Dual-component synaptic potentials in the lamprey mediated by excitatory amino acid receptors

The synaptic mechanisms underlying amino acid-mediated excitation in the lamprey spinal cord have been investigated. Fine stimulating electrodes were used to stimulate single axons in the spinal cord and evoke unitary EPSPs in lamprey motoneurons and one type of premotor interneuron, the CC interneuron. Three types of EPSP, distinguished by their time course and sensitivity to amino acid antagonists, were seen. Fast EPSPs had a fast rise time (mean, 6.5 msec) and a short half-decay time (mean, 22.5 msec). Slow EPSPs lasted at least 200 msec, had a slow rise time (mean, 28 msec), and a long half-decay time (mean, 109 msec). The third type of unitary potential, called "mixed" EPSP, also lasted at least 200 msec, had a fast rise time (mean, 12 msec), and a long half-decay time (mean, 105 msec). Lamprey neurons were found to possess 3 types of excitatory amino acid receptor: N-methyl-D-aspartate (NMDA), kainate, and quisqualate receptors. 2-Amino-5-phosphonovaleric acid (APV) or Mg2+ blocked the depolarizations caused by N-methyl-D,L-aspartate (NMA) but not those of kainate or quisqualate. Cis-2, 3-piperidine dicarboxylic acid (PDA) blocked the depolarizations caused by NMA and kainate but not those of quisqualate. Fast EPSPs were unaffected by the bath application of APV or Mg2+ but were greatly reduced by PDA, suggesting that these EPSPs were mediated by non-NMDA, possibly kainate receptors. Both APV and Mg2+ blocked the slow EPSPs, suggesting that they were mediated by NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for excitatory amino acid transmission between mesencephalic nucleus of V afferents and jaw-closer motoneurons in the guinea pig

Previous studies have suggested that monosynaptic transmission between spinal primary afferent fibers and motoneurons is mediated by an excitatory amino acid, most likely glutamate or aspartate. No such comparable studies have been carried out in the trigeminal system. In an attempt to elucidate the neurotransmitter(s) mediating monosynaptic transmission between mesencephalic of V nucleus afferents (Mes V) and trigeminal jaw-closer motoneurons, the effect of iontophoretic application of excitatory amino acid antagonists on the Mes V-induced field potential, recorded in the trigeminal motor nucleus (Mot V), was examined. Application of DL-2-amino-4-phosphonobutyrate (APB) and the broad spectrum amino acid antagonists, kynurenic acid (KYN) and gamma-D-glutamylglycine (DGG), for 3-4 min reversibly reduced the amplitude of the Mes V induced field potential. The effect of APB was much greater than any of the other compounds tested. On the other hand, the specific N-methyl-D-aspartate (NMDA) receptor blocker DL-2-amino-5-phosphonovaleric acid (APV), was without effect on the field potential. Based on current-response curves for each antagonist tested, the order of potency was determined to be APB greater than KYN greater than DGG greater than APV. These antagonists were also compared with respect to their efficacy in blocking individual jaw-closer motoneuron activity induced by iontophoretic application of amino acid receptor excitants glutamate (Glut), aspartate (Asp), kainate (K), and quisqualate (Q). NMDA application was without effects on these motoneurons. The profile of activity of these antagonists on these amino acid excitants was similar to that found in other areas of the CNS by other investigators. KYN and DGG both significantly reduced responses induced by all excitants tested, whereas APB had more modest effects on K and Q excitation and was without effect on Glut and Asp excitations in most cells tested. The data suggest that an excitatory amino acid, activating non-NMDA receptors, mediates some component of synaptic transmission between Mes V afferents and jaw-closer motoneurons. The data is also consistent with the proposal made in other systems that APB blocks synaptic transmission by a mechanism other than postsynaptic receptor blockade.     

Reticulospinal neurones activate excitatory amino acid receptors

Paired intracellular recordings were used to study the monosynaptic excitatory postsynaptic potentials (EPSP) in lamprey motoneurones evoked by stimulation of single reticulospinal Müller and Mauthner cells. The chemical component of the synaptic potentials was depressed by both application of the non-selective excitatory amino acid antagonists kynurenic acid and cis-2,3-piperidine dicarboxylate. The N-methyl-D-aspartate (NMDA) antagonists Mg2+ and 2-amino-5-phosphonovalerate caused a selective depression of a late component of the EPSP. Thus, fast-conducting reticulospinal neurones appear to release an excitatory amino acid acting at both NMDA and non-NMDA receptors.     

