CNQX increases GABA-mediated synaptic transmission in the cerebellum by an AMPA/kainate receptor-independent mechanism.

GABA(A) receptor-mediated inhibitory synaptic transmission within the CNS is often studied in the presence of glutamate receptor antagonists. However, for nearly a decade it has been known that, in the hippocampus, one of the most commonly used alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), can increase the frequency of spontaneous GABA(A) receptor-mediated postsynaptic currents (sIPSCs). In the present study we examined the effect of CNQX and related compounds on GABA-mediated synaptic transmission in the cerebellum. At various stages of development, low concentrations of CNQX increased the frequency of sIPSCs recorded from granule cells. This effect was independent of the blocking action of CNQX on ionotropic glutamate receptors, as it was not observed with the broad-spectrum glutamate receptor antagonist kynurenate. No increase in sIPSC frequency was observed with the NMDA receptor antagonists D-AP5 or 7-ClK, the selective AMPA receptor antagonists GYKI 52466 or GYKI 53655, or the kainate receptor antagonist NS-102. In contrast, two other quinoxaline derivatives, NBQX and DNQX, were capable of increasing sIPSC frequency. These results demonstrate that the novel excitatory action of CNQX, unrelated to blockade of ionotropic glutamate receptors, is not restricted to the hippocampus and can be observed with structurally related compounds.     

Reactive oxygen species are involved in the apoptosis induced by nonsteroidal anti-inflammatory drugs in cultured gastric cells

We have examined the pharmacology of kainate receptors in cultured hippocampal neurons (6-8 days in vitro (DIV)) from embryonic rats (E17). Cultured neurons were pre-treated with concanavalin A to remove kainate receptor desensitization and whole-cell voltage clamp electrophysiology employed to record inward currents in response to glutamatergic agonists and antagonists. N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor responses were blocked using MK801 (3 microM) and the 2,3-benzodiazepine, LY300168 (GYKI53655, 50 microM), respectively. Inward currents were recorded in hippocampal neurons upon application of kainate and the 2S,4R isomer of 4-methyl glutamic acid (SYM2081) with EC50 values of 3.4 +/- 0.4 microM and 1.6 +/- 0.5 microM, respectively (n = 6 cells). The GluR5 selective agonists, LY339434 (100 microM) and (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl) propionic acid (ATPA) (100 microM), did not evoke detectable inward currents in any cell responding to kainate. LY293558 and the selective GluR5 antagonist, LY382884, had weak antagonist effects on responses evoked by either kainate or (2S,4R)-4-methyl glutamate (IC50 > 300 microM). The quinoxalinedione, 2,3-dihyro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), blocked both kainate and (2S,4R)-4-methyl glutamate-activated currents at much lower concentrations (IC50 approximately 10 microM). These results provide pharmacological evidence that ion channels comprised of GluR6 kainate receptor subunits mediate kainate receptor responses in hippocampal neurons cultured 6-8 DIV.     

Pre- and postsynaptic effects of kainate on layer II/III pyramidal cells in rat neocortex

Kainate receptors mediate both direct excitatory and indirect modulatory actions in the CNS. We report here that kainate has both pre- and postsynaptic actions in layer II/III pyramidal neurons of rat prefrontal cortex. Application of low concentration of kainate (50-500 nM) increased the amplitude of evoked excitatory postsynaptic currents (EPSCs) whereas higher concentrations (3 microM) caused a decrease. The frequency of spontaneous and miniature (action potential-independent) EPSCs was increased by low concentrations of kainate without affecting their amplitudes, suggesting a presynaptic mechanism of action. The facilitatory and inhibitory effects of kainate were mimicked by the GluR5 subunit selective agonist ATPA. In addition to decreasing EPSC amplitudes, high concentrations of kainate and ATPA induced an inward current which was not blocked by AMPA- or NMDA-receptor antagonists GYKI52466 and D-APV, respectively. The inward currents were blocked by the AMPA/KA receptor antagonist CNQX, indicating the presence of postsynaptic kainate receptors. Single shock stimulation in the presence of GYKI52466 and D-APV evoked an EPSC which was blocked by CNQX. The GluR5 antagonist LY382884 changed paired-pulse facilitation to paired pulse depression, indicating that synaptically released glutamate can activate presynaptic kainate receptors. These results suggest that kainate receptors containing GluR5 subunits play a major role in glutamatergic transmission in rat neocortex, having both presynaptic modulatory and direct postsynaptic excitatory actions.     

