Selective AMPA Receptor Antagonist Research Articles

Sustained ethanol inhibition of native AMPA receptors on medial septum/diagonal band (MS/DB) neurons.

The direct impact of ethanol on native, non-NMDA glutamate receptors was examined in acutely isolated MS/DB neurons from rat. The impact of ethanol functional tolerance and physical dependence on non-NMDA receptor function was also determined. Non-NMDA receptors were defined pharmacologically as predominantly the AMPA subtype, because both AMPA- or kainate-activated currents were blocked by GYKI 52466, a selective AMPA receptor antagonist. The relative magnitude of potentiation of AMPA-activated currents by 10 or 100 microM cyclothiazide was consistent with recombinant AMPA flop-subtype receptors. Finally, the selective kainate receptor agonist, SYM 8021, induced little current in MS/DB neurons. AMPA receptor currents when activated by kainate were sensitive to ethanol, showing inhibition of approximately 5 - 50% when 10 - 300 mM ethanol and kainate were briefly co-applied (3 s). Ethanol (100 mM) also inhibited both the initial transient peak and sustained currents activated by AMPA. Inhibition was sustained during continuous ethanol superfusions of 5 min, suggesting a lack of acute tolerance to ethanol-induced AMPA receptor blockade. Rapid application of 3 - 3000 microM kainate activated concentration-dependent currents in MS/DB neurons from Control and Ethanol Dependent animals that were not significantly different. Also, direct ethanol inhibition (300 mM) of kainate-activated currents was not reduced by ethanol dependence, suggesting a lack of functional tolerance. These results suggest that native AMPA receptors on MS/DB neurons are inhibited by pharmacologically-relevant concentrations of ethanol. However, these receptors, unlike NMDA receptors, do not undergo adaptation with sustained ethanol exposure sufficient to induce physical dependence. British Journal of Pharmacology (2000) 129, 87 - 94

Late embryonic expression of AMPA receptor function in the CA1 region of the intact hippocampus in vitro.

Studies in slices suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic currents are not present in CA1 (Cornu ammonis) pyramidal neurons at birth (P0). We have re-examined this issue in the rat intact hippocampal formation (IHF) in vitro. Injections of biocytin or carbocyanine show that the temporo-ammonic, commissural and Schaffer collateral pathways are present at birth in the marginal zone of CA1. Electrical stimulation of these pathways evoked field excitatory postsynaptic potentials (fEPSPs) in the marginal zone of CA1 from embryonic day 19 (E19) to postnatal day 9 (P9). These fEPSPs are mediated by synaptic AMPA receptors as they are reduced or completely blocked by: (i) tetrodotoxin; (ii) high divalent cation concentrations; (iii) the adenosine A1 receptor agonist CPA; (iv) anoxic episodes; (v) the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI-53655) or the mixed AMPA-kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). The amplitude of the fEPSPs is also reduced by D(-)-2-amino-5-phosphonopentanoic acid (D-APV) and its duration is increased by bicuculline suggesting the participation of N-methyl-D-aspartate (NMDA) and GABAA (gamma-aminobutyric acid) receptors. Finally, AMPA receptor-mediated fEPSPs are also recorded in P0 slices, but they are smaller and more labile than in the IHF. Our results suggest that in embryonic CA1 neurons, glutamate acting on AMPA receptors already provides a substantial part of the excitatory drive and may play an important role in the activity-dependent development of the hippocampus. Furthermore, the IHF may be a convenient preparation to investigate the properties of the developing hippocampus.

AMPA receptors: a better target for ischaemic brain damage?

