Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors

A series of ω-phosphono-α-car☐ylic acids were tested as antagonists of excitatory amino acid depolarizations and long-term potentiation (LTP) in region CA1 of rat hippocampal slices. The 5- and 7-phosphono compounds (±AP5and±AP7) blocked N-methyl-D-aspartate (NMDA) depolarizations and prevented the induction of LTP of the synaptic field potential and population spike components of the Schaffer collateral response.±AP5and±AP7 did not reduce kainate or quisqualate depolarizations and did not affect unpoten synaptic response amplitude.±AP5, ±AP6and±AP8 did not block amino acid excitant responses or LTP.These results demonstrate that NMDA receptors present in hippocampal region CA1 are not necessary for normal synaptic transmission, but are involved in the initiation of long-term synaptic plasticity.     

Receptor types mediating the excitatory actions of exogenousl-aspartate andl-glutamate in rat olfactory cortex

Changes in potential between the pial and cut surfaces of rat olfactory cortex slices evoked by N-methyl-d-aspartate (NMDA), quisqualate, kainate,l-glutamate andl-aspartate and also by γ-aminobutyric acid (GABA) have been monitored using extracellular electrodes. All agonists produced a pial-negative potential response when superfused onto the pial surface, GABA,l-aspartate andl-glutamate being less potent than the others. Repeated applications of NMDA, but not of the other agonists, led to a progressive reduction in response to approximately 30% of the initial depolarization. The responses to NMDA (100 μM) were selectively abolished by(±)2-amino-5-phosphonopentanoic acid (APP; 100 μM) while depolarizations evoked byl-glutamate andl-aspartate (both at 10 mM) were only antagonized by21 ± 2 (n = 12) and36 ± 3 (n = 12) percent respectively (means ± S.E.M.). γ-d-Glutamylglycine (γ-DGG; 1 mM) and(±)cis-2,3-piperidine dicarboxylate (cis-PDA; 2 and 5 mM), in addition to antagonizing responses to NMDA, also partially blocked quisqualate- and kainate-evoked depolarizations. When a mixture of APP (100 μM), γ-DGG (1 mM) and cis-PDA (5 mM) was applied to preparations, although NMDA receptors were completely blocked and responses to both quisqualate and kainate antagonized by approximately 80%,l-glutamate andl-aspartate evoked depolarizations were only reduced by51 ± 7 (n = 4) and 49 ± 4 (n = 4) percent respectively (means ± S.E.M.). The results are discussed in terms of the contributions made by NMDA, quisqualate and kainate receptors to the composite responses evoked byl-aspartate andl-glutamate.     

Autoradiographical analysis of excitatory amino acid binding sites in rat hippocampus during the development of hippocampal kindling

Binding sites for excitatory amino acids have been determined by autoradiographical procedures in the rat hippocampus and striatum during hippocampal kindling. The binding sites measured were the N-methyl-D-aspartate (NMDA)-sensitive sites for L-[3H]glutamate and [3H]MK-801 sites (transmitter recognition site and ion channel of the NMDA receptor, respectively), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) sites (quisqualate receptor), [3H]kainate sites (kainate receptor) and NMDA-insensitive sites for L-[3H]glutamate. In general, little change was apparent in the hippocampus or striatum for any of these binding sites when assessed 48 h after attaining stages 1/2, 3 or 5 of kindling. These results suggest that hippocampal kindling does not bring about a change in the excitatory amino acid receptor binding sites examined, and that the appearance of an NMDA receptor-mediated component to synaptic responses in the hippocampus produced by kindling, cannot be explained on this basis.     

l-[H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

The anatomical distribution ofl-[³H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding ofl-[³H]glutamate is accounted for the presence of 3 distinct binding sites when measured in the absence of Ca²⁺, Cl⁻ and Na⁺ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labeled byd-[³H]2-amino-5-phosphonopentanoate (d-[³H]AP5), [³H]kainate ([³H]KA) and [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([³H]AMPA) which are thought to be selective ligands for the N-methyl-d-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.     