Unmyelinated cutaneous afferent neurons activate two types of excitatory amino acid receptor in the spinal cord of Xenopus laevis embryos

The trunk and tail skin of Xenopus laevis embryos near the time of hatching is innervated by the mechanoreceptive free nerve endings of Rohon-Beard neurons, a homogeneous class of cutaneous primary afferent fibers. Rohon-Beard neurons have cell bodies and axons in the dorsal spinal cord, where they monosynaptically excite a population of dorsolaterally situated interneurons (Clarke and Roberts, 1984). EPSPs can be recorded in these dorsolateral interneurons following electrical stimulation of the unmyelinated neurites of Rohon-Beard neurons in the skin. The EPSPs are dual component, consisting of separate fast and slow potentials that are usually evoked synchronously and that closely resemble those described previously in Xenopus and lamprey motoneurons (Dale and Roberts, 1985; Dale and Grillner, 1986). The excitation of dorsolateral interneurons by Rohon-Beard neurons is reduced by the bath application of excitatory amino acid antagonists. Kynurenic acid suppresses both the fast and slow components of the EPSPs, while both (+/-)-2-amino-5-phosphonovaleric acid (APV) and 1 mM magnesium reduce the slow component but have little or no effect on the peak amplitude of the EPSPs. These data suggest that Rohon-Beard neurons release an excitatory amino acid neurotransmitter, which acts simultaneously at both N-methyl-D-aspartate (NMDA) and non-NMDA receptor types. This is the first direct demonstration of dual-component excitatory amino acid-mediated synaptic transmission from cutaneous primary afferent neurons in the vertebrate spinal cord. The bath application of the agonists NMDA, kainate, or quisqualate in salines containing 1 microM TTX depolarized the interneurons and reduced their input resistance, which suggests that the interneurons possess all 3 types of excitatory amino acid receptor. Kynurenic acid strongly inhibits responses to NMDA and kainate, but is relatively less effective against the larger responses of quisqualate in this system.     

Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors

Dose-response curves for activation of excitatory amino acid receptors on mouse embryonic hippocampal neurons in culture were recorded for 15 excitatory amino acids, including the L-isomers of glutamate, aspartate, and a family of endogenous sulfur amino acids. In the presence of 3 microM glycine, with no extracellular Mg, micromolar concentrations of 11 of these amino acids produced selective activation of N-methyl-D-aspartate (NMDA) receptors. L-Glutamate was the most potent NMDA agonist (EC50 2.3 microM) and quinolinic acid the least potent (EC50 2.3 mM). Dose-response curves were well fit by the logistic equation, or by a model with 2 independent agonist binding sites. The mean limiting slope of log-log plots of NMDA receptor current versus agonist concentration (1.93) suggests that a 2-site model is appropriate. There was excellent correlation between agonist EC50S determined in voltage clamp experiments and KdS determined for NMDA receptor binding (Olverman et al., 1988). With no added glycine, and 1 mM extracellular Mg, responses to NMDA were completely blocked; responses to kainate and quisqualate were unchanged. Under these conditions, glutamate and the sulfur amino acids activated a rapidly desensitizing response, similar to that evoked by micromolar concentrations of quisqualate and AMPA, but mM concentrations of L-aspartate, homoquinolinic acid, and quinolinic acid failed to elicit a non-NMDA receptor-mediated response. Except for L-glutamate (EC50 480 microM), the low potency of the sulfur amino acids prevented the study of complete dose-response curves for the rapidly desensitizing response at quisqualate receptors. Small-amplitude nondesensitizing quisqualate receptor responses were activated by much lower concentrations of all quisqualate receptor agonists. Full dose-response curves for the nondesensitizing response were obtained for 9 amino acids; L-glutamate was the most potent endogenous agonist (EC50 19 microM). Domoate (EC50 13 microM) and kainate (EC50 143 microM) activated large-amplitude, nondesensitizing responses.     

Action of l-DOPA and acidic amino acids on isolated neurons of spinal cord in the lamprey

Ionic currents induced by application of L-3, 4-dihydroxyphenylalanine (L-DOPA) and acidic amino acids: L-glutamate, kainate, N-methyl-D-aspartate (NMDA) were investigated in experiments on isolated spinal cord neurons of the lamprey by means of whole-cell recording and concentration clamp methods. L-DOPA was found to activate glycine, but not excitatory amino acid receptors.     

