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.     

Multiple ionic mechanisms are activated by the potent agonist quisqualate in cultured cerebellar Purkinje neurons.

Current clamp recordings were used to analyze responses of cultured cerebellar Purkinje neurons to quisqualate and several other selective non-N-methyl- D-aspertate (NMDA) agonists. Quisqualate, a potent agonist in the cerebellar Purkinje neuron, evoked both short- and long-term changes in excitability, that activated within seconds and lasted for several minutes. Two components of the response were activated differentially by subtype selective agonists, and differed in their mechanism of expression and time course. The initial component of the response was activated by ionotropic agonists ((RS)-d-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) domoate), and by quisqualate and glutamate which are effective at both the ionotropic and metabotropic quisqualate receptor subtypes, but not by the metabotropic agonist trans (+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD). This component was dependent on extracellular Na+, and characterized by a rapid depolarization with a short latency (less than 1-2 s) and a decrease in membrane resistance as expected for an ionotropic reponse. The rapid depolarization extended into an agonist-dependent plateau phase, which could not be evoked by depolarization alone. The second ('late') phase of the response was a slowly-activating, long-lasting change in membrane excitability, accompanied by little or no change in the membrane potential. The late phase, marked by an increase in voltage-dependent bursting spike activity, was induced by the metabotropic agonist, ACPD, and by quisqualate and glutamate, but not by ionotropic selective agonists such as AMPA. Little or no bursting was evoked by AMPA, domoate, kainate or homocysteate. This late phase was also accompanied by increases in the magnitude and duration of the complex spikes and in the afterhyperpolarization following brief current-driven depolarizations. The slower time course of the late component is consistent with a pathway involving second messenger systems. Our results support the hypothesis that coregulation of both ionotropic and metabotropic mechanisms produces the complex and prolonged excitatory response characteristic of the Purkinje neuron.     

Characterization of l-2-Amino-4-Phosphonobutanoate Action Following Sensitization by Quisqualate in Rat Hippocampal Slice Cultures

An excitatory action of l-2-amino-4-phosphonobutanoate (l-AP4), a glutamate analogue, is observed following pre-exposure of tissue to quisqualate. We have studied the mechanism of sensitization of l-AP4 responses by quisqualate in voltage-clamped CA3 pyramidal cells in rat hippocampal slice cultures in the presence of tetrodotoxin. Prior to quisqualate addition, CA3 cells did not respond to l-AP4 (50 - 1000 microM). Following brief application of quisqualate (500 nM for 30 s), l-AP4 (50 - 200 microM) induced a complex excitatory response which could be obtained for >1 h. l-AP4 caused an ionotropic inward current associated with a conductance increase. This response was in part sensitive to 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and in part sensitive to d-2-amino-5-phosphonovalerate (d-AP5) and Mg2+ ions. At depolarizing potentials, in the presence of CNQX and d-AP5, l-AP4 caused excitation by depressing K+ currents, mimicking the metabotropic action of glutamate. This indicates that the action of l-AP4 is mediated by three different receptor types: N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors, and glutamatergic metabotropic receptors. The l-AP4 response persisted in solutions containing low Ca2+ and high Mg2+ concentrations or 100 - 200 microM Cd2+, suggesting that it is independent of extracellular Ca2+. We were unable to identify any substance other than quisqualate capable of sensitizing the l-AP4 action. This effect also occurred when quisqualate was applied in Ca2+-free solution or in solutions containing low concentrations of Na+ or Cl-. Sensitization of l-AP4 responses by quisqualate was not observed in acutely dissociated pyramidal cells recorded by means of the whole-cell recording mode, although ionotropic quisqualate responses were present. Sensitization was readily reversed by short applications of the endogenous excitatory amino acids glutamate, aspartate and homocysteate at concentrations of 10 - 100 microM. Our data are consistent with the hypothesis that the excitatory action of l-AP4 results from a Ca2+-independent release of endogenous excitatory amino acids from some presynaptic neuronal or glial site.     

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.     

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.     

'Metabotropic' glutamate receptors in rat hypothalamus: characterization and developmental profile.