Parallel antagonism of synaptic transmission and kainate/quisqualate responses in the hippocampus by piperazine-2,3-dicarboxylic acid analogs

A new series of potent antagonists of excitatory neurotransmission in the rat hippocampus has been identified. These derivatives of piperazine-2,3-dicarboxylate (PzDA) include the most potent acidic amino acid antagonists yet described for Schaffer collateral-commissural EPSPs. These antagonists also effectively block excitatory synaptic responses recorded in the lateral and medial perforant pathways and in the mossy fiber pathway. The PzDA derivatives also block focal depolarizations produced by kainate, quisqualate, and N-methyl-D-aspartate. N-methyl-D-aspartate responses are more susceptible to inhibition by PzDA derivatives, although the spectrum of antagonism of N-methyl-D-aspartate and synaptic responses by PzDA derivatives is not parallel. However, the antagonism of kainate and quisqualate responses by PzDA derivatives shows the same rank order of potency as synaptic responses. These data indicate that synaptic receptors in the hippocampus have a pharmacologic profile similar to that of kainate or quisqualate receptors.     

Functional N-Methyl-D-Aspartate and Non-N-Methyl-D-Aspartate Receptors are Expressed by Rat Supraoptic Neurosecretory Cells in vitro

A considerable amount of evidence has accumulated to support a role for excitatory glutamatergic transmission in the regulation of the hypothalamo-neurohypophysial system. Glutamate immunoreactivity has been found in axon terminals forming asymmetric synapses on to magnocellular neurosecretory cells and kynurenic acid, a broad spectrum glutamate receptor antagonist inhibits 1) spontaneous electrical activity in vivo, 2) excitatory postsynaptic potentials in hypothalamic slices, and 3) osmotically-evoked vasopressin release from hypothalamic explants. While this provides strong evidence for glutamatergic regulation of hypothalamic magnocellular neurosecretory cells, the subtypes of glutamate receptors expressed by these cells have not been defined. We have, therefore, obtained current and voltage clamp recordings from supraoptic magnocellular neurosecretory cells in vitro to investigate the functional and pharmacological properties of their glutamate receptors. Application of micromolar concentrations of L-glutamate, or of the agonists kainate, quisqualate and N-methyl-D-aspartate (NMDA), produced reversible and dose-dependent depolarizations in all cells tested. These responses were mediated by postsynaptic receptors since they persisted during chemical synaptic blockade with Ca^{2 +} -free or tetrodotoxin-containing solutions. The inward current induced by NMDA showed a marked Mg²⁺-sensitive voltage dependence, and was blocked by D, L-2-amino-5-phosphonovalerate. In contrast, currents induced by kainate and quisqualate showed linear current-voltage properties and were antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione. We conclude that both NMDA and non-NMDA receptors are expressed by magnocellular neurosecretory cells of the rat supraoptic nucleus.     

Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors

A series of ω-phosphono-α-car?ylic acids were tested as antagonists of excitatory amino acid depolarizations and long-term potentiation (LTP) in region CA1 of rat hippocampal slices. The 5- and 7-phosphono compounds (±AP5and±AP7) blocked N-methyl-D-aspartate (NMDA) depolarizations and prevented the induction of LTP of the synaptic field potential and population spike components of the Schaffer collateral response.±AP5and±AP7 did not reduce kainate or quisqualate depolarizations and did not affect unpoten synaptic response amplitude.±AP5, ±AP6and±AP8 did not block amino acid excitant responses or LTP.These results demonstrate that NMDA receptors present in hippocampal region CA1 are not necessary for normal synaptic transmission, but are involved in the initiation of long-term synaptic plasticity.     