Ethanol inhibition of synaptically evoked kainate responses in rat hippocampal CA3 pyramidal neurons.

Many studies have demonstrated that intoxicating concentrations of ethanol (10-100 mM) can selectively inhibit the component of glutamatergic synaptic transmission mediated by N-methyl-D-aspartate (NMDA) receptors while having little or no effect on excitatory synaptic transmission mediated by non-NMDA receptors [i.e., alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and/or kainate (KA) receptors]. However, until the recent development of highly selective AMPA receptor antagonists, it was not possible to assess the relative contribution of AMPA and KA receptors to non-NMDA receptor-mediated synaptic transmission or to determine whether these glutamate receptor subtypes differed in their sensitivity to ethanol. In the present experiments, we used the highly selective AMPA receptor antagonist LY 303070 to pharmacologically isolate KA receptor-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal CA3 pyramidal neurons and tested their sensitivity to ethanol. Concentrations of ethanol as low as 20 mM significantly and reversibly depressed KA EPSCs. Ethanol also inhibited KA currents evoked by direct pressure application of KA in the presence of LY 303070, suggesting that this inhibition was mediated by a postsynaptic action. In contrast, ethanol had no effect on AMPA EPSCs in these cells, even at the highest concentration tested (80 mM). Ethanol significantly inhibited NMDA EPSCs in these neurons, but these responses were less sensitive to ethanol than KA EPSCs. These results suggest that in addition to its well-described depressant effect on NMDA receptor-mediated synaptic transmission, ethanol has an even greater inhibitory effect on glutamatergic synaptic transmission mediated by KA receptors in rat hippocampal CA3 pyramidal neurons.     

Differential modulation of glutamatergic transmission by 3,5-dibromo-L-phenylalanine

An increasing body of evidence supports the hypothesis that diminished function of N-methyl-D-aspartate (NMDA) receptors and the associated increase in glutamate release and overstimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors are critical elements of the pathophysiology of schizophrenia. Here, we describe a halogenated derivative of the aromatic amino acid L-phenylalanine that 1) activates NMDA receptors, 2) depresses presynaptic glutamate release, and 3) blocks AMPA/kainate receptors. The experiments were conducted in rat cerebrocortical cultured neurons by using the patch-clamp technique. 3,5-Dibromo-L-phenylalanine (3,5-DBr-L-Phe) augmented NMDA miniature excitatory postsynaptic currents (mEPSCs) and activated the steady-state current, effects that were eliminated by NMDA receptor antagonists DL-2-amino-5-phosphonopentanoic acid and MK-801 (dizocilpine maleate; 5H-dibenzo[a,d]cyclohepten-5,10-imine). 3,5-DBr-L-Phe was a partial agonist at the glutamate-binding site of NMDA receptors with an EC50 of 331.6 +/- 78.6 microM and with an efficacy of 30.5 +/- 4.7% compared with NMDA. 3,5-DBr-L-Phe depressed both amplitude and frequency of AMPA/kainate mEPSCs. The IC50 of 3,5-DBr-L-Phe to inhibit AMPA/kainate mEPSC frequency was 29.4 +/- 4.3 microM. 3,5-DBr-L-Phe significantly decreased paired pulse depression of AMPA/kainate EPSCs and attenuated current activated by AMPA with higher efficacy at lower concentration of AMPA. 3,5-DBr-L-Phe neither affected GABA miniature inhibitory postsynaptic currents nor elicited action potentials. By enhancing NMDA receptor function, reducing glutamate release and blocking AMPA/kainate receptors 3,5-DBr-L-Phe represents a new type of polyvalent modulator of glutamatergic synaptic transmission with potential therapeutic applications.     