The excitatory transmitter glutamate accumulates in the interstitial space of brain tissue following ischaemic insult, and facilitates neuronal cell death via excessive stimulation of glutamate receptors. Pre-clinical data suggest that glutamate blockers might be effective for preventing neurodegeneration following brain ischaemia. The prevalent assumption used to be that damage to neurons is mediated mainly following excessive activation of N-methyl-d-aspartate (NMDA)-type glutamate receptors. However, NMDA-receptor blockade did not effectively reduce ischaemic brain damage in several animal models. Another glutamate receptor subtype, the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor, is more closely associated with neuronal cell death following ischaemic stroke. The study by Schielke et al.1 Schielke G.P. et al. The neuroprotective effect of the novel AMPA receptor antagonist PD152247 (PNQX) in temporary focal ischaemia in the rat. Stroke. 1999; 30: 1472-1477 Crossref PubMed Scopus (23) Google Scholar shows that 1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido [3,4-f]quinoxaline-2,3-dione (PNQX), a potent and selective AMPA receptor antagonist, reduced ischaemic brain damage following temporary focal ischaemia in the rat. Temporary ischaemia was induced by occluding the middle cerebral artery and both carotid arteries for 3 h. Brain lesion volume, determined by haematoxylin and eosin staining after three days, was reduced by half in rats receiving PNQX within 30 min of the insult 1 Schielke G.P. et al. The neuroprotective effect of the novel AMPA receptor antagonist PD152247 (PNQX) in temporary focal ischaemia in the rat. Stroke. 1999; 30: 1472-1477 Crossref PubMed Scopus (23) Google Scholar .

Enhancement of AMPA-Mediated Current after Traumatic Injury in Cortical Neurons

Overactivation of ionotropic glutamate receptors has been implicated in the pathophysiology of traumatic brain injury. Using an in vitro cell injury model, we examined the effects of stretch-induced traumatic injury on the AMPA subtype of ionotropic glutamate receptors in cultured neonatal cortical neurons. Recordings made using the whole-cell patch-clamp technique revealed that a subpopulation of injured neurons exhibited an increased current in response to AMPA. The current-voltage relationship of these injured neurons showed an increased slope conductance but no change in reversal potential compared with uninjured neurons. Additionally, the EC(50) values of uninjured and injured neurons were nearly identical. Thus, current potentiation was not caused by changes in the voltage-dependence, ion selectivity, or apparent agonist affinity of the AMPA channel. AMPA-elicited current could also be fully inhibited by the application of selective AMPA receptor antagonists, thereby excluding the possibility that current potentiation in injured neurons was caused by the activation of other, nondesensitizing receptors. The difference in current densities between control and injured neurons was abolished when AMPA receptor desensitization was inhibited by the coapplication of AMPA and cyclothiazide or by the use of kainate as an agonist, suggesting that mechanical injury alters AMPA receptor desensitization. Reduction of AMPA receptor desensitization after brain injury would be expected to further exacerbate the effects of increased postinjury extracellular glutamate and contribute to trauma-related cell loss and dysfunctional synaptic information processing.

Effects of YM90K, a selective AMPA receptor antagonist, on amygdala-kindling and long-term hippocampal potentiation in the rat

To investigate the role of α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) type glutamate receptors in epileptic seizures, we examined the antiepileptogenic and anticonvulsant effects of YM90K [6-(1 H-imidazol-1-yl)-7-nitro-2,3-(1 H,4 H)-quinoxalinedione hydrochloride], a potent and selective new AMPA receptor antagonist, in the rat amygdala-kindling model of epilepsy. Pretreatment with YM90K (7.5–30 mg/kg i.p.) markedly retarded the evolution of kindling. Once kindling was established, administration of YM90K (7.5–30 mg/kg i.p.) significantly and dose-dependently suppressed fully kindled seizures. The maximal effects were observed 15–30 min after injection. When the intensity of electrical stimulation was increased to twice the generalized seizure-triggering threshold, the anticonvulsant effects of YM90K were reversed, suggesting that they were due to elevation of the generalized seizure-triggering threshold. Furthermore, an anticonvulsant dose (15 mg/kg) of YM90K affected neither field potentials nor long-term potentiation in the hippocampus in vivo. These results indicate that AMPA receptors play an important role in the seizure expression mechanism and the development of kindling-induced epileptogenesis, and suggest the possible clinical usefulness of AMPA receptor antagonists as antiepileptic drugs.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. Giant depolarizing potentials (GDPs) are generated by the interplay of the depolarizing action of GABA and glutamate. In this study, single and dual whole cell recordings (in current-clamp configuration) were performed from CA3 pyramidal cells in hippocampal slices obtained from postnatal (P) days P1- to P6-old rats to evaluate the role of ionotropic glutamate receptors in GDP generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-40 microM) completely blocked GDPs. However, in the presence of CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 microM) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 microM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50-100 microM). In the presence of GYKI 53655, both kainic or domoic acid (0.1-1 microM) were unable to induce GDPs. In contrast, bath application of D-(-)-2-amino-5-phosphonopentanoic acid (50 microM) or (+)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 microM) produced only a 37 +/- 9% (SE) and 36 +/- 11% reduction in GDPs frequency, respectively. Cyclothiazide, a selective blocker of AMPA receptor desensitization, increased GDP frequency by 76 +/- 14%. Experiments were also performed with an intracellular solution containing KF to block GABAA receptor-mediated responses. In these conditions, a glutamatergic component of GDP was revealed. GDPs could still be recorded synchronous with those detected simultaneously with KCl-filled electrodes, although their amplitude was smaller. Similar results were found in pair recordings obtained from minislices containing only a small portion of the CA3 area. These data suggest that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals.