Excitatory amino acid-evoked membrane currents and excitatory synaptic transmission in lamprey reticulospinal neurons

The characteristics of excitatory amino acid-evoked currents and of excitatory synaptic events have been examined in lamprey Müller neurons using voltage clamp and current clamp recording techniques. Application of glutamate evoked depolarizations associated with a decrease in input resistance. The reversal potential of the responses was -35 mV. Under voltage clamp conditions, a series of excitatory amino acid agonists evoked inward currents associated with little or no increase in baseline current noise. The order of potency of the excitatory amino acid agonists was quisqualate greater than kainate greater than glutamate greater than aspartate, while N-methyl-D-aspartic acid (NMDA) was inactive. Inward currents evoked by glutamate, as well as by kainate and quisqualate were attenuated reversibly by 1 mM kynurenic acid (KYN). In contrast, glutamate-evoked currents were not affected by 100 microM D(-)-2-amino-5-phosphonovaleric acid (APV), a selective NMDA antagonist. Spontaneously occurring and stimulus-evoked excitatory postsynaptic events were antagonized reversibly by 1 mM KYN. At this concentration, KYN had no effect on membrane potential, input resistance, or excitability of the cells. In contrast, excitatory postsynaptic currents were unaffected by APV. It is concluded that both glutamate responses and excitatory synaptic transmission in lamprey Müller neurons are mediated by non-NMDA-type receptors and that these receptors are associated with ionic channels with a low elementary conductance. The combined pharmacological and biophysical characteristics of these responses are therefore different from those previously reported in other preparations. Spontaneous (but not stimulus-evoked) inhibitory synaptic events in Müller neurons were blocked reversibly by 1 mM KYN but not by 100 microM APV, suggesting that excitation of interneurons inhibitory to Müller cells was also mediated by non-NMDA receptors.     

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.     

Activation of 5-HT1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and alpha-methyl-5-HT (alpha-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 microM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or alpha-Me-5-HT (both 100 microM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of L-glutamate and L-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. alpha-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT1C/2 antagonist ketanserin and the 5-HT1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA-depolarizations.(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.     

A pharmacological characterization of the mGluR1α subtype of the metabotropic glutamate receptor expressed in a cloned baby hamster kidney cell line

The pharmacological specificity of the mGluR1α subtype of the metabotropic glutamate receptor (mGluR) was examined in a cloned baby hamster kidney cell line (BHK-ts13) measuring [³H]glutamate binding and inositol phosphate (PI) hydrolysis. PI-hydrolysis was maximally stimulated by quisqualate (1112±105%of basal), glutamate (1061±70%of basal), ibotenate (1097±115%of basal) andβ-N-methylamino-l-rmalanine (BMAA) (1010±104%of basal). In contrast, the maximal stimulation of PI-hydrolysis by(1S,3R)-1-amino-cyclopentane-1,3-dicar☐ylic acid (t-ACPD) was only673±78% of the basal level. The relative order of potency was quisqualate > glutamate > ibotenate > t-ACPD > BMAA. Agonist-stimulated PI-hydrolysis was attenuated (25±4%inhibition) byl-2-amino-3-phosphonopropionic acid and partially blocked (44±7%) by pertussis toxin treatment. Saturation binding studies with [³H]glutamate on membranes prepared from BHK-ts13 cells expressing the mGluR1α subtype showed that glutamate binds to a single affinity state of this receptor with a limited capacity (K_d = 296nM, B_max = 0.8pmol/mg protein). In competition experiments, [³H]glutamate was displaced by quisqualate, glutamate, ibotenate, t-ACPD and BMAA with a rank order of potency similar to that found for stimulation of PI-hydrolysis.     

Glutamate receptors in striatum and substantia nigra: Effects of medial forebrain bundle lesions

We examined NMDA-sensitive [³H]glutamate, [³H]AMPA, [³H]kainate and metabotropic-sensitive [³H]glutamate binding sites in neostriatum and substantia nigra pars reticulata (SNr) in rats after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. One week after the lesion, NMDA, AMPA, kainate and metabotropic receptors were decreased in the ipsilateral neostriatum, whereas at three months NMDA receptors were increased while AMPA, kainate and metabotropic receptors were not changed. In the SNr at one week, only AMPA and metabotropic receptors were significantly decreased whereas three months after the lesion NMDA, AMPA and kainate binding sites were decreased. The early decrease of excitatory amino acid receptors in the striatum is likely to reflect degeneration of dopaminergic fibers, suggesting that specific subpopulations of excitatory amino acid binding sites are located on dopaminergic terminals.     

