The glycine coagonist site of the NMDA receptor.
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Animals↗
Biomedical subjects
R Dingledine
Publications and source records attributed to R Dingledine.
Excitatory amino acid receptors in epilepsy.
Excitatory amino acid transmitters participate in normal synaptic transmission throughout the CNS (see Headley and Grillner, May TiPS), so it comes as no surprise that such excitatory pathways are involved in the initiation of seizures and their propagation. Most attention has been directed to synapses using NMDA receptors, although more recent evidence indicates potential roles for the AMPA receptors as well. In this article--the first of two to focus on the neurological dangers inherent in excitatory amino acid pathways--Raymond Dingledine, Chris McBain and James McNamara consider their involvement in epilepsy; next month's article will cover brain damage following ischemia and hypoxia.
Animals↗
Molecular biology of mammalian amino acid receptors.
The amino acid receptor proteins are ubiquitous transducers of most excitatory and inhibitory synaptic transmission in the brain. In July 1987 two reports appeared describing the molecular cloning of a pair of subunits of the GABAA receptor (7) and one subunit of the glycine receptor (13). These papers sparked wide interest and led quickly to the concept of a ligand-gated receptor-ion channel superfamily that includes nicotinic acetylcholine receptors as well as certain amino acid receptors. The identification of additional subunits of each receptor followed; with the recent cloning of a kainate receptor subunit (14), only the NMDA receptor remains elusive. Several disciplines have been brought to bear on these receptor clones, including in situ hybridization and functional expression in Xenopus laevis oocytes and mammalian cell lines. In this review we compare cloning strategies that have been used for amino acid receptors and discuss structural similarities among the receptor subunits. Two findings that have arisen from molecular cloning and expression of these receptors receive special attention. First, the molecular heterogeneity of GABAA receptors is larger than expected from pharmacological studies of native receptors. Second, although the native receptors are thought to be heterooligomers, much like the model proposed for the nicotinic receptors, some individual amino acid receptor subunits can form functional receptor channels, presumably in a homomeric configuration. This review focuses, therefore, on what we have learned from cloning efforts about amino acid receptors and what might lie ahead in this field.
Amino Acid Sequence↗
Multiple components of both transient and sustained barium currents in a rat dorsal root ganglion cell line.
1. Currents through voltage-activated Ca2+ channels in rat dorsal root ganglion (DRG) x mouse neuroblastoma hybrid (F-11) cells were studied using the whole-cell patch clamp technique with 30 mM-Ba2+ as charge carrier. Two components of the inward Ba2+ current were distinguished on the basis of voltage dependence and time course. Each component could be further subdivided based on pharmacology. 2. A transient inward current activated at test potentials positive to -40 mV, peaked within 20 ms and then decayed during a 200 ms depolarization. The peak amplitude of the transient current occurred between -10 and +10 mV. With a 300 ms conditioning pulse, half-inactivation of the transient current occurred at -30 mV. A sustained inward current activated at test potentials positive to -30 mV and reached a maximum at +20 to +30 mV. The sustained current showed little voltage-dependent inactivation over 200 ms. The amplitudes of both the transient and sustained currents were increased by perfusing with Ba2+ instead of Ca2+. 3. Most F-11 cells had both the transient and sustained Ba2+ currents although the relative amount of the two currents varied with culture conditions. The transient current was more prominent in cells fed with a 'growth' medium (15-20% serum) whereas the sustained current was increased in cells fed with a 'differentiation' medium (1% serum plus growth factors). F-11 cells can be used to study transient current in relative isolation from sustained Ca2+ current under certain culture conditions. The neuroblastoma parent of the F-11 cell line, N18TG-2 cells, exhibited little or no voltage-dependent Ba2+ current. 4. Brief application of omega-conotoxin fraction GVIA (10 microM) produced a long-lasting block of 81% of the sustained current and 27% of the transient current. 5. The transient and sustained Ba2+ currents in F-11 cells were reversibly blocked by brief exposure to Cd2+ or Ni2+. Block of the sustained current was evident with 100 nM-Cd2+ whereas the threshold concentration for Ni2+ block was 1 microM. Cd2+ and Ni2+ were equipotent blockers of the transient current. Dose-response curves for Cd2+ and Ni2+ block of both sustained and transient currents had shallow slopes suggesting that the block was more complex than a simple bimolecular interaction between blocker and one blocking site. Dose-response curves were fitted by a model that included two binding sites for each divalent blocker.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Different responses of CA1 and CA3 regions to hypoxia in rat hippocampal slice.