Depressant actions of γ-d-glutamylaminomethyl sulfonate (GAMS) on amino acid-induced and synaptic excitation in the cat spinal cord

We have investigated the effects of iontophoretically administered gamma-D-glutamylaminomethyl sulfonate (GAMS) on excitation of dorsal horn neurons and Renshaw cells of the cat spinal cord induced by exogenous excitants and by synaptic activation following stimulation of low threshold primary afferent fibers. Comparisons were made between the synaptic depressant effects of GAMS and those of gamma-D-glutamylglycine (gamma DGG) and (+/-)-2-amino-5-phosphonovalerate (APV). At low iontophoretic ejection currents, GAMS showed clear selectivity in antagonizing responses to excitatory amino acids in the order kainate greater than quisqualate greater than L-aspartate greater than NMDA greater than L-glutamate. This selectivity was decreased at high ejection currents, when acetylcholine-induced excitation of Renshaw cells was also reduced. GAMS was equieffective with gamma DGG in depressing both APV-sensitive polysynaptic excitation and APV-resistant monosynaptic excitation of spinal neurons. Ventral root evoked excitation of Renshaw cells was not reduced by GAMS. In some cells a depression of synaptic excitation by GAMS was observed in the absence of an effect on either L-glutamate- or L-aspartate-induced excitation. This raises the possibility that some other endogenous substance may be a transmitter acting at kainate/quisqualate type receptors in the cat spinal cord. However, other factors are discussed which may explain this observation.     

Excitatory synaptic inputs to pyramidal neurons of the lateral amygdala

Whole-cell patch clamp recordings were made from pyramidal neurons in the rat lateral amygdala (LA). Synaptic currents were evoked by stimulating in either the external capsule (ec), internal capsule (ic) or basolateral nucleus (BLA). Stimulation of either the ic, ec or BLA evoked a glutamatergic excitatory synaptic current (EPSC) which was mediated by both non-NMDA and NMDA (N-methyl-d-aspartic acid) receptors. The ratio of the amplitude of the NMDA receptor-mediated component measured at +40 mV to the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component measured at -60 mV was similar regardless of whether EPSCs were evoked in the ec, ic or BLA. At resting membrane potentials, excitatory synaptic potentials evoked from either the ec or putative thalamic inputs were unaffected by application of the NMDA receptor antagonist APV. Spontaneous glutamatergic currents had two components to their decay phase. The slow component was selectively blocked by the NMDA receptor antagonist D-APV, indicating that AMPA and NMDA receptors are colocalized in spiny neurons. We conclude that pyramidal cells of the LA receive convergent inputs from the cortex, thalamus and basal nuclei. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalized in the postsynaptic density.     

CNQX and DNQX block non-NMDA synaptic transmission but not NMDA-evoked locomotion in lamprey spinal cord

The motor pattern underlying locomotion in the lamprey is activated and maintained by excitatory amino acid neurotransmission. The quinoxalinediones 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) are potent and selective antagonists of non-N-methyl-D-aspartate (NMDA) receptors in the mammalian central nervous system. In the lamprey, these compounds are now shown to block fast excitatory synaptic potentials elicited in neurones of the spinal ventral horn. They selectively antagonise responses to the application of selective kainate and quisqualate receptor agonists (kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA)) but do not influence NMDA receptor-mediated responses. Additionally, it is shown that the activation of NMDA receptors is sufficient to elicit and maintain fictive locomotion after blockade of non-NMDA receptors with either DNQX or CNQX. Conversely, activation of quisqualate receptors with AMPA, but not quisqualate leads to fictive locomotion with properties much like that activated by kainate.     

Glutamate, kainate and quisqualate enhance GABA-dependent chloride uptake in cortex

Several lines of evidence indicate a possible interaction between the major inhibitory and excitatory cortical neurotransmitters, GABA and glutamate. To assess the neurochemical basis for such an interaction, we examined the effects of glutamate and several analogs on GABA-dependent chloride uptake in a mouse cortical synaptoneurosome preparation. L-Glutamate and the specific receptor subtype ligands kainate and quisqualate led to a small but significant enhancement in chloride uptake in the presence, but not the absence, of the GABA analog muscimol (5 microM). Enhancement was seen at excitatory amino acid (EAA) concentrations of 2-10 microM, but not at higher concentrations. D-Glutamate, NMDA, the NMDA-related antagonists APV and MK801, and the kainate/quisqualate antagonist CNQX, had no effect on chloride uptake. However, CNQX (50 microM) but not APV (50 microM) blocked the increase in chloride uptake due to kainate or quisqualate (10 microM). In addition, depolarization of synaptoneurosomes using high potassium (40 mM KC1) or ouabain pretreatment (5 microM) blocked the effects of kainate and quisqualate. Glutamate, kainate, and quisqualate had no effect on binding at the benzodiazepine, TBPS, or GABA sites on the GABAA receptor complex.     