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.

Characterisation of kainate receptor mediated whole-cell currents in rat cultured cerebellar granule cells

Whole-cell voltage clamp recordings have been used to identify and characterise inward currents mediated by native kainate receptors in rat cultured cerebellar granule cells. While the selective AMPA receptor antagonist GYKI 53655 (50 μM) completely abolished inward currents evoked by AMPA (10–100 μM) in the presence of cyclothiazide (100 μM), kainate evoked currents in cells pretreated with concanavalin A (Con A) always showed a component (35–140 pA, n=13) resistant to blockade. The majority (73±7%, n=5) of GYKI 53655-resistant kainate-evoked inward currents remained in the presence of 100 μM AMPA. However, these currents were reversibly blocked by the competitive AMPA/kainate receptor antagonist NBQX (100 μM). (2 S, 4 R)-4-methylglutamate (SYM 2081, 10 μM) evoked inward currents in Con A treated cells (15–60 pA, n=7), which were resistant to complete blockade by GYKI 53655 (50 μM) but antagonised by NBQX (100 μM). Kainate-evoked responses in the presence of GYKI 53655 (50 μM) had linear or slightly outwardly rectifying current–voltage (I–V) relationships in all cells examined ( n=5) and were resistant to blockade by Joro spider toxin (JsTx, 1 μM; n=5). These results provide evidence that rat cultured cerebellar granule cells express functional kainate receptors made up of subunits which are edited at the Q/R site, and that SYM 2081 is an agonist at these native kainate receptors with a greater selectivity than kainate itself.

Synthesis and Resolution of ATPO, a Potent and Selective Ampa Receptor Antagonist

The excitatory amino acid (EAA) receptors mediate one of the major signaling systems in the central nervous system, and they are thought to be implicated in some neurodege-nerative disorders, e.g. Alzheimer's disease and stroke. It is of key importance to design selective ligands for every group of EAA receptors. The AMPA receptors are one such group of EAA receptors. (RS)-ATPO is a potent antagonist at the AMPA receptors, but it has hitherto been pharmacologically characterized only as the racemate.[1, 2]

Effects of YM872 on atrophy of substantia nigra reticulata after focal ischemia in rats.

Middle cerebral artery (MCA) occlusion causes atrophy in the ipsilateral substantia nigra reticulata (SNR). The effects of glutamate AMPA receptor antagonism on SNR atrophy, which is supposed to inhibit excitatory inputs from the subthalamic nucleus to the SNR, was investigated in rats with permanent MCA occlusions. Histological examination revealed marked atrophy two weeks after MCA occlusion in the saline-treated control group. However, constant i.v. infusion of YM872, a selective AMPA receptor antagonist, for 2 weeks significantly reduced SNR atrophy; neurological deficits also decreased. These results suggest that the AMPA receptor may be involved in the pathogenesis of SNR atrophy during the subacute phase of focal cerebral ischemia.