1. To study the effects of brief periods of hypoxia on cellular functions in the rat hippocampal slice, extracellular and intracellular recordings were made from pyramidal neurons, and interstitial potassium activity ([K+]o) was measured in the pyramidal cell layers. Slices were perfused in an interface chamber at 36-37 degrees C with medium containing 8.5 mM [K+]o. Hypoxia was induced by switching the overflow gas from O2-CO2 to N2-CO2. 2. Brief periods of hypoxia (5-60 s) produced electrographic seizures with typical tonic and clonic components in 53% of 293 slices that generated spontaneous interictal bursts. Hypoxia-induced seizures were usually initiated in and restricted to the Ca1 region; only 2.5% of these slices generated seizures in CA3. In contrast to the CA1 region, the CA3 region could undergo spreading depression during hypoxia. The probability of seizure generation in CA1 was increased with increasing duration of hypoxia and was greatly reduced by lowering the bath temperature a few degrees. 3. [K+]o gradually increased in the CA1 and CA3 cell layers during the 20 s leading up to an hypoxia-induced seizure. [K+]o rose to approximately 9.8 mM (from a base line of 8.5 mM) in CA1 just before a seizure and to 11.4 mM during the seizure. After hypoxia, [K+]o reached a higher level in CA1 than in CA3, regardless of whether 1 microM tetrodotoxin was present to eliminate differences in cell firing in the two regions. CA1 pyramidal cells and glia gradually depolarized by several millivolts during and after hypoxia; no initial hyperpolarizing phase was detected. 4. Burst input from CA3 was necessary for hypoxia-induced seizures. The frequency and intensity of spontaneous burst-firing in CA3 remained steady in the period leading up to a CA1 seizure episode. In contrast, the intensity of synaptically driven bursts in CA1 grew markedly just before seizure onset. N-methyl-D-aspartate (NMDA) receptors participated in the crescendo of increasingly synchronous activity in CA1, because the competitive NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (D-APV, 30 microM), stereoselectively reduced seizure intensity. 5. Hypoxia-induced seizures were followed by a depressant phase, which was manifested most prominently by a prolonged (up to several minutes) reduction in the frequency and intensity of burst-firing in the CA3 region, hyperpolarization of CA1 neurons, and undershoot of [K+]o. In normal (3.5 mM) [K+]o, synaptically driven population spikes in CA1 were only reduced in amplitude by hypoxia; hypoxia did not induce seizures in 3.5 mM [K+]o.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Role of EPSPs in initiation of spontaneous synchronized burst firing in rat hippocampal neurons bathed in high potassium.
1. Spontaneous discharges that resemble interictal spikes arise in area CA3 b/c of rat hippocampal slices bathed in 8.5 mM [K+]o. Excitatory postsynaptic potentials (EPSPs) also appear at irregular intervals in these cells. The role of local synaptic excitation in burst initiation was examined with intracellular and extracellular recordings from CA3 pyramidal neurons. 2. Most (70%) EPSPs were small (less than 2 mV in amplitude), suggesting that they were the product of quantal release or were evoked by a single presynaptic action potential in another cell. It is unlikely that most EPSPs were evoked by a presynaptic burst of action potentials. Indeed, intrinsic burst firing was not prominent in CA3 b/c pyramidal cells perfused in 8.5 mM [K+]o. 3. The likelihood of occurrence and the amplitude of EPSPs were higher in the 50-ms interval just before the onset of each burst than during a similar interval 250 ms before the burst. This likely reflects increased firing probability of CA3 neurons as they emerge from the afterhyperpolarization (AHP) and conductance shunt associated with the previous burst. 4. Perfusion with 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a potent quisqualate receptor antagonist, decreased the frequency of EPSPs in CA3 b/c neurons from 3.6 +/- 0.9 to 0.9 +/- 0.3 (SE) Hz. Likewise, CNQX reversibly reduced the amplitude of evoked EPSPs in CA3 b/c cells. 5. Spontaneous burst firing in 8.5 mM [K+]o was abolished in 11 of 31 slices perfused with 2 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Model of synchronized epileptiform bursts induced by high potassium in CA3 region of rat hippocampal slice. Role of spontaneous EPSPs in initiation.