Receptor types mediating the excitatory actions of exogenous L-aspartate and L-glutamate in rat olfactory cortex

Changes in potential between the pial and cut surfaces of rat olfactory cortex slices evoked by N-methyl-D-aspartate (NMDA), quisqualate, kainate, L-glutamate and L-aspartate and also by gamma-aminobutyric acid (GABA) have been monitored using extracellular electrodes. All agonists produced a pial-negative potential response when superfused onto the pial surface, GABA, L-aspartate and L-glutamate being less potent than the others. Repeated applications of NMDA, but not of the other agonists, led to a progressive reduction in response to approximately 30% of the initial depolarization. The responses to NMDA (100 microM) were selectively abolished by (+/-)2-amino-5-phosphonopentanoic acid (APP; 100 microM) while depolarizations evoked by L-glutamate and L-aspartate (both at 10 mM) were only antagonized by 21 +/- 2 (n = 12) and 36 +/- 3 (n = 12) percent respectively (means +/- S.E.M.). gamma-D-Glutamylglycine (gamma-DGG; 1 mM) and (+/-)cis-2,3-piperidine dicarboxylate (cis-PDA; 2 and 5 mM), in addition to antagonizing responses to NMDA, also partially blocked quisqualate- and kainate-evoked depolarizations. When a mixture of APP (100 microM), gamma-DGG (1 mM) and cis-PDA (5 mM) was applied to preparations, although NMDA receptors were completely blocked and responses to both quisqualate and kainate antagonized by approximately 80%, L-glutamate and L-aspartate evoked depolarizations were only reduced by 51 +/- 7 (n = 4) and 49 +/- 4 (n = 4) percent respectively (means +/- S.E.M.). The results are discussed in terms of the contributions made by NMDA, quisqualate and kainate receptors to the composite responses evoked by L-aspartate and L-glutamate.     

Hippocampal cells primed with quisqualate are depolarized by AP4 and AP6, ligands for a putative glutamate uptake site

The glutamate analog 2-amino-4-phosphonobutyrate (AP4), which in control slices has little effect on Schaffer synaptic responses in hippocampal area CA1, reduces Schaffer responses in slices treated with quisqualate. We have shown that this effect of AP4 is associated with depolarization of CA1 neurons and a persisting small reduction in Schaffer response amplitude. 2-Amino-6-phosphonohexanoate also depressed Schaffer responses following priming with quisqualate, but 2-amino-7-phosphonoheptanoate did not. Treatment with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) or N-methyl-D-aspartate (NMDA) did not sensitize slices to AP4. The pharmacology of this 'priming effect' of quisqualate corresponds to that of a putative uptake site. We suggest the effects of AP4 (and AP6) result from exchange for previously accumulated quisqualate.     

Characterization of specific, high-affinity binding sites for l-[H]glutamic acid in rat brain membranes

l-[³H]Glutamic acid binds reversibly to rat brain membranes with high affinity. Specific binding is linear with tissue concentration and has a pH optimum at neutrality. Saturation isotherms reveal anomalous kinetics of specific binding with an high affinity site with a K_D of 11 nM and a lower affinity site with a K_D of 80 nM; the Scatchard plots intercept at a common bound/free ratio. Hill plots of the complete saturation isotherms have a slope of 1.0. There are marked regional differences in the distribution of binding sites in rat brain: parietal cortex, frontal cortex, hippocampus striatum thalamus cerebellum, pons-medulla and hypothalamus. Except for a small amount of specific binding in heart, other peripheral tissues do not exhibit specific binding of l-[³H]glutamic acid. Several amino acids with neuroexcitatory effects inhibit the specific binding: l-glutamic acid l-aspartic acid and d,l-homocysteic acid d-glutamic acid and l-cysteine sulfinic acid; related amino acids without neuroexcitatory effects do not inhibit specific binding. Reputed antagonists of glutamate-induced neuronal depolarization block specific binding: α-aminoadipic acid 2-amino,4-phosphonobutyric acid glutamate diethylester. Prior kainate lesion of the neurons intrinsic to the striatum results in a 45% decrement in specific binding of l-[³H]glutamic acid whereas cortical ablation, which causes degeneration of a cortical-striatal glutamatergic projection and reduces striatal glutamate synaptosomal uptake, does not affect specific binding. These results are compatible with the interpretation that the binding of [³H]glutamic acid occurs at excitatory receptors on neurons.     