Pharmacology and toxicology of ATOA, an AMPA receptor antagonist and a partial agonist at GluR5 receptors

( RS)-2-Amino-3-[3-(carboxymethoxy)-5- tert-butyl-4-isoxazolyl]propionic acid (ATOA) has previously been described as an antagonist at ( RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors with an IC 50 value of 150 μM towards AMPA-induced depolarisation in the rat cortical wedge preparation. ATOA has now been shown also to be a partial agonist at recombinant GluR5 receptors, expressed in Xenopus oocytes, with an EC 50 value of 170 μM and a relative efficacy of 0.17±0.04 compared with responses produced by kainic acid (1.0). Using cultured cerebral cortical neurones as a test system and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage, ATOA was shown to be cytotoxic (ED 50 >300 μM), though much less toxic than the structurally related dual AMPA and GluR5 agonist, ( RS)-2-amino-3-(3-hydroxy-5- tert-butyl-4-isoxazolyl)propionic acid (ATPA) (ED 50=14±2 μM). The toxic effect of ATPA was sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX) but was not significantly reduced by the selective AMPA receptor antagonist, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). The toxicity of ATOA (1 mM) could not be significantly attenuated by co-administration of AMOA (300 μM) or DNQX (25 μM). A structure–activity analysis indicates that the tert-butyl group of ATPA and ATOA facilitates the interaction of these compounds with GluR5 receptors.

Pharmacological differentiation of kainate receptors on neonatal rat spinal motoneurones and dorsal roots

The objectives of this study, conducted on neonatal rat spinal cord and dorsal roots in vitro, were to characterise the actions of a range of willardiine analogues on GluR5-containing kainate receptors present in dorsal roots, to determine whether GluR5-containing receptors are also present on motoneurones, and to differentiate responses mediated by kainate receptors from those mediated by AMPA receptors on motoneurones. ( S)-5-Trifluoromethyl-willardiine, ( S)-5-iodowillardiine, ( S)-5-iodo-6-azawillardiine and ATPA were found to be potent agonists of kainate receptors on dorsal roots (EC 50 values 0.108±0.002, 0.127±0.010, 0.685±0.141 and 1.3±0.3 μM, respectively) being more potent but of lower efficacy than kainate (EC 50 value 14.8±1.8 μM). ( S)-5-Iodo-6-azawillardiine blocked kainate-induced depolarisations of the dorsal root, probably via its desensitising action. Kainate-induced responses of dorsal roots were weakly antagonised by ( RS)-3,5-dicarboxyphenylglycine (DCPG) (apparent K D 1.5±0.4 mM). Kainate receptors containing GluR5 subunits do not appear to be present on motoneurones since ( RS)-3,5-DCPG (1 mM) potentiated rather than antagonised kainate-induced depolarisations of motoneurones. Although ( S)-5-iodowillardiine (a potent and selective agonist at GluR5-containing kainate receptors) depolarised motoneurones (EC 50 value 5.8±0.6 μM), such depolarisations were antagonised by both ( RS)-3,4- and ( RS)-3,5-DCPG, which are selective AMPA receptor antagonists at motoneurones, showing a K D value of 73 μM (Schild slope, 0.96±0.09) and an apparent K D value of 123±38 μM, respectively. This accords with the previously reported activity of willardiine analogues at AMPA receptors. Since neither ( RS)-3,4- nor ( RS)-3,5-DCPG antagonised kainate-induced motoneuronal depolarisations but cyclothiazide enhanced and GYKI53655 blocked these responses it is possible that a component of the kainate response may be mediated by a population of DCPG-insensitive AMPA receptors on motoneurones. However, it is also possible that a population of kainate receptors other than those containing GluR5 subunits, are responsible for these effects. The new compounds introduced in this study are likely to be useful tools for studying the physiological role of kainate receptors in CNS function.

Binding characteristics of a potent AMPA receptor antagonist [3H]Ro 48-8587 in rat brain.

A new AMPA receptor antagonist, Ro 48-8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [3H]AMPA, [3H] kainate, and [3H] MK-801 binding to rat brain membranes and on AMPA- or NMDA-induced depolarization in rat cortical wedges. [3H]Ro 48-8587 bound with a high affinity (KD = 3 nM) to a single population of binding sites with a Bmax of 1 pmol/mg of protein in rat whole brain membranes. [3H]Ro 48-8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48-8587 > 6-nitro-7-sulphamoylbenzo[f] quinoxaline-2,3-dione (NBQX) > YM 90K > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (approximately 95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3-6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 nM, the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (approximately 2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.