1. We constructed a computer model of the in vitro CA3 region of the rat hippocampal slice bathed in a high-potassium medium. Our aim was to understand better the mechanisms of initiation of synchronized bursts and the processes that regulate the interburst interval in the experimental system. 2. Our model began with a previously published model of the longitudinal CA3 hippocampal slice. The model contains three interconnected cell populations: 9,000 (excitatory) pyramidal cells; 450 inhibitory cells whose postsynaptic action is somatic and decays quickly, corresponding to chloride-dependent inhibition mediated by gamma-aminobutyric acid (GABA)A channels, and 450 inhibitory cells whose postsynaptic action is dendritic, of delayed onset and long lasting, that corresponds to K-dependent inhibition mediated by GABAB channels. 3. The model was then modified to account for specific features of the high-K experimental system: 1) the pyramidal cells do not generate intrinsic bursts; 2) EIPSP(CI) and EK are both shifted in a depolarizing direction; 3) spontaneous (i.e., not caused by presynaptic firing) excitatory postsynaptic potentials (EPSP)s were included; and 4) a steady current was injected into the pyramidal cells to depolarize them. 4. This model generates synchronized population bursts with interburst intervals of approximately 1.0-1.5 s. Bursts in individual pyramidal cells are preceded by barrages of EPSPs. These results agree with experiment. 5. Our model agrees with the following additional experiments: 1) synchronized bursts are abolished by partial blockade of excitatory synapses; 2) burst frequency is increased by partial blockade of a slow-intrinsic-K conductance; and 3) blockade of chloride-dependent inhibition leads to bursts of longer duration with longer interburst intervals. 6. The basic structural features of this model are similar to, but not identical to, the model of the disinhibited hippocampal slice. Spontaneous EPSPs appear to be critical in the high-K system for initiating, but not for synchronizing, population bursts. The experimental data and simulation results raise interesting questions about the role of spontaneous EPSPs in initiating synchronized discharges in other epileptic systems and on the possible role of spontaneous EPSPs in the normal brain.
Action Potentials↗
Homomeric GluR1 excitatory amino acid receptors expressed in Xenopus oocytes.
The GluR1 cDNA clone encodes a functional excitatory amino acid receptor (Hollmann et al., Nature 342: 643-648 (1989]. We have studied the pharmacological properties of this homomeric (single subunit) receptor expressed in Xenopus oocytes and compared these properties with those of receptors encoded by rat forebrain mRNA. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate, quisqualate, and glutamate were partial agonists at both GluR1 and forebrain non-N-methyl-D-aspartate (-NMDA) receptors. The potency of the agonists kainate, domoate, and glutamate was higher, and that of the antagonists 6-cyano-7-nitro-quinoxalinedione and 6,7-dichloro-3-hydroxy-2-quinoxaline carboxylic acid lower, for GluR1 receptors as compared with forebrain non-NMDA receptors. The GluR1 receptor differed strikingly from forebrain-derived non-NMDA receptors, however, in that it exhibited slow, calcium-dependent desensitization. Thus, most properties of the GluR1 receptor are similar but not identical to those of non-NMDA receptors expressed from forebrain mRNA. These results indicate that the ligand recognition sites on GluR1 homomeric receptors are subtly different from those of non-NMDA receptors expressed from a mixture of forebrain mRNA.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Dual effect of glycine on NMDA-induced neurotoxicity in rat cortical cultures.