Electrophysiological evidence for the presence of ionotropic and metabotropic excitatory amino acid receptors on dopaminergic neurons of the rat mesencephalon: an in vitro study.

The actions of the ionotropic and metabotropic excitatory amino acid agonists AMPA, quisqualate, kainate, NMDA and trans-ACDP were studied by means of intracellular electrophysiological recordings from dopaminergic neurons of rat mesencephalon in brain slices. It was observed that all these agents evoked an inward current in cells which were voltage-clamped near the resting potential (-50, -60 mV). The membrane responses produced by AMPA, kainate and quisqualate were associated with an increase of the apparent input conductance while the responses induced by NMDA and trans-ACDP were associated with a decrease in the apparent input conductance. Therefore, stimulation of ionotropic and metabotropic amino acid receptors activates inward currents in the dopaminergic cells by different mechanisms.     

Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro.

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.     

Glutamate-induced inhibition of paired pulse facilitation of monosynaptic excitatory post-synaptic potentials in frog spinal motoneurons.

To evaluate actions of glutamate on excitatory synaptic transmission in the central nervous system, we examined glutamate-induced changes in the paired pulse facilitation of monosynaptic excitatory post-synaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Glutamate (1 mM) depolarized motoneurons both in the presence and absence of Mg2+. In most cells perfused with Mg(2+)-free or high Ca(2+)-Mg2+ solutions, the glutamate potential was accompanied by a reduction in peak amplitude of EPSPs, although the degree of change varied with the cells. Glutamate enhanced the EPSP amplitude in a few cells with Mg(2+)-free and high Ca(2+)-Mg2+ solutions, and in most cells with high Mg2+ medium. In 3/5 cells tested, the paired pulse facilitation of EPSPs was reduced by glutamate when the EPSP amplitude either increased or decreased. NMDA (50 microM), kainate (50-100 microM), quisqualate (5-50 microM) and L-2-amino-4-phosphonobutyrate (L-AP4, 1 mM) also decreased the facilitation in about half of the cells tested. The glutamate-induced decrease in the facilitation was observed in both the presence and absence of Mg2+ and was not affected by the concomitant application of glutamate and antagonists for non-NMDA or NMDA receptors, such as 6-cyano-7-nitro-quinoxalinediones (CNQX, 60 microM) or 2-amino-5-phosphonovalerate (APV, 250 microM). Glutamate reduced the facilitation of excitatory post-synaptic currents (EPSCs) recorded at a constant membrane potential under voltage clamp, when the EPSC amplitude either increased or decreased and when the input conductance either increased or decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of the glutamate receptor in cultured astrocytes

Cultured astrocytes from neonatal rat cerebral hemispheres are depolarized by the excitatory neurotransmitter glutamate. In this study we have used selective agonists of different neuronal glutamate receptor subtypes, namely, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate type, to characterize pharmacologically the glutamate receptor in astrocytes. The agonists of the neuronal quisqualate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) and quisqualate, depolarized the membrane. Kainate, an agonist of the neuronal kainate receptor, depolarized astrocytes more effectively than quisqualate. Combined application of kainate and quisqualate depolarized astrocytes to a level which was intermediate to that evoked by quisqualate and kainate individually. Agonists activating the neuronal NMDA receptor, namely NMDA and quinolinate, were ineffective. Application of NMDA did not alter the membrane potential even in combination with glycine or in Mg2+-free solution, conditions under which neuronal NMDA receptor activation is facilitated. The nonselective agonists L-cysteate, L-homocysteate, and beta-N-oxalylamino-L-alanine (BOAA) mimicked the effect of glutamate. Dihydrokainate, a blocker of glutamate uptake, did not, and several antagonists of neuronal glutamate receptors only slightly affect the glutamate response. These findings suggest that astrocytes express one type of glutamate receptor which is activated by both kainate and quisqualate, lending further support to the notion that cultured astrocytes express excitatory amino acid receptors which have some pharmacological similarities to their neuronal counterparts.     