Excitatory amino acids (EAAs) are known to stimulate neurohormone release through the activation of ion-channel-linked receptors (ionotropic receptors). Here we report that a receptor for EAAs linked to polyphosphoinositide hydrolysis (metabotropic receptor) is also present at the hypothalamus where its expression is developmentally regulated. Stimulation of [3H]inositolmonophosphate ([3H]InsP) formation by quisqualate (EC50 = 1.5 microM), ibotenate (EC50 = 100 microM) and trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD; EC50 = 30 microM) is extremely high (up to 50-fold) in the first 10 days of postnatal life, progressively declines during maturation and is virtually absent in the adult. Stimulation of phosphoinositide hydrolysis by quisqualate, ibotenate and t-ACPD is more pronounced than that induced by classical neurotransmitters that stimulate inositol phosphate formation such as norepinephrine and carbamylcholine. Agonists of the ionotropic glutamate receptor such as kainate, NMDA and alpha-amino-3-hydroxy-5-methyl-5-isoxazolpropionate (AMPA), do not modify inositol phosphate accumulation in hypothalamic slices. The selective antagonist of quisqualate metabotropic receptor, D,L-2-amino-3-phosphonopropionate (AP3), produces a slight stimulation of phosphoinositide hydrolysis, but potently antagonizes the stimulation produced by quisqualate and t-ACPD.     

Development and regulation of excitatory amino acid receptors involved in the release of arachidonic acid in cultured hippocampal neural cells

Release of [3H]arachidonic acid mediated by excitatory amino acid (EAA) receptors was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. Treatment with N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate resulted in age- and dose-dependent stimulation of [3H]arachidonic acid release. During development, the maximum response for NMDA was observed relatively earlier (at 7 days) than those for QA and kainate (at 14 days) in the hippocampal neuronal cultures. The half maximal effects were obtained at about 15 microM NMDA at all ages studied and about 0.5 microM QA at 14 and 20 days. At optimum concentrations NMDA- and QA-induced releases were additive. Unlike with neurons, treatment with all the 3 EAA receptor agonists, NMDA, QA and kainate, had no significant effect on [3H]arachidonate release in hippocampal astroglial cells. In cultured 14-day-old neurons, the increases in NMDA- and QA-mediated [3H]arachidonic acid release were completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and the ionotropic QA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. But the iontropic QA receptor agonist alpha-amino-3-hydroxy-5-methyl-isoxazole-4- propionic acid (AMPA) had no significant effect on [3H]arachidonate release, indicating that interaction between ionotropic QA and metabolotropic QA receptors may be essential for optimal QA-mediated arachidonic acid release. At physiological concentrations of Mg2+ (1.2 mM), AMPA was found to potentiate NMDA-induced release of [3H]arachidonic acid; the effect appeared to be related to a removal of Mg2+ blockade mediated by mild depolarisation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitatory amino acid binding sites in the caudate nucleus and frontal cortex of Huntington's disease.

Huntington's disease is a dominantly inherited, progressive neurodegenerative disorder causing marked pathology in the basal ganglia. The pathophysiology of the selective neuronal death is as yet unknown, but evidence suggests that the neurotoxicity may result from endogenous substances acting at excitatory amino acid receptors. Previous data have shown a selective decrease in binding to one class of glutamate receptors, the N-methyl-D-aspartate (NMDA) receptor in the putamen of Huntington's disease. The present study was undertaken to determine the relative density of binding to all of the currently defined subpopulations of excitatory amino acid receptors in the caudate nuclei and frontal cortex of patients with Huntington's disease and of control subjects, using quantitative in vitro autoradiography. NMDA, MK-801, glycine, kainate, and alpha-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor binding were all decreased to a similar extent (50-60%). Binding to the metabotropic quisqualate receptor and to the non-NMDA, nonkainate, nonquisqualate (NNKQ) site was decreased nonsignificantly by 31% and 26%, respectively. Autoradiograms of NMDA, MK-801, AMPA, kainate, metabotropic, and NNKQ receptors in caudates revealed an inhomogeneous pattern of binding that is different from the binding pattern seen in control caudates. Binding to all receptor subtypes was the same in the frontal cortex from Huntington's disease patients and control subjects. The data suggest that no single excitatory amino acid receptor is selectively decreased in the caudate of Huntington's disease.     

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.     