Effect of cerebral hypoxia on NMDA receptor binding characteristics after treatment with 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) in newborn piglets

Previous studies have shown that hypoxia modifies the NMDA receptor/ion channel complex in cortical brain cell membranes of newborn piglets. The present study tests the hypothesis that blockade of the glutamate recognition site of the NMDA receptor with the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP) prevents modification of the receptor during hypoxia. Twenty seven anesthetized, ventilated newborn piglets were randomized into four groups: 7 normoxic (Nx), 6 CPP-treated normoxic (CPP-Nx), 8 hypoxic (Hx) and 6 CPP-treated hypoxic (CPP-Hx). Treatment groups received CPP 2 mg/kg i.v. The CPP-Hx group received CPP 30 min: prior to hypoxia, which was induced by lowering the FiO₂, to 5–7% for 1 h. Physiologic data showed no change in heart rate, blood pressure, arterial blood gas values, glucose or lactate following CPP administration. During hypoxia there was a significant decrease in PaO₂, pH and an increase in lactate compared to baseline values. The CPP-Hx group had significantly higher lactate levels than the Hx group during hypoxia. P₂ membrane fractions were prepared and thoroughly washed. Characteristics of the NMDA receptor ion channel were determined by [³H]MK-801 binding assays and characteristics of the glutamate recognition site by specific NMDA-displaceable [³H]glutamate binding assays. Brain tissue ATP and PCr levels confirmed tissue hypoxia, and were not preserved by CPP administration. [³H]MK-801 binding assays revealed that CPP treatment attenuated the hypoxia-induced decrease in the number of receptors (B_max) and receptor binding affinity (K_d) during hypoxia. CPP treatment also decreased receptor affinity (increasedK_d) for [³H]MK-801 binding during normoxia and hypoxia. Assays of [³H]glutamate binding revealed that hypoxia decreased both theB_max and the Kd of the NMDA receptor for [³H]glutamate and both were preserved by CPP treatment prior to hypoxia. CPP had no effect on [³H]glutamate B_max or K_d during normoxia. We conclude that hypoxia decreases theB_max andK_d of the NMDA receptor glutamate recognition site for [³H]glutamate and the ion channel site for [³H]MK-801 in newborn piglets. These changes are prevented by CPP administration prior to hypoxia. The different effects of CPP binding during normoxia and hypoxia suggest a use-dependent mechanism for CPP binding during hypoxia, possibly through an hypoxia-induced alteration of the high-affinity binding site for CPP. During both normoxia and hypoxia CPP binding appeared to induce a conformational change in the receptor causing a decrease in binding affinity for [³H]MK-801. CPP administration did not preserve brain tissue ATP or PCr levels during hypoxia and may alter cellular metabolism in addition to its action at the NMDA receptor. However, even with depletion of the energy precursors ATP and PCr, and with higher lactate levels in the CPP-Hx group, CPP was able to maintain NMDA receptor binding characteristics during hypoxia and may decrease excitotoxic cellular damage from hypoxia.     