AMPA, kainate, and quisqualate activate a common receptor-channel complex on embryonic chick motoneurons

The actions of the putative quisqualate-selective agonist DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) were examined in identified embryonic chick motoneurons using gigaseal recording techniques and compared with properties of the selective non-NMDA excitatory amino acid agonists kainate and quisqualate. Pressure application of AMPA induces an inward going current when neurons are voltage-clamped at negative membrane potentials. The current-voltage relationship for this response is linear with reversal near 0 mV. Over the range of 1 microM-10 mM, the AMPA-induced current is dose-dependent with an ED50 of 40 microM. AMPA currents are insensitive to the selective NMDA receptor antagonist, 2-amino-5-phosphonovalerate, and the putative quisqualate selective blocker, glutamate diethyl ester, but are partially inhibited by kynurenic acid. In competition experiments, applications of saturating concentrations of AMPA and either kainate or quisqualate produce responses intermediate between the response to either agonist alone, indicating commonality in the mechanism of these agents. Applications of AMPA with the NMDA-selective agonist aspartate give an additive response. Analysis of current fluctuations indicates that AMPA, quisqualate, and kainate gate a channel with a primary conductance near 20 pS. Differences in maximal macroscopic current evoked by saturating concentrations of AMPA, kainate, and quisqualate cannot be explained by differences in mean channel open time as the most efficacious agonist, kainate, has the shortest channel open time (AMPA = 5.9 +/- 0.4 msec, kainate = 2.7 +/- 0.1 msec, quisqualate = 5.0 +/- 0.5 msec). Rather, kainate induces a greater frequency of channel opening. This finding contrasts with results obtained at the nicotinic ACh receptor, where the most efficacious agonists have the longest mean channel open time. Our results suggest that AMPA acts at the same receptor-channel complex as kainate and quisqualate on chick motoneurons and support the hypothesis that only 2 classes of excitatory amino acid receptor complexes exist in this preparation.     

Quantitative physiological characterization of a quinoxalinedione non-NMDA receptor antagonist

The effects of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, or FG 9065) on excitatory amino acid responses in cultured neurons from rat hippocampus were studied using tight-seal whole-cell recording techniques. CNQX reduced the magnitude of peak inward currents produced by exogenously applied kainate, quisqualate, and N-methyl-D-aspartate (NMDA) with Ki's of 2.5, 3.5, and 96 microM, respectively. The antagonism was competitive against kainate and quisqualate, but noncompetitive against NMDA. Glycine markedly reduced CNQX antagonism of NMDA responses. The same recording technique using pairs of monosynaptically connected neurons demonstrated reversible diminution of excitatory postsynaptic potentials in 7 of 7 pairs, using CNQX at concentrations as low as 10 microM. CNQX applied alone did not evoke inward or outward currents at membrane potentials near the resting membrane potential and did not affect the current-voltage relationship at membrane potentials between -90 and -30 mV. These observations represent the first quantitative characterization of glutamate receptor antagonism by CNQX with respect to physiological rather than biochemical parameters and demonstrate that CNQX is far more potent and more selective than currently available non-NMDA antagonists. The results suggest that CNQX will be a useful pharmacologic tool for the study of synaptic transmission in a variety of systems in which glutamate or related excitatory amino acids are involved.     

The distribution of kainate and quisqualate receptors in the rat cerebellum

Sensitivity of isolated Purkinje neurons from rat cerebellum to agonists of excitatory amino acids was studied. Kainate and quisqualate activated inward currents in the most studied neurons. NMDA and APB were ineffective. Distribution of kainate and quisqualate receptors was different. Currents activated by kainate and quisqualate were not additive. Hypotheses allowing one to explain the observed results were discussed.     

Characterization of quisqualate receptor desensitization in cultured postnatal rat hippocampal neurons.