Activity of 2,3-benzodiazepines at Native Rat and Recombinant Human Glutamate Receptors In Vitro: Stereospecificity and Selectivity Profiles

The activity and selectivity of the glutamate receptor antagonists belonging to the 2,3-benzodiazepine class of compounds have been examined at recombinant human non-NMDA glutamate receptors expressed in HEK293 cells and on native rat NMDA and non-NMDA receptors in vitro. The racemic 2,3-benzodiazepines GYK152466, LY293606 (GYKI53405) and LY300168 (GYKI53655) inhibited AMPA (10 μM)-mediated responses in recombinant human GluR1 receptors expressed in HEK293 cells with approximate ₅₀ values of 18 μM, 24 μM and 6 μM, respectively and AMPA (10 μM) responses in recombinant human GluR4 expressing HEK293 cells with approximate ₅₀ values of 22 μM, 28 μM and 5 μM, respectively. GYKI 52466, LY293606 and LY300168 were non-competitive antagonists of AMPA receptor-mediated responses in acutely isolated rat cerebellar Purkinje neurons with approximate ₅₀ values of 10 μM, 8 μM and 1.5 μM, respectively. The activity of racemic compounds LY293606 and LY300168 was established to reside in the (−) isomer of each compound. At a concentration of 100 μM, GYKI52466, LY293606 and LY300168 produced <30% inhibition of kainate-activated currents evoked in HEK293 cells expressing either human homomeric GluR5 or GluR6 receptors or heteromeric GluR6+KA2 kainate receptors. The activity of the 2,3-benzodiazepines at 100 μM was weak at kainate receptors, but was stereoselective. Similar levels of inhibition were observed for kainate-induced currents in dorsal root ganglion neurons. Intact tissue preparations were also used to examine the stereoselective actions of the 2,3-benzodiazepines. In the cortical wedge preparation, the active isomer of LY300168, LY303070, produced a non-competitive antagonism of AMPA-evoked depolarizations with smaller changes in depolarizations induced by kainate and no effect on NMDA-dependent depolarizations. LY303070 was also effective in preventing 30 μM AMPA-induced depolarizations in isolated spinal cord dorsal roots with an approximate ₅₀ value of 1 μM. Synaptic transmission in the hemisected spinal cord preparation was stereoselectively antagonized by the active isomers of LY300168 and LY293606. In summary, these results indicate that 2,3-benzodiazepines are potent, selective and stereospecific antagonists of the AMPA subtype of the non-NMDA glutamate receptor. © 1997 Elsevier Science Ltd. All rights reserved.     

Electrophysiological Characterization of AMPA and NMDA Receptors in Rat Dorsal Striatum

The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at -70 and +40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between +50 and -50 mV. NMDA/AMPA ratio was 0.20±0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26±0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32±0.03. The rectification index (control 2.39±0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02±0.11 and 0.93±0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.     

The selective AMPA receptor antagonist GYKI 53784 blocks action potential generation and excitotoxicity in the guinea pig cochlea

The role of AMPA receptors in cochlear synaptic transmission and excitotoxicity was investigated by comparing the actions of a selective AMPA antagonist GYKI 53784 (LY303070) with additional AMPA/kainate antagonists, GYKI 52466 and DNQX, and the NMDA antagonist, D-AP5, in several electrophysiological, neurotoxicological and histochemical tests. GYKI 53784 had the same potency as DNQX and was 10 times more potent than GYKI 52466 in reducing auditory nerve activity. The NMDA antagonist D-AP5 had no effect on auditory nerve activity. When single-fiber activity was blocked with GYKI 53784, the effects of AMPA or kainate were also antagonized. GYKI 53784 completely blocked excitotoxicity (i.e. destruction of the afferent nerve endings) induced by AMPA and kainate. The histochemical detection of Co(2+) uptake was used to study Ca(2+) influx within the primary auditory nerve cells. Application of AMPA induced no significant Co(2+) uptake into the cells, suggesting that these receptors normally have a very low permeability to Ca(2+). Application of kainate induced significant Co(2+) uptake that was blocked by the AMPA receptor antagonist GYKI 53784 suggesting that kainate stimulated Ca(2+) entry through AMPA receptor channels. Results suggest that AMPA-preferring receptors are functionally located at the sensory cell-afferent synapse whereas NMDA and kainate receptors are not.     

Competitive inhibition by NBQX of kainate/AMPA receptor currents and excitatory synaptic potentials: importance of 6-nitro substitution.

