Characterization of AMPA receptors on isolated amacrine-like cells in carp retina

In amacrine-like cells freshly dissociated from crucian carp (Carassius auratus) retina, we recorded whole-cell responses to rapid application of glutamate and kainate. Currents induced by glutamate, but not kainate, usually showed extremely rapid desensitization, and the mean time constant for the decay of the responses to 10 mM glutamate was 2.77 ms. N-methyl-D-aspartate (NMDA) failed to induce any current even with coapplication of glycine and removal of extracellular Mg2 +. 1-(4-aminophenyl)-3-Methylcarbamyl-4-methyl-7,8-methylenedioxy-3, 4-dihydro-5H-2,3-benzodiazepine (GYKI 53655), a selective alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, was found to completely block glutamate-induced currents, suggesting that the glutamate receptors on these cells are AMPA preferring. The value of EC50 for glutamate and kainate was determined to be 2.73 mM and 97.5 microM, respectively. Noise analysis of fluctuation of whole-cell currents induced by kainate of different concentrations indicated that the mean conductance of the AMPA receptor channels was 5.70 pS. Splice variant analysis of the AMPA receptors was also conducted by comparing the effects of cyclothiazide, a flip receptor-preferring modulator and 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difluoro-phenoxyaceta mide (PEPA), a flop receptor-preferring modulator, on glutamate-induced responses. PEPA was much more potent than cyclothiazide at these receptors with a EC50 of 17.3 microM. The mean ratio of the potentiation by PEPA versus cyclothiazide (P/C ratio) was 4.39. These modulatory effects of cyclothiazide and PEPA were rather similar to those obtained at AMPA receptors assembled from flop variants expressed in Xenopus oocytes, suggesting that the AMPA receptor of the carp amacrine cells may predominantly consist of the flop splice variants.     

GYKI 52466 has positive modulatory effects on AMPA receptors

The 2,3-benzodiazepine derivative GYKI 52466 has been well characterized as a negative modulator of AMPA-type glutamate receptors. The present study re-examined the effects of GYKI 52466 on AMPA receptor-mediated currents in patches excised from pyramidal neurons in the hippocampal CA1 field and found that this drug has positive modulatory effects in addition to its receptor blocking action. A low concentration of GYKI 52466 (10 microM) reliably increased the steady-state current by about three-fold, while the peak current was reduced by 30% only. Higher drug concentrations produced parallel reductions in both the steady-state and peak currents. The increase in the steady-state current was not accompanied by a change in the deactivation time constant and thus, is more likely to result from a change in desensitization than a slowing of channel closing. The results indicate that GYKI 52466 modulates AMPA receptor-mediated currents in a complex manner, perhaps by acting through more than one binding site.     

In vivo, the direct and seizure-induced neuronal cytotoxicity of kainate and AMPA is modified by the non-competitive antagonist, GYKI 52466

The 2,3-benzodiazepine GYKI 52466, administered intracerebrally or systemically, was assessed for its ability to protect against the neuronal death in the brain caused by intra-hippocampal injections of the non-N-methyl-D-aspartate (NMDA) receptor agonists, kainate and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). In contrast to a previous report, a low intra-hippocampal dose of GYKI 52466 (25 nmol) did not protect against kainate toxicity. In order to achieve higher doses of GYKI 52466, solubilization in 2-hydroxypropyl-beta-cyclodextrin was used, and limited protection against AMPA, but not kainate toxicity was found. There was a commensurate reduction in seizure-related neuronal loss in the limbic regions of the brain. When diazepam was used to prevent seizures, GYKI 52466 had no effect on hippocampal neuronal loss caused by the direct toxicity of AMPA and kainate on hippocampal neurons. Systemic administration of GYKI 52466 had only a minimal effect on preventing neuronal death caused by AMPA. In vivo, GYKI 52466 is only weakly effective as a neuroprotective agent.     

Modulation of kainate--activated currents by diazoxide and cyclothiazide analogues (IDRA) in cerebellar granule neurons

1. Patch-clamp technique was used in primary cultures of cerebellar granule neurons to study the modulation of the cyclothiazide analogue (IDRA21) and of the diazoxide derivative (IDRA 5) on KA-evoked currents. 2. The dose-response of kainic acid (KA) reveals an EC50=90 microM and an Hill coefficient of 1.3. IDRA 21 and cyclothiazide potentiate KA-evoked current in a dose dependent way, being cyclothiazide more potent but less efficacious than IDRA 21. Conversely IDRA 5 acts as a negative modulator of KA evoked -current. 3. Application of IDRA 21 and cyclothiazide results in a current potentiation of 125+/-18% and 80+/-12% respectively, while IDRA 5 decreases KA-current (-21+/-5%). Coapplication of cyclothiazide and IDRA 21 produces a potentiation of 110+/-17%, suggesting a competition of the two drugs for the same site. 4. In the same experimental model we studied the ability of IDRA compounds of promoting toxicity through AMPA-receptor activation. Under basal conditions AMPA treatment (50 microM for 1 hour) results in a negligible excitotoxicity. 5. In contrast similar treatment with AMPA + IDRA 21 (1 mM) or + IDRA 5 (1 mM) or + cyclothiazide (100 microM) induces citotoxicity. The neurotoxic damage induced by IDRA 21 and cyclothiazide is blocked by GYKI 53655 (50 microM) and by NBQX (10 microM). Interestingly GYKI and NBQX are ineffective in reducing IDRA 5 toxicity.     