To examine the roles of glycine in neurotoxicity caused by NMDA, primary rat cortical cultures were exposed to 100-300 microM NMDA plus glycine (0-3000 microM) or other glycine analogs in a simple saline solution, and toxicity was assessed by the amount of lactate dehydrogenase (LDH) released from the cultures. NMDA-induced neurotoxicity was abolished by 100 microM D-2-amino-5-phosphonovaleric acid (D-APV), phencyclidine (IC50, 4.1 microM), and Mg (IC50, 7.5 mM), or by reducing [Ca]0 to 0.1 mM. NMDA-induced neurotoxicity could also be abolished by 7-chlorokynurenic acid (IC50, 8.6 microM), suggesting the presence of residual glycine in the culture medium (confirmed by high-performance liquid chromatography measurement). Moreover, in the presence of 30 microM 7-chlorokynurenic acid, glycine, D-serine, D-alanine, beta-fluoro-D-alanine, and 1-aminocyclopropanecarboxylic acid could restore the neurotoxic action of NMDA, and their relative potencies and relative efficacies were the same as measured in electrophysiological assays in Xenopus oocytes or cultured neurons. The addition of greater than 100 microM glycine doubled the excitotoxic effect of NMDA. The potency of glycine was low (EC50, 27 microM), and this effect was not due to a direct action on the NMDA receptor. The above-mentioned agonists were unable to substitute for glycine, even at high concentrations (1 mM). On the other hand, beta-alanine, taurine, and GABA (1 mM) did potentiate NMDA neurotoxicity, and strychnine (IC50, 550 nM) could greatly reduce neurotoxicity in the presence of 1 mM glycine plus 300 microM NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)
2-Amino-5-phosphonovalerate↗
Effect of kindling on potassium-induced electrographic seizures in vitro.
The properties of high [K+]o-induced spontaneous bursting and electrographic seizures in hippocampal slices prepared from rats subjected to kindling from either the lateral entorhinal cortex or the angular bundle were compared to those in control slices. Kindling enhanced the frequency of K+-induced burst-firing in the CA3 region and the duration of triggered bursts in the dentate gyrus, as previously reported. However, kindling had no influence on the characteristics or occurrence of electrographic seizures in the CA1 region of slices bathed in elevated [K+]o. In addition, the development of electrographic seizures in slices from control animals did not require a preconditioning period of burst input from the CA3 region.
Alkaloids↗
Control of epileptiform burst rate by CA3 hippocampal cell afterhyperpolarizations in vitro.
Spontaneous interictal bursts occurred at a high rate in rat hippocampal slices bathed in raised [K]o (7-10.5 mM) and at a slower rate in slices bathed in low [Cl]o (53 mM) solutions. Intracellular recordings from CA3b/c neurons bathed in high [K]o showed that the amplitude of the afterhyperpolarization that followed each spontaneous epileptiform burst varied -50 +/- 7 mV per decade change in [K]o (n = 7). Afterhyperpolarization duration was highly correlated with the interburst interval in 7-10.5 mM [K]o but not in low [Cl]o (3.5 mM [K]o). The duration of the afterhyperpolarization accounted for 79 +/- 2% of the interburst interval (1.4 +/- 0.1 s) in 8.5 mM [K]o but only 51 +/- 8% in low [Cl]o (interval 6.6 +/- 1.2 s). Treatment of slices bathed in 8.5 mM [K]o with phorbol 12,13-diacetate (2-20 microM) caused a decrease in the amplitude and duration of the afterhyperpolarization and a concomitant 41% increase in burst rate. In addition, spontaneous discharges that resembled the tonic phase of electrographic seizures appeared in the CA3 region. The GABAB receptor blocker phaclofen (0.2-1 mM) had no effect on the burst rate. We suggest that the epileptiform burst afterhyperpolarization limits the maximum rate of spontaneous interictal-like bursting and that a decrease in its duration allowed the subsequent expression of electrographic seizure activity in area CA3.
Action Potentials↗
Modification of potassium-induced interictal bursts and electrographic seizures by divalent cations.
Reduction of external calcium and magnesium from 1.5 to 1.2 mM intensified potassium-induced interictal bursts, increased the likelihood of electrographic seizure occurrence in CA1, and rendered seizure initiation independent of N-methyl-D-aspartate (NMDA) receptor activation. In contrast to slices bathed in 1.5 mM divalent cations, in 1.2 mM divalents spontaneous CA1 seizures still occurred in CA1 minislices that contained at least 1500 neurons after removal of the CA3 burst generator, suggesting that divalent cations critically modulate the dependence of CA1 seizure initiation on interictal input. Since this slight reduction in external divalent cations enhanced tissue excitability, similar changes might promote epileptogenesis in situ.