We evaluated the inhibitory potencies at excitatory amino acid receptors of 2,3-dihydroxy-7-sulfamoyl-benzo[f]quinoxaline (BQX) and its 6-nitro derivative, NBQX. Currents activated by kainate or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in two-electrode voltage-clamp recordings of Xenopus oocytes injected with rat cortex mRNA were inhibited by BQX and NBQX: the apparent Ki values versus kainate were 14 microM and 78 nM, respectively, and versus AMPA were 23 microM and 63 nM, respectively. Thus, to a degree even more marked than with other quinoxalinedione derivatives, 6-nitro substitution of BQX to yield NBQX increases potency (200-fold) at the non-NMDA ionotropic receptor, but does not confer selectivity for kainate or AMPA. Schild analysis of the NBQX inhibition of the kainate and AMPA currents yielded pA2 values of 7.17 +/- 0.05 and 7.05 +/- 0.10, respectively, and slopes near unity confirming the competitive nature of the inhibition. Neither BQX nor NBQX significantly inhibited the current activated by glycine plus NMDA. The selectivity ratio of NBQX (greater than 5000-fold) is by far the greatest of any quinoxalinedione derivative antagonist of the kainate/AMPA receptor. BQX and NBQX also inhibited the excitatory postsynaptic field potentials mediated by kainate/AMPA receptors in the CA1 region of hippocampal slices after stimulation of the Schaffer collateral-commissural pathways with IC50 values of 130 and 0.90 microM, respectively. The 10-fold differences between the IC50 values in hippocampal slices and the Ki values in Xenopus oocytes correlate closely with data for other quinoxalinedione derivative antagonists.     

Ionotropic and metabotropic glutamate receptor antagonism attenuates cue-induced cocaine seeking.

Neuroanatomical and pharmacological evidence implicates glutamate transmission in drug-environment conditioning that partly controls drug seeking and relapse. Glutamate receptors could be targets for pharmacological attenuation of the motivational properties of drug-paired cues and for relapse prevention. The purpose of the present study was therefore to investigate the involvement of ionotropic and metabotropic glutamate receptor subtypes in cue-induced reinstatement of cocaine-seeking behavior. Rats were trained to self-administer cocaine using a second-order schedule of reinforcement (FR4(FR5:S)) under which a compound stimulus (light and tone) associated with cocaine infusions was presented contingently. Following extinction, the effects of the competitive NMDA receptor antagonist CGP 39551 (0, 2.5, 5, 10 mg/kg intraperitoneally (i.p.)), two competitive AMPA/kainate antagonists, CNQX (0, 0.75, 1.5, 3 mg/kg i.p.) and NBQX (0, 1.25, 2.5, 5 mg/kg i.p.), the NMDA/glycine site antagonist L-701,324 (0, 0.63, 1.25, 2.5 mg/kg i.p.), and the mGluR5 antagonist MPEP (0, 1.25, 2.5, 5 mg/kg i.p.) on cue-induced reinstatement of cocaine seeking were examined. The AMPA/kainate receptor antagonists CNQX and NBQX, the NMDA/glycine site antagonist L-701,324, and the mGluR5 antagonist MPEP attenuated significantly cue-induced reinstatement. The NMDA antagonist CGP 39551 failed to affect reinstatement. Additional control experiments indicated that attenuation of cue-induced reinstatement by CNQX, NBQX, L-701,324, and MPEP was not accompanied by significant suppression of spontaneous locomotor activity. These results suggest that conditioned influences on cocaine seeking depend on glutamate transmission. Accordingly, drugs with antagonist properties at various glutamate receptor subtypes could be useful in prevention of relapse induced by conditioned stimuli.     

Allosteric interactions between cyclothiazide and AMPA/kainate receptor antagonists.