AMPA receptor activation potentiated by the AMPA modulator 1-BCP is toxic to cultured rat hippocampal neurons

The benzoylpiperidine 1-(1,3-benzodioxol-5-ylcarbonyl)-piperidine (1-BCP), and related compounds, potentiate α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acidergic (AMPAergic) synaptic currents in central neurons, and improve performance of rodents and humans on learning and memory tasks. Their physiological actions are similar but not identical to thiazides, which also enhance AMPAergic synaptic responses and improve performance of rats in water-maze and passive- avoidance tests. Thiazides also dramatically increase AMPA receptor-mediated neuronal death in vitro and in vivo. Here it was evaluated whether 1-BCP potentiated AMPA receptor-mediated excitotoxicity in hippocampal neuron cultures. Glutamate+MK 801 (to block NMDA receptors)+1 mM 1-BCP produced neuronal death that was reversed by 10 μM 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), a selective AMPA receptor antagonist. 1-BCP and drugs with similar activities can facilitate AMPA receptor-mediated excitotoxicity.

P-369 - Neuroprotective Effect of YM872, a Novel Selective AMPA Receptor Antagonist, on the Middle Cerebral Artery (MCA)-Occluded Model in F344 Rats

P-369 - Neuroprotective Effect of YM872, a Novel Selective AMPA Receptor Antagonist, on the Middle Cerebral Artery (MCA)-Occluded Model in F344 Rats

Slow modulation of depolarizing afterpotentials through activation of CaM KII in pyramidal cells of rat frontal cortex

Inhibition of histone deacetylases (HDACs) is a promising approach for the treatment of mood disorders. However, the effects of HDAC inhibition on the serotonin (5-HT) system, a common target for psychiatric disorders, are poorly understood. Here, we show that a broad-spectrum HDAC inhibitor, trichostatin A (TSA), enhances the function of 5-HT neurons in organotypic raphe slice cultures. Sustained treatment with TSA (1 μM) for 2 or 4 days significantly increased the 5-HT tissue content and tryptophan hydroxylase 2 (TPH2) expression, which were accompanied by hyper-acetylation of histone H3 in the promoter region of the TPH2 gene. TSA treatment for 4 days increased the extracellular 5-HT level, which was significantly suppressed in the presence of the selective AMPA receptor (AMPAR) antagonist NBQX. Moreover, the expression of both the AMPAR subunit GluA2 and Ca2+/calmodulin-dependent kinase II α (CaMKIIα) mRNAs were significantly increased by TSA treatment. Co-treatment with the CaMKII inhibitors KN-62 and KN-93 prevented the TSA-induced increase in 5-HT release, but had no effect on the increases in 5-HT tissue content. These results suggest that inhibition of HDACs increases 5-HT synthesis and release by epigenetic mechanisms, and that 5-HT release is mediated by the enhancement of AMPAR-mediated excitatory inputs and CaMKII signaling.

Bioisosterically modified dipeptide excitatory amino acid receptor antagonists containing 3-oxygenated isothiazole ring systems

The AMPA receptor agonist Thio-AMPA, the 3-isothiazolol analogue of AMPA was converted into the selective NMDA antagonist, 2, in which a 3-isothiazolone unit is a bioisosteric analogue of the peptide bond of the NMDA antagonist, γ-( R)-Glu-Gly. The isomeric 3-oxygenated isothiazole amino acid, 3, and the corresponding isothiazole phosphono amino acid 4 were also synthesized, and were shown to be selective AMPA receptor antagonists. Compound 1, in which the peptide bond of γ-( R)-Glu-Gly is replaced by an ester group, was synthesized and shown to be unstable in the test buffer system.

The synaptic activation of kainate receptors.