Ionotropic glutamate receptors in isolated horizontal cells of the rabbit retina

With the use of the whole-cell voltage-clamp technique, we have recorded the currents induced by ionotropic glutamate receptor agonists on isolated axonless horizontal cells (HC) of rabbit retina. Bath application of the non-N-methyl-D-aspartate receptor agonists: kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and L-glutamate (GLU) produced an increase in the conductance for non-selective cations. All the isolated horizontal cells responded to GLU, AMPA and KA. Responses elicited by GLU and AMPA but not KA exhibited a concentration-dependent desensitization. Application of N-methyl-D-aspartate (NMDA) evoked no responses. The rank order affinities of the agonists as estimated from EC50 values were AMPA > GLU > KA. Whereas KA had the lowest affinity of the agonists tested, it produced the largest currents. Hill coefficients of the concentration-response data were near 1 for AMPA, and 2 for KA and GLU. Coapplication of AMPA with cyclothiazide (CTZ) blocks AMPA receptor desensitization, and enhanced its effects on conductance. However, CTZ did not change the KA -induced conductances. In all cells tested, 6,7-dinitroquinoxaline (DNQX) completely and reversibly blocked the effects of KA and AMPA. The KA- and AMPA-induced currents were also completely blocked by 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), a selective AMPA receptor antagonist. These results indicate that the responses to glutamate agonists in HC were mediated almost exclusively by AMPA receptors. Our study indicates that AMPA receptors play a fundamental role in mediating the synaptic input into rabbit horizontal cells.     

Pharmacological characterization of voltage-clamped catfish rod horizontal cell responses to kainic acid

Excitatory amino acid-induced currents were examined in voltage-clamped rod horizontal cells dissociated from the catfish retina. The cells responded to glutamate (GLU) and the GLU analogues kainate (KA), quisqualate (QA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), while N-methyl-D-aspartate (NMDA) produced inconsistent responses. Of the effective agonists, only KA produced large, concentration-dependent current responses. While QA, AMPA, GLU, and NMDA were poor agonists, these compounds were able to block rod horizontal cell responses to KA. The rank order potency for this inhibition was: QA greater than AMPA greater than or equal to L-GLU much greater than D-GLU = NMDA. Several excitatory amino acid receptor antagonists were also able to inhibit rod horizontal cell responses to KA. The rank order potency for the inhibition by the compounds tested was: kynurenate greater than cis-piperidine-dicarboxylic acid much greater than D,L-alpha-amino-adipate. Comparison of the potency of several ligands to inhibit rod and cone horizontal cell responses to KA suggested similarities in the KA binding sites of both cell types.     

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.     

Glutamate receptors in the enteric nervous system: ionotropic or metabotropic?

Intracellular recording methods were used to investigate actions of glutamate on morphologically identified neurones in the myenteric and submucous plexuses of guinea-pig small intestine. Glutamate evoked a tetrodotoxin-resistant, slowly activating depolarizing response in most of the submucous neurones (86 of 125, 69%) and a smaller number of myenteric neurones (6 of 60, 10%). The depolarizing responses were restricted to S-type neurones with uniaxonal morphology. The group I metabotropic glutamate receptor (mGluRs) agonists quisqualate, 1S, 3R-ACPD and DHPG mimicked the depolarizing action of glutamate. A group I mGluRs antagonist, S-4-carboxyphenylglycine (S-4CPG), suppressed the glutamate responses with an IC50 of 357 microM at 30 microM glutamate. Group II or III mGluRs agonists did not produce depolarizing responses and group II or III mGluRs antagonists did not alter glutamate-evoked depolarization. The ionotropic glutamate receptor (iGluRs) agonists NMDA, AMPA, or kainate did not evoke depolarizing responses and glutamate-evoked depolarization was unaffected by the iGluRs antagonists D-APV, MK-801, or DNQX. No rapidly activating fast depolarizing responses reminiscent of fast excitatory postsynaptic potentials (EPSPs) were ever observed during application of glutamate or AMPA and stimulus-evoked fast EPSPs were unaffected by DNQX. The results suggest that the excitatory action of glutamate on enteric neurones is mediated by group I metabotropic glutamate receptors and that ionotropic glutamate receptors are not involved. The results also suggest that glutamate-mediated fast EPSPs may not be present in myenteric and submucous neurones in guinea-pig small bowel.     