Effects of a spider toxin (JSTX) on hippocampal CA1 neurons in vitro

The effect of a toxin (JSTX) obtained from Nephila clavata (Joro spider) on the CA1 pyramidal neurons of the hippocampus was studied using slice preparations. JSTX blocked the excitatory postsynaptic potentials (EPSPs) in the pyramidal neuron evoked by Schaffer collateral stimulation but was without effect on the antidromic action potentials or on the resting conductance. Depolarization induced by ionophoretic application of glutamate was readily suppressed by JSTX but aspartate-induced depolarization was much less sensitive to the toxin. Among preferential agonists activating 3 receptor subtypes for excitatory amino acids, quisqualate responses were most effectively suppressed by JSTX. Kainate responses were similarly suppressed but in some cells higher concentration of the toxin was needed to block the responses. N-methyl-D-aspartate (NMDA) responses were the least sensitive to JSTX but they were suppressed by +/- 2-amino-5-phosphonovaleric acid (APV). Long term potentiation (LTP) once it had taken place was not completely inhibited by APV. In the presence of JSTX, however, LTP was blocked and tetanic stimuli produced only a short-lived potentiation. In Mg2+ free solution, an orthodromic stimulation evoked repetitive spike responses which were superimposed on the depolarization following the initial spike. APV suppressed the depolarization and associated spikes leaving an orthodromic response which was sensitive to JSTX. The results suggest that JSTX blocks EPSPs in CA1 pyramidal neurons which are mediated by non-NMDA type receptors.     

Characterization of the excitatory amino acid receptor-mediated release of [H]acetylcholine from rat striatal slices

The pharmacological nature of the interaction of excitatory amino acids with striatal cholinergic neurons was investigated in vitro. Agonists of excitatory amino acid receptors evoked the release of [³H]acetylcholine from slices of rat striatum, in the presence of magnesium (1.2 mM). Removal of magnesium from the medium markedly increased the release of [³H]acetylcholine evoked by all excitatory amino acid receptor agonists tested, with the exception of kainate. In the absence but not the presence of magnesium, a clear rank order of potency was found: N-methyl-dl-aspartate = ibotenate >l-glutamate >l-aspartate cysteate > kainate = quisqualate.The excitatory amino acid receptor mediating [³H]acetylcholine release resembles the N-methyl-d-aspartate preferring (N-type) receptor, as previously characterized electrophysiologically, according to 3 criteria: (1) rank order of potency of agonists; (2) magnesium-sensitivity; and (3) antagonism by 2-amino-5-phosphonovalerate.The release of [³H]acetylcholine evoked by N-methyl-dl-aspartate was blocked by tetrodotoxin (0.5 μM). Moreover, N-methyl-dl-aspartate failed to evoke [³H]acetylcholine release from slices of hippocampus, where cholinergic afferents, rather than interneurons, are found. These results suggest that excitatory amino acids act at receptors on the dendrites of striatal cholinergic interneurons, giving rise to action potentials and release of acetylcholine from cholinergic nerve terminals.     

Postnatal development of the excitatory amino acid system in visual cortex of the rat. Changes in ligand binding to NMDA, quisqualate and kainate receptors

The postnatal development of the ligand binding to N-methyl-D-aspartate (NMDA), quisqualate and kainate receptor sites was examined in whole homogenates of the visual cortex of rats, aged 2-360 days. As selective ligands, [3H]CPP (3-(2-carboxypyperazine-4-yl)-propyl-1-phosphonic acid, [3H]AMPA (RS-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) and [3H]KA (kainic acid) were used, respectively. The binding of CPP was low in newborns, rapidly increased from the second postnatal week, reached its maximum between weeks 2 and 3, then slowly declined up to the age of 1 year. In contrast, the binding of AMPA and kainate was high perinatally, increased rapidly up to day 6 after birth to reach an early maximum value, then gradually decreased to adult values which were attained at an age of 3-4 weeks. These age-related changes were derived from alterations in the density of binding sites, which, in the case of AMPA, was accompanied by an increase in binding affinity. The results, compared with the developmental time-course of excitatory synapses, indicate that, in the immature cerebral cortex, NMDA receptors may be primarily involved in synaptic transmission, whereas quisqualate and kainate receptors may play some other (e.g. trophic) roles.