2-Amino-5-phosphonovalerate↗
Role of extracellular space in hyperosmotic suppression of potassium-induced electrographic seizures.
1. Focal electrographic seizures arose in the CA1 region of rat hippocampal slices bathed in elevated (8.5 mM) external potassium [( K+]o). High [K+]o also induced spontaneous interictal bursts that originated in area CA3 and propagated to CA1. To examine the contribution to electrographic seizure initiation of excitatory mechanisms that are influenced by extracellular volume, we studied the effect of hyperosmotic expansion of interstitial volume on seizure occurrence, interictal bursts, and excitatory synaptic transmission. The tissue electrical resistance was also measured leading up to and during seizures. 2. Media made 5-30 mosmol/kg hyperosmotic by addition of agents restricted to the extracellular space (mannitol, sucrose, raffinose, L-glucose, dextran) rapidly and reversibly abolished [K+]o-induced spontaneous CA1 seizures in 86% of slices tested. However, similar increases in osmolality effected by agents that access the intracellular compartment (D-glucose, glycerol) did not influence electrographic seizure occurrence. Hyperosmotic changes with plasma membrane impermeable compounds, but not permeable compounds, produced significant concentration-dependent decreases (1-10%) in the electrical resistance of CA1 stratum pyramidale. Because tissue resistance is proportional to extracellular volume, these results suggest that hyperosmotic suppression of electrographic seizures is associated with expansion of the extracellular space in hippocampal slices. 3. Measurement of electrical resistance of the CA1 stratum pyramidale during spreading depression and electrographic seizure revealed an increase in tissue resistance to 122% and 108% of control, respectively. Furthermore, a slight (approximately 2%) but significant increase in electrical resistance gradually occurred over the 20 s immediately preceding seizure generation. The observed increase in tissue resistance suggests extracellular space is decreased during these events. 4. Hyperosmolality did not alter CA3 interictal burst frequency. However, burst intensity, estimated from the total length of the burst waveform, was significantly reduced in both the CA3 (83% control) and CA1 region (67% control) when osmotic changes were imposed by plasma membrane impermeant compounds. Additionally, media made hypoosmotic by removal of 7.5 mM NaCl reversibly increased burst intensity. 5. High [K+]o potentiated excitatory synaptic transmission and excitatory postsynaptic potential (EPSP) spike coupling.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Selectivity of quinoxalines and kynurenines as antagonists of the glycine site on N-methyl-D-aspartate receptors.
Xenopus oocytes injected with rat brain mRNA were used to identify and characterize the effects of compounds that are antagonists at both the glycine site on N-methyl-D-aspartate (NMDA) receptors and the quisqualate/kainate receptor. Oocytes were voltage-clamped at -60 mV and inward currents were measured at equilibrium following perfusion with agonists and antagonists. Application of 7-chlorokynurenic acid (7-Cl-Kyn) or 6,7-dichloro-3-hydroxy-2-quinoxaline carboxylic acid (6,7-diCl-HQC), each at 15 microM, produced a parallel shift to the right of the glycine concentration-response curve. Schild analysis indicated a KB of 300 nM for 6,7-diCl-HQC and 350 nM for 7-Cl-Kyn. The slopes of the Schild plots were 1.01-1.02 in each case, suggesting that both compounds are competitive glycine antagonists. Both compounds also blocked the receptor mediating kainate-induced inward current. Schild analysis of 6,7-diCl-HQC (KB = 3.0 microM, slope = 0.98) indicated competitive antagonism of kainate currents, but with a potency 10-fold lower than at the glycine site. 7-Cl-Kyn antagonized kainate-evoked currents (KB = 14.1 microM), but the slope of the Schild regression was less than 1 (0.72 +/- 0.11; p less than 0.05). Thus, 7-Cl-kyn was approximately 40-fold more potent at the glycine site than at the receptor mediating kainate currents but is probably not entirely competitive at the latter receptor. Omission of the Cl groups from these antagonists drastically reduced activity at both glycine and kainate sites. 6,7-Dinitro- and 6-cyano-7-nitro-quinoxalinedione were both more potent antagonists of kainate than glycine, but substitution of Cl at the 6-position and especially the 6- and 7-positions increased potency at the glycine site. These results suggest that the glycine coagonist site of the NMDA receptor and the agonist binding site of the quisqualate/kainate receptor have some structural similarity. Halogenated derivatives of quinoxalines and kynurenines should be useful in evaluating the function of the glycine site in synaptic transmission mediated by NMDA receptors. In this regard we found that 7-Cl-kyn (5 and 15 microM) selectively attenuated NMDA receptor-mediated epileptiform bursts in the CA1 region of hippocampal slices perfused with zero-Mg medium, without reducing the amplitude of the primary population spike. This block could be overcome by 300 microM D-serine, which alone did not influence bursting. These results together indicate that the glycine site plays a role in epileptiform bursting mediated by NMDA receptors in adult rat hippocampus.