Distribution of immunoreactive GABA and glutamate receptors in the gustatory portion of the nucleus of the solitary tract in rat

The distribution of glutamate (GLU) and gamma-aminobutyric acid (GABA) receptors within the gustatory portion of the rat nucleus of the solitary tract (gNST) was investigated using immunohistochemical, histological and neural tract tracing techniques. Numerous somata throughout the gNST were immunoreactive for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors, while few were labeled for kainate receptors. AMPA and NMDA receptors were particularly abundant in the rostral central (RC) subdivision of the gNST, which receives most of the primary afferent input from the oral cavity and contains most of the gNST neurons that project to the parabrachial nuclei (PBN). This finding supports electrophysiological evidence that AMPA and NMDA receptors are involved in responses to orosensory input and indicates that their action may influence ascending taste signals as well. Compared to the ionotropic GLU receptors, few cell bodies were immunoreactive for metabotropic GLU receptors. Somata immunoreactive for GABA(A) and GABA(B) receptors were located throughout the nucleus. The densest neuropil labeling was for GABA(A) receptors in the ventral (V) subnucleus, the gNST subdivision that sends output to brainstem oromotor centers. The distributions of immunolabeling for GLU and GABA receptors imply that different functional roles may exist for specific receptors within this nucleus.     

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)     

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.     

Excitatory amino acid receptors on sustained retinal ganglion cells in the kitten during the critical period of development

Effects of iontophoretically applied excitatory amino acid analogues, kainate, quisqualate and N-methyl-D-aspartate (NMDA) and their receptor antagonists on the sustained class of retinal ganglion cells were studied in the optically intact eye of pentobarbitone-anaesthetized kittens (7-9 weeks of age). These results were compared with the effects obtained in adult cats. All 3 excitatory amino acid agonists had excitatory actions on the majority of On- and Off-sustained ganglion cells in the kitten but at higher current levels than those required for adult cells, suggesting all 3 types of receptors of weaker sensitivity are present on the kitten cells. Whilst the relative potency of kainate, quisqualate and NMDA was 15:3:1 in the adult cells, it was 5:2:1 in the kitten cells. As for other neurones in the CNS, an increase in the potency of kainate receptors and a decrease in that of NMDA receptors appear, therefore, to characterize the postnatal development of the excitatory amino acid receptors on the retinal ganglion cells. In accordance with the agonist results, a broadband receptor antagonist, kynurenate, powerfully antagonised responses of kitten cells as well as those of adult cells. The pure NMDA receptor antagonist, 3((+-)-2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), however, only suppressed spontaneous firing of kitten cells. Furthermore, in kitten cells, the visually-driven firing was depressed while the level of firing was raised by these excitatory amino acid analogous, and a long period of inhibition of firing followed the agonist-induced excitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells

Ionotropic glutamate receptors mediate fast forms of excitatory synaptic transmission in mature neurons and may play critical roles in neuronal development. However, the developmental stage at which neuronal cells begin to express functional receptors and their roles in lineage progression remain unclear. In the present study, neural precursor cells were isolated from the cortical neuroepithelium of embryonic day 13 rats, and rapidly expanded in serum-free medium in response to basic fibroblast growth factor. RT-PCR revealed the presence of mRNAs encoding AMPA(A), AMPA(C), KA(1), KA(2), NMDA(1), and NMDA(2D) subunits after 3 days in culture. The functional expression of AMPA/kainate and NMDA receptors was investigated using Ca(2+) imaging and whole-cell patch-clamp recording techniques in cells pulse-labeled with bromodeoxyuridine (BrdU) for 1-4 hr. The recorded cells were then double-immunostained for BrdU incorporation and neuron-specific beta-tubulin (TuJ1). The results show that AMPA/kainate and NMDA induced increases in cytosolic Ca(2+) and inward currents only in differentiating neurons. In contrast, proliferating (BrdU(+)TuJ1(-)) cells failed to respond to any ionotropic glutamate receptor agonists. Interestingly, Ca(2+) imaging revealed that a subpopulation of BrdU(+)TuJ1(+) cells also responded to AMPA, indicating the emergence of functional ionotropic AMPA/kainate receptors during terminal cell division and the earliest commitment to neuronal cell lineage. These in vitro results were supported by flow cytometric sorting of AMPA-responsive cells pulse-labeled with BrdU for 1 hr in vivo, which revealed that functional AMPA receptors appear in BrdU(+)TuJ1(+) cells under physiological conditions and may play a role in terminal cell division.