1. Cyclothiazide blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization and potentiates AMPA receptor gated currents. Interactions between cyclothiazide, and the non-competitive antagonist GYKI52466 (GYKI) and competitive antagonist 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo (F) quinoxaline (NBQX) were studied at native and recombinant AMPA/kainate receptors using whole-cell recording in order to characterize the modulation by cyclothiazide of these two antagonist sites. 2. GYKI 100 microM, which is sufficient to eliminate virtually hippocampal kainate (100 microM) currents, failed to prevent access of cyclothiazide to its site of potentiation, and was unable to enhance removal of cyclothiazide potentiation. However, cyclothiazide reduced GYKI (30 microM) block from 84 +/- 8.3% to 38 +/- 12%, and slowed the onset of the block with a time course much faster than the time course for onset and offset of potentiation induced by cyclothiazide. Cyclothiazide had qualitatively similar effects upon antagonism by NBQX 1 microM. 3. Kainate activated desensitizing currents in dorsal root ganglion (DRG) neurones, which were unaffected by cyclothiazide. GYKI blocked these kainate currents with lower affinity (IC50 > 120 microM) than for hippocampal neurones (IC50 < 30 microM), and cyclothiazide did not affect GYKI antagonism. 4. Steady-state AMPA currents from homomeric GluRA-Dflip receptors in HEK 293 cells were dramatically potentiated (up to 216 fold) by cyclothiazide via reduction of desensitization. In contrast, kainate-gated currents in HEK 293 cells expressing GluR6R receptors exhibited pronounced desensitization that was unaffected by cyclothiazide. GYKI retains its inhibition at both recombinant AMPA and kainate receptors. 5. These results indicate that cyclothiazide allosterically influences two important antagonist sites on AMPA receptors. In addition, AMPA/kainate receptor subunit composition influences the affinity of GYKI for the receptor.     

Synaptic activation of a presynaptic kainate receptor facilitates AMPA receptor-mediated synaptic transmission at hippocampal mossy fibre synapses.

The development of GluR5-selective kainate receptor ligands is helping to elucidate the functions of kainate receptors in the CNS. Here we have further characterised the actions of a GluR5 selective agonist, ATPA, and a GluR5 selective antagonist, LY382884, in the CA3 region of rat hippocampal slices. In addition, we have used LY382884 to study a novel synaptic mechanism. This antagonist substantially reduces frequency facilitation of mossy fibre synaptic transmission, monitored as either AMPA or NMDA receptor-mediated EPSCs. This suggests that GluR5-containing kainate receptors on mossy fibres function as autoreceptors to facilitate the synaptic release of L-glutamate, in a frequency-dependent manner.     

Ionotropic glutamate receptor types leading to adenosine-mediated inhibition of electrically evoked [3H]-noradrenaline release in rabbit brain cortex slices.

1. Glutamate inhibits the electrically evoked release of noradrenaline in rabbit brain cortex slices; the inhibition is mediated by adenyl compounds, presumably adenosine. The aim of the present study was to identify the receptors involved in this indirect inhibitory effect of glutamate. Slices of the occipitoparietal cortex were preincubated with [3H]-noradrenaline and then superfused and stimulated by trains of 6 pulses, 100 Hz. 2. The ionotropic glutamate receptor agonists alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AM-PA; 10-100 microM), kainate (10-100 microM) and N-methyl-D-aspartate (NMDA; 30-300 microM) but not the metabotropic glutamate receptor agonist, 1-amino-1,3-cyclopentanedicarboxylate (ACPD; 10-100 microM) reduced the electrically evoked overflow of tritium. 3. The effects of AMPA, kainate and NMDA were attenuated or abolished by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as well as by adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as well as by adenosine deaminase but not by the alpha 2-adrenoceptor antagonist yohimbine, the gamma-aminobutyric acid (GABA) receptor antagonists, bicuculline and 2-hydroxysaclofen and the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). 4. The NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5) blocked the inhibitory effect of NMDA but not that of AMPA and kainate. The non-NMDA-receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the effect of AMPA but not of kainate and NMDA. 5. In addition to decreasing the electrically evoked overflow of tritium, AMPA, kainate and NMDA but not ACPD caused a steep but transient rise of basal tritium efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of glutamatergic agonists and antagonists at recombinant human homomeric and heteromeric kainate receptors in vitro