The quisqualate class of glutamate receptors is thought to play an important role in excitatory synaptic transmission, synaptic plasticity, and neuronal death. Since desensitization is a prominent feature of the responses mediated by this class of receptors, we have characterized the rapidly desensitizing quisqualate response in cultured postnatal rat hippocampal neurons using the whole-cell patch-clamp technique. Quisqualate and its structural analogs elicit a peak current that rapidly decays to a steady-state level. In contrast, currents induced by kainate, NMDA, and their structural analogs exhibit either no decay or a much slower decay. The biophysical and pharmacological properties of the peak and steady-state quisqualate currents indicate that both are mediated by an ionotropic quisqualate receptor. Quisqualate currents desensitized monoexponentially by approximately 70% with a time constant near 80 msec. Both the rate and percentage of desensitization showed slight voltage dependence and were concentration dependent, reaching maximal values at saturation. Additionally, the overlap of the dose-response curves for activation of the steady-state current and desensitization of the peak current by a conditioning dose suggests that the two processes are related. Furthermore, desensitizing quisqualate currents were observed when Ca2+, Mg2+, Na+, K+, and Cl- were removed from the extracellular solution or their concentrations greatly reduced. These results suggest that the decline in the response is not caused by a simple open channel block mechanism. Despite the lack of desensitization by kainate, our observations are consistent with the hypothesis that quisqualate and kainate act at a single receptor-channel complex. Kainate and quisqualate appeared to interact competitively when applied simultaneously and noncompetitively when quisqualate was applied first. In addition, saturating doses of quisqualate and kainate gave steady-state currents of equal amplitude in neurons treated with the lectin WGA, an inhibitor of quisqualate receptor desensitization.     

Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists

The antagonist pharmacology of glutamate neurotoxicity was quantitatively examined in murine cortical cell cultures. Addition of 1-3 mM DL-2-amino-5-phosphonovalerate (APV), or its active isomer D-APV, acutely to the exposure solution selectively blocked the neuroexcitation and neuronal cell selectively blocked the neuroexcitation and neuronal cell loss produced by N-methyl-D-aspartate (NMDA), with relatively little effect on that produced by either kainate or quisqualate. As expected, this selective NMDA receptor blockade only partially reduced the neuroexcitation or acute neuronal swelling produced by the broad-spectrum agonist glutamate; surprisingly, however, this blockade was sufficient to reduce glutamate-induced neuronal cell loss markedly. Lower concentrations of APV or D-APV had much less protective effect, suggesting that the blockade of a large number of NMDA receptors was required to acutely antagonize glutamate neurotoxicity. This requirement may be caused by the amplification of small amounts of acute glutamate-induced injury by subsequent release of endogenous NMDA agonists from injured neurons, as the "late" addition of 10-1000 microM APV or D-APV (after termination of glutamate exposure) also reduced resultant neuronal damage. If APV or D-APV were present both during and after glutamate exposure, a summation dose-protection relationship was obtained, showing substantial protective efficacy at low micromolar antagonist concentrations. Screening of several other excitatory amino acid antagonists confirmed that the ability to antagonize glutamate neurotoxicity might correlate with ability to block NMDA-induced neuroexcitation: The reported NMDA antagonists ketamine and DL-2-amino-7-phosphono-heptanoate, as well as the broad-spectrum antagonist kynurenate, were all found to attenuate glutamate neurotoxicity substantially; whereas gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds reported to block predominantly quisqualate or kainate receptors, did not affect glutamate neurotoxicity. The present study suggests that glutamate neurotoxicity may be predominantly mediated by the activation of the NMDA subclass of glutamate receptors--occurring both directly, during exposure to exogenous compound, and indirectly, due to the subsequent release of endogenous NMDA agonists. Given other studies linking NMDA receptors to channels with unusually high calcium permeability, this suggestion is consistent with previous data showing that glutamate neurotoxicity depends heavily on extracellular calcium.     

Action of 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic aci (CPP): a new and highly potent antagonist of N-methyl-d-aspartate receptors in the hippocampus

A new compound, 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), has been evaluated as an excitatory amino acid receptor antagonist using electrophysiological assays and radioligand binding. In autoradiographic preparations, CPP reduces l-[³H]glutama binding in regions of the hippocampus rich in N-methyl-d-aspartate (NMDA) receptors, but not in regions richin kainate sites. In isolated membrane fraction preparations, CPP displaces l-[³H]glutamate binding to NMDA sites, but does not compete with the binding of selective kainate or quisqualate site ligands. CPP potently reduces depolarizations produced by application of NMDA but not depolarizations produced by quisqualate or kainate. Its order of potency against excitatory amino acid-induced responses in the hippocampus is NMDA > homocysteate > aspartate > glutamate > quisqualate. CPP has no efect on lateral perforant path responses or on inhibition of these responses by 2-amino-4-phosphonobutyrate. Finally, at doses that do not affect Schaffer collateral synpatic transmission, CPP reversibly blocks the induction of long-term potentiation of Schaffer synaptic responses. This new compounds is, therefore, a higly selective brain NMDA receptor blocker, and the most potent such by nearly an order of magnitude.