 1021 alone did not alter spontaneous lactate dehydrogenase (LDH) release. Treatment with ACEA 1021 (0.1–10 µ M ) before 500 µ M glutamate, 30 µ M NMDA, or 300 µ M kainate exposure was found to reduce LDH release in a concentration-dependent fashion. These effects were altered by adding glycine to the medium. Glycine (1 m M ) partially reversed the effect of ACEA 1021 on kainate cytotoxicity. Glycine (100 µ M –1 m M ) completely blocked the effects of ACEA 1021 on glutamate and NMDA cytotoxicity. The glycine concentration that produced a half-maximal potentiation of excitotoxin-induced LDH release in the presence of 1.0 µ M ACEA 1021 was similar for glutamate and NMDA (18 ± 3 and 29 ± 9 µ M , respectively). ACEA 1021 also reduced kainate toxicity in cultures treated with MK-801. The effects of glycine and ACEA 1021 on glutamate-induced LDH release were consistent with a model of simple competitive interaction for the strychnine-insensitive NMDA receptor glycine recognition site, although nonspecific effects at the kainate receptor may be of lesser importance.     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-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. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

Role of excitatory amino acids in the regulation of dopamine synthesis and release in the neostriatum

Summary We have explored the role of excitatory amino acids in the increased dopamine (DA) release that occurs in the neostriatum during stress-induced behavioral activation. Studies were performed in awake, freely moving rats, usingin vivo microdialysis. Extracellular DA was used as a measure of DA release; extracellular 3,4-dihydroxyphenylalanine (DOPA) after inhibition of DOPA decarboxylase provided a measure of apparent DA synthesis. Mild stress increased the synthesis and release of DA in striatum. DA synthesis and release also were enhanced by the intra-striatal infusion of N-methyl-D-aspartate (NMDA), an agonist at NMDA receptors, and kainic acid, an agonist at the DL-a-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA)/kainate site. Stress-induced increase in DAsynthesis was attenuated by co-infusion of 2-amino-5-phosphonovalerate (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), antagonists of NMDA and AMPA/kainate receptors, respectively. In contrast, intrastriatal APV, CNQX, or kynurenic acid (a non-selective ionotropic glutamate receptor antagonist) did not block the stress-induced increase in DArelease. Stress-induced increase in DA release was, however, blocked by administration of tetrodotoxin along the nigrostriatal DA projection. It also was attenuated when APV was infused into substantia nigra. Thus, glutamate may act via ionotropic receptors within striatum to regulate DA synthesis, whereas glutamate may influence DA release via an action on receptors in substantia nigra. However, our method for monitoring DA synthesis lowers extracellular DA and this may permit the appearance of an intra-striatal glutamatergic influence by reducing a local inhibitory influence of DA. If so, under conditions of low extracellular DA glutamate may influence DA release, as well as DA synthesis, by an intrastriatal action. Such conditions might occur during prolonged severe stress and/or DA neuron degeneration. These results may have implications for the impact of glutamate antagonists on the ability of patients with Parkinson's disease to tolerate stress.