An increasing body of evidence suggests that native kainate receptors form ion channels from homomeric and heteromeric combinations of five receptor subunits: GluR5, GluR6, GluR7, KA1 and KA2. We have examined the activity of agonists and antagonists at recombinant human kainate receptors expressed in HEK293 cells, using both whole-cell electrophysiological recording and 96-well plate fluo-3 based calcium microfluorimetry (FLIPR). Both homomeric (GluR5 and GluR6) and heteromeric (GluR5/6, GluR5/KA2 and GluR6/KA2) receptors were examined. Heteromeric receptor assemblies showed electrophysiological and pharmacological profiles which were distinct from homomeric channels. Several agonists, including AMPA, ATPA and (S)-5-iodowillardiine, and antagonists, including gamma-D-glutamylaminomethylsulphonic acid (GAMS) and the decahydroisoquinoline compounds LY293558, LY377770 and LY382884, were found to act at GluR5-containing channels while having no effect at GluR6 homomers. AMPA, ATPA and (S)-5-iodowillardiine did activate GluR6/KA2 heteromers, but only as partial agonists. Additionally, ATPA was shown to act as an antagonist at homomeric GluR6 receptors at high concentrations (IC50 approximately 2 mM). Kynurenic acid was also found to differentiate between GluR6 and GluR6/KA2 receptors, antagonizing glutamate at GluR6 (IC50 = 0.4 mM), while having no effect at GluR6/KA2 channels. The results of the current study provide a broad pharmacological characterization of both homomeric and heteromeric recombinant human kainate receptors, and identify which compounds are likely to be useful tools for studying these various receptor subtypes.     

Novel AMPA receptor potentiators LY392098 and LY404187: effects on recombinant human AMPA receptors in vitro

The present study describes the activity of two novel potent and selective AMPA receptor potentiator molecules LY392098 and LY404187. LY392098 and LY404187 enhance glutamate (100 μM) stimulated ion influx through recombinant homomeric human AMPA receptor ion channels, GluR1-4, with estimated EC₅₀ values of 1.77 μM (GluR1_i), 0.22 μM (GluR2_i), 0.56 μM (GluR2_o), 1.89 μM (GluR3_i) and 0.20 μM (GluR4_i) for LY392098 and EC₅₀ values of 5.65 μM (GluR1_i), 0.15 μM (GluR2_i), 1.44 μM (GluR2_o), 1.66 μM (GluR3_i) and 0.21 μM (GluR4_i) for LY404187. Neither compound affected ion influx in untransfected HEK293 cells or GluR transfected cells in the absence of glutamate. Both compounds were selective for activity at AMPA receptors, with no activity at human recombinant kainate receptors. Electrophysiological recordings demonstrated that glutamate (1 mM)-evoked inward currents in human GluR4 transfected HEK293 cells were potentiated by LY392098 and LY404187 at low concentrations (3–10 nM). In addition, both compounds removed glutamate-dependent desensitization of recombinant GluR4 AMPA receptors. These studies demonstrate that LY392098 and LY404187 allosterically potentiate responses mediated by human AMPA receptor ion channels expressed in HEK 293 cells in vitro.     

Complex effects of CNQX on CA1 interneurons of the developing rat hippocampus

We have investigated the effect of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), on spontaneous GABA(A) receptor-mediated transmission in the hippocampal CA1 subfield. On average, simultaneous recordings from CA1 str. radiatum interneurons and pyramidal cells showed that CNQX application doubled the frequency of bicuculline sensitive spontaneous inhibitory postsynaptic currents (sIPSCs) without apparently changing their amplitude. However, despite the increase in sIPSC frequency, current-clamp recording showed that CNQX application was sufficient in most cases to depolarize interneurons to firing threshold. In contrast, CNQX application could not induce firing in pyramidal cells. In the presence of tetrado-toxin (TTX), CNQX increased interneuron membrane conductance, and depolarized interneurons from resting potentials. The axons of the studied interneurons ramify widely in the CA1 region and suggest that the cells of our sample are mostly involved with control of dendritic excitability.Our results indicate that CNQX-induced increase of sIPSC frequency is not limited to excitatory cells, but also impacts GABAergic interneurons. However, despite the increase of sIPSC frequency, CNQX-induced depolarization is sufficient to selectively generate firing in interneurons and thus modify the network properties mediated by GABA(A) receptors in the hippocampus.     