Animals↗
Structural requirements for activation of the glycine coagonist site of N-methyl-D-aspartate receptors expressed in Xenopus oocytes.
Five structural features important for activation of the glycine recognition site on N-methyl-D-aspartate (NMDA) receptors were identified by structure-activity studies of more than 60 glycine analogues in voltage-clamped Xenopus oocytes injected with rat brain mRNA. First, sterically unhindered and ionized carboxyl and amino termini were essential for action at this site. Second, an increase in the interterminal separation by greater than one carbon (e.g., beta-alanine) markedly attenuated activity at this site. Third, activity at the glycine site was stereoselective. The D-isomers of alanine and serine were approximately 20 and 30 times more potent than the L-isomers. Fourth, only small sterically unobtrusive substitutions at the alpha-carbon could be tolerated. alpha-Methyl (D-alanine) and alpha-cyclopropyl (1-amino-cyclopropane carboxylic acid) (ACC) substitutions were effective as agonists but most larger aliphatic and aromatic alpha-carbon substitutions were simply inactive. Glycine, D-alanine, and ACC probably have only a two-point attachment to the receptor. Finally the alpha-carbon substituent of D-serine is envisioned as binding to a third site on the receptor probably via hydrogen bonding of the omega-terminal hydroxyl group. Thus, serine, an hydroxymethyl substitution of glycine, permitted activation of NMDA receptor-mediated currents, whereas isosteric substitutions incapable of hydrogen bonding (e.g., 2-aminobutyric acid) were inactive. Additionally, the position and size of the hydroxyl-containing group is critical for agonist action; D-threonine, DL-homoserine, and hydroxyphenolic substitutions at the alpha-carbon were all inactive. Halogenated analogs of a size comparable to D-serine but capable only of proton acceptance at the omega-terminus (beta-fluoro-D-alanine and beta-chloro-D-alanine) possessed agonist action, whereas an analog capable of only proton donation (1,2-diaminopropionic acid) was inactive. Full concentration-response curves were constructed for those analogs displaying greater than 25% of the effect of glycine when tested at 3 microM. With the exception of (R)-(+)cycloserine and beta-fluoro-D-alanine, all compounds were nearly full agonists and had Hill coefficients not significantly different from unity. The order of relative potency of the active analogs was ACC greater than glycine greater than D-serine greater than D-alanine greater than beta-fluoro-D-alanine greater than (R)-(+)-cycloserine greater than L-serine greater than L-alanine. Molecular modelling of a series of active and inactive analogs with close structural relation to glycine was undertaken. These results were complementary to those data obtained from the electrophysiological investigation.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acids↗
N-methyl-D-aspartate/glycine and quisqualate/kainate receptors expressed in Xenopus oocytes: antagonist pharmacology.