L-Glutamate, the principal excitatory neurotransmitter in the vertebrate central nervous system, acts on three classes of ionotropic glutamate receptors, named after the agonists AMPA, NMDA and kainate. AMPA receptors are known to mediate fast synaptic responses and NMDA receptors to mediate slow synaptic responses at most excitatory synapses in the brain. Kainate receptors are formed from a separate set of genes (GluR5-7, KA-1 and KA-2) and are widely distributed throughout the brain. They are implicated in epileptogenesis and cell death. However, the physiological functions of kainate receptors are not known. The development of 2,3-benzodiazepine antagonists that are selective for AMPA receptors enables kainate receptors to be specifically activated by exogenous ligands, such as kainate. Here we demonstrate that high-frequency stimulation of mossy fibres in rat hippocampal slices, in the presence of the highly selective AMPA receptor antagonist GYKI 53655 plus NMDA- and GABA-receptor antagonists, activates an inward current in CA3 neurons that has a pharmacology typical of kainate receptors. The finding that kainate receptors can be activated synaptically adds to the diversity of information transfer at glutamatergic synapses.

Inhibition of [ 3H]γ-aminobutyric acid release by kainate receptor activation in rat hippocampal synaptosomes

We studied the modulation of γ-aminobutyric acid (GABA) release by activation of kainate receptor in rat whole hippocampal synaptosomes. Kainate (10–300 μM) inhibited [ 3H]GABA release in a concentration-dependent manner with an EC 50 of 25 μM. This effect of kainate (30 μM) was prevented by the ionotropic non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM) and by the selective kainate receptor antagonist 5-nitro-6,7,8,9-tetrahydrobenzo( g)indole-2,3-dione-3-oxime (NS-102, 10 μM), but not by the selective non-competitive AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5 H-2,3-benzodiazepine (GYKI 52466, 100 μM). Other kainate receptor agonists, such as domoic acid (0.3–10 μM) and (2 S,4 R)-4-methylglutamic acid (MGA, 0.3–3 μM), also inhibited [ 3H]GABA release in a concentration-dependent manner with EC 50 values of 4.0 μM and 0.90 μM, respectively, whereas α-amino-3-hydroxy-5-methyl-4-oxazolepropionate (AMPA, 10–100 μM) was devoid of effect. These inhibitory effects of both domoic acid (3 μM) and MGA (1 μM) were antagonized by CNQX (10 μM). These results indicate that GABA release can be modulated directly by presynaptic high-affinity kainate heteroreceptors. © 1997 Elsevier Science B.V. All rights reserved.

A role for presynaptic AMPA receptors in the enhancement of neuronal glutamate release by kainic acid.

Kainic acid (KA), an analogue of glutamic acid, is a powerful neuroexcitant and neurotoxin. It has been proposed that an increased release of excitatory amino acids (EAAs) from synaptic terminals substantially contributes to the central neurotoxic action of this compound [1,2]. We have recently reported the existence of presynaptic, cyclothiazide (CYZ)-sensitive AMPA receptors on glutamatergic nerve terminals in rat forebrain slices [3]. These receptors appear to mediate a positive feedback control on synaptic glutamate release. We now report that these novel autoreceptors may play a role in KA-mediated enhancement of neuronal glutamate release. Serial rat forebrain coronal sections were cut and loaded with [3H]D-aspaxtate (PHID-asp) as previously described [4]. Following loading with PHID-asp, slices were superfused with oxygenated Krebs buffer and the influence of agonists and antagonists on basal and electrically stimulated (36mA, 2msec pulses, 20Hz for 5min) release of label was examined using a standard protocol [4]. Electrically stimulated release in this preparation is calcium-dependent and tetrodotoxin-insensitive, confirming that it arises principally from direct stimulation of presynaptic nerve terminals [ 5 ] . The results are summarized in Figure 1 and Table 1. KA (1-1selectively enhanced elecmcally-stimulated efflux of [3H]D-asp with a bell-shaped dose-response curve (max. 2.7 fold enhancement at 10 pM). Basal efflux of label was not significantly increased. In the presence of a low (1OpM) concentration of the AMPA receptor desensitization inhibitor CYZ, responses to KA, 1-100pM remained u~changed, wheieas responses to KA, ImM were markedly potentiated (approx. 4.2 fold enhancement). Consistent with this, the selective AMPA receptor antagonists NBQX [13] and GYKI 52466 114,151 (1100pM) failed to influence responses to KA, 100pM plus CYZ,