Non-N-methyl-D-aspartate (NMDA) receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(f)quinoxaline-7-sulphonamide (NBQX) decreases functional disorders in cytotoxic brain oedema

N-methyl-D-aspartate (NMDA) and non-NMDA receptors were found to be involved in development of functional disorders caused by hexachlorophene. In order to specify the role of glutamate receptors we studied the protective effects of the selective antagonist of the kainate/(+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor/channel 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulphonamide disodium (NBQX) and of the non-competitive NMDA receptor antagonist ifenprodil tartrate on coordinative motor behaviour of adult male Wistar rats as assessed in a simple 'ladder-test'. Neurotoxic injury of the cerebrum after hexachlorophene administration and putative amelioration after treatment with test substances was demonstrated histologically. Hexachlorophene-induced motor disturbance remitted spontaneously when stopping the noxis, but remittance occurred significantly earlier when NBQX [0.45 and 0.6 mg/kg intraperitoneal (i.p.)] was applied as well. Ifenprodil (0.15 to 1.2 mg/kg) did not improve the motor function. Vacuolation of white matter of the whole cerebrum was observed after 3 weeks of treatment with hexachlorophene. These morphological alterations caused by hexachlorophene treatment [central nervous system (CNS) vacuolation] spontaneously revert only after 5-6 weeks. The 5-day duration with test substances was too short for remission of vacuolation which thus may not apply to the situation after treatment with glutamate antagonists, despite improvement of motor function. The results suggest that kainate/AMPA receptor channels are at least partially involved in the mechanism of brain damage induced by hexachlorophene, however, the polyamine binding site of the NMDA receptor evidently is not involved.     

Topiramate selectively protects against seizures induced by ATPA, a GluR5 kainate receptor agonist

Although the mechanism of action of topiramate is not fully understood, its anticonvulsant properties may result, at least in part, from an interaction with AMPA/kainate receptors. We have recently shown that topiramate selectively inhibits postsynaptic responses mediated by GluR5 kainate receptors. To determine if this action of topiramate is relevant to the anticonvulsant effects of the drug in vivo, we determined the protective activity of topiramate against seizures induced by intravenous infusion of various ionotropic glutamate receptor agonists in mice. Topiramate (25–100 mg/kg, i.p.) produced a dose-dependent elevation in the threshold for clonic seizures induced by infusion of ATPA, a selective agonist of GluR5 kainate receptors. Topiramate was less effective in protecting against clonic seizures induced by kainate, a mixed agonist of AMPA and kainate receptors. Topiramate did not affect clonic seizures induced by AMPA or NMDA. In contrast, the thresholds for tonic seizures induced by higher doses of these various glutamate receptor agonists were all elevated by topiramate. Unlike topiramate, carbamazepine elevated the threshold for AMPA- but not ATPA-induced clonic seizures. Our results are consistent with the possibility that the effects of topiramate on clonic seizure activity are due to functional blockade of GluR5 kainate receptors. Protection from tonic seizures may be mediated by other actions of the drug. Together with our in vitro cellular electrophysiological results, the present observations strongly support a unique mechanism of action of topiramate, which involves GluR5 kainate receptors.     

Pharmacological effects of AMPA receptor potentiators LY392098 and LY404187 on rat neuronal AMPA receptors in vitro

The present study describes the pharmacological activity of two novel positive allosteric modulators at AMPA receptors in acutely isolated rat cerebellar Purkinje neurons and cultured rat hippocampal neurons. Currents elicited by application of glutamate (100 μM) to isolated cerebellar Purkinje neurons were potentiated by LY392098, LY404187, cyclothiazide, CX516 and aniracetam. The rank order of potency was LY404187> LY392098> cyclothiazide > CX516> aniracetam. LY392098 displayed a higher maximal efficacy than the other compounds examined. AMPA-activated inward currents in cultured rat hippocampal neurons were potentiated by LY392098, LY404187 and cyclothiazide in a reversible and concentration-dependent manner although considerable heterogeneity in the magnitude of response from cell to cell was observed. LY392098 was ineffective in potentiating AMPA receptor responses when dialyzed via the intracellular solution. The selectivity profiles of the two novel AMPA receptor potentiators were examined. LY392098 or LY404187 had minimal activity on NMDA receptor responses, on voltage-gated calcium channel currents in cultured hippocampal neurons and on GluR5 kainate receptor currents in acutely isolated rat dorsal root ganglion neurons.