Quantitative pharmacological studies were done to determine the properties of excitatory amino acid receptors expressed in Xenopus oocytes injected with rat brain mRNA. Smooth currents with properties indicative of N-methyl-D-aspartate (NMDA) and quisqualate/kainate receptors were observed in mRNA-injected oocytes. Schild analysis of currents evoked by NMDA indicated that the EAA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) exerted a competitive block of the oocyte NMDA receptor, because the Schild regression was linear with a slope not significantly different from unity (1.03 +/- 0.025) up to 100 microM D-APV. The pA2 estimated for D-APV antagonism of NMDA currents (5.87 +/- 0.043) was nearly identical to that for D-APV as an L-aspartate antagonist (pA2 = 5.86 +/- 0.073, slope = 0.97 +/- 0.036), suggesting that these two agonists are selective for NMDA receptors in oocytes up to concentrations well above 1 mM. 6-Nitro-7-cyano-quinoxaline-2,3-dione (CNQX) reduced the maximum NMDA response significantly (70% reduction by 15 microM CNQX) but had no effect on the NMDA EC50. CNQX exerted a mixed competitive-noncompetitive block of the glycine site on NMDA receptors; 15 microM CNQX increased the glycine EC50 by 5-fold and reduced the maximum glycine response by 35%. In addition, CNQX exerted a potent and competitive antagonism of currents evoked by kainate. The Schild regression was linear up to 30 microM CNQX with a slope of 1.02 +/- 0.014 and a pA2 of 6.53 +/- 0.029. The block of kainate or NMDA currents by 2 microM CNQX was not voltage dependent. D-APV exerted a weak antagonism of kainate-evoked currents, with a pA2 of 3.39 +/- 0.044, but the slope of the Schild regression was slightly less than 1 (0.90 +/- 0.03). These data demonstrate a clear pharmacological distinction between receptors that mediate the kainate- and NMDA-induced currents and quantify the potency of CNQX and D-APV acting at NMDA/glycine and quisqualate/kainate receptors. The implications of these data for the identification of EAA receptors in oocytes and the classification of neuronal EAA receptors are discussed.
2-Amino-5-phosphonovalerate↗
Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes.
Receptors for N-methyl-D-aspartate (NMDA) are involved in many plastic and pathological processes in the brain. Glycine has been reported to potentiate NMDA responses in neurons and in Xenopus oocytes injected with rat brain messenger RNA. Glycine is now shown to be absolutely required for activation of NMDA receptors in oocytes. In voltage-clamped oocytes, neither perfusion nor rapid pressure application of NMDA onto messenger RNA-injected oocytes caused a distinct ionic current without added glycine. When glycine was added, however, NMDA evoked large inward currents. The concentration of glycine required to produce a half-maximal response was 670 nanomolar, and the glycine dose-response curve extrapolated to zero in the absence of glycine. Several analogs of glycine could substitute for glycine, among which D-serine and D-alanine were the most effective. The observation that D-amino acids are effective will be important in developing drugs targeted at the glycine site.
Amino Acids↗
GABAergic inhibition and the induction of spontaneous epileptiform activity by low chloride and high potassium in the hippocampal slice.
Intracellular recordings from CA3b/c neurons in rat hippocampal slices showed that reduction of the extracellular Cl- concentration from 136 to 53 mM produced a positive (+10 mV) shift in the reversal potential of GABAergic inhibitory postsynaptic potentials (IPSPs). This shift was not significantly different from the shift produced by raising K+ from 3.5 to 8.5 mM. Spontaneous interictal bursting occurred in both low Cl- and high K+. Extracellular recordings from the pyramidal cell layer in the CA3b/c region of hippocampal slices showed that bursts in 56 mM Cl- were of the same waveform and intensity as bursts produced by high K+. However the frequency of spontaneous bursting was much lower (6.6 +/- 1.2/min, n = 10) in low Cl- compared to high K+ (42.2 +/- 3.0/min, n = 33). Burst frequency was a linear function of the shift in IPSP reversal potential produced by high K+, but not low Cl-. Replacing 60% of the Cl- with methylsulfate or isethionate was sufficient to produce spontaneous bursting, whereas it was necessary to replace 80% of the Cl- when propionate was used as a substitute. All 3 Cl- substitutes lowered the ionized Ca2+ concentration, but raising the extracellular Ca2+ concentration back to normal did not change the burst frequency. Since the amplitude of IPSPs is reduced to a similar extent in low Cl- and high K+ solutions, whereas bursting is much faster in high K+, we suggest that impaired GABAergic inhibition is insufficient to fully account for spontaneous interictal bursting that is produced in hippocampal slices by raised extracellular K+.
Animals↗