AMPA receptors on developing medial septum/diagonal band neurons are sensitive to early postnatal binge-like ethanol exposure

The impact of binge-like, early postnatal ethanol treatment on AMPA or kainate whole cell currents was examined in acutely isolated medial septum/diagonal band (MS/DB) neurons. AMPA (10 or 100 microM) current was inhibited by GYKI 52466, a selective AMPA receptor (AMPAR) antagonist, in all neurons isolated on postnatal day (PD) 5-8, PD 12-15 or PD 32-35. Cyclothiazide, a selective inhibitor of AMPAR desensitization, also effectively potentiated AMPA currents. This suggests that non-NMDA, ionotropic glutamate receptors on immature MS/DB neuron are predominantly AMPARs. Concentration-dependent kainate (10-1000 microM) application evoked nondesensitizing currents that exhibited an increase in the maximum response by the end of first postnatal month, consistent with developmental regulation of AMPAR function. Acute 3 s ethanol application (100 mM) consistently blunted AMPA- and kainate currents approximately 20-30% across age groups. Inhibition was sustained during continuous ethanol superfusion lasting 10-12 min without evidence of acute tolerance. Repeated oral intubation of rat pups with ethanol (5.25 g/kg/day on PD 4-9), which models third trimester human binge drinking, resulted in peak blood ethanol levels of approximately 350 mg/dl (measured 90 min after PD 6 dosing). AMPA or kainate currents were upregulated in neurons isolated on PD 32-35 by earlier ethanol intubation suggesting that binge-like intoxication augments developing AMPAR function. Despite this augmentation of AMPAR function, no significant changes were found in the sensitivity of AMPA currents to GYKI 52466, cyclothiazide or acute ethanol (100 mM) sensitivity or in the levels of GluR1/GluR2 subunit proteins from MS/DB tissue. These results indicate that non-NMDA ionotrophic glutamate receptors on immature MS/DB neurons, which are largely of the AMPAR subtype, are moderately sensitive to immediate inhibition by ethanol. Repeating this inhibition during early postnatal binge-like intoxication can augment normal development of AMPAR function.     

AMPA receptor antagonists, GYKI 52466 and NBQX, do not block the induction of long-term potentiation at therapeutically relevant concentrations

The involvement of alpha-amino-3-hydroxy-5-methylizoxazole-4-propionic acid (AMPA) receptors in induction of long-term potentiation (LTP) was examined in rat hippocampal slice preparation. Using conventional extracellular recording, excitatory postsynaptic potentials (EPSPs) and population action potentials (PSs), evoked by low-frequency stimulation of the Schaffer collateral-commissural fibres, were recorded in the CA1 region. The effects of a competitive AMPA receptor antagonist, 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2, 3-dione (NBQX), and that of a non-competitive blocker, 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466) have been examined. 0.25-0.5 microM of NBQX and 20-40 microM of GYKI 52466 did not suppress the induction of LTP. LTP was attenuated only at the highest concentrations tested (1 microM NBQX or 80 microM GYKI 52466). These in vitro concentrations, however, exceed the brain levels needed for in vivo anticonvulsant action. Furthermore, even at the highest concentrations both compounds suppressed only the expression but not the induction of LTP. Namely after their washout LTP reappeared. Thus, at pharmacologically relevant concentrations these AMPA receptor antagonists apparently do not suppress LTP, a cellular mechanism underlying memory formation. These experiments suggest that in clinical practice AMPA receptor blockade may have some advantage over N-methyl-D-aspartate receptor antagonism, which is accompanied by severe memory impairment.     

2,3-benzodiazepine AMPA antagonists

The 2,3-benzodiazepine GYKI 52466 (1-(4-aminophenyl)-4 methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) and its analogues represent a family of selective AMPA antagonists. They modulate AMPA channel functions through an allosteric site on the receptor, which is probably different from the ones involved in the actions of cyclothiazide and aniracetam. These compounds are frequently used as research tools to elucidate glutamate receptor-mediated functions. The most effective members of the family inhibit AMPA-induced currents in the submicromolar range. In addition, they are active at low systemic doses in various in vivo experimental models and also possess a good oral bioavailability. In vitro and in vivo pharmacological results with 2,3-benzodiazepine AMPA antagonists indicate their potential therapeutical value in treating a great variety of central nervous system diseases, of which epilepsy and neurodegenerative disorders are regarded as the most important.     

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.     

Ionotropic glutamate receptor modulation preferentially affects NMDA receptor expression in rat hippocampus

Electrophysiological data suggest that alterations in the function of one glutamate receptor subtype may affect the function of other subtypes. Further, previous studies have demonstrated that NMDA receptor antagonists affect NMDA and kainate receptor expression in rat hippocampus. In order to address the mutual regulation of NMDA, AMPA, and kainate receptor expression in rat hippocampus, we conducted two experiments examining the effects of NMDA and non-NMDA glutamate receptor modulators on NMDA, AMPA, and kainate receptor expression using in situ hybridization and receptor autoradiography. NMDA receptor expression was preferentially affected by systemic treatments, as all drugs significantly altered [(3)H]MK-801 binding, and several drugs increased [(3)H]ifenprodil binding. GYKI52466 and aniracetam treatments resulted in changes in both [(3)H]ifenprodil binding and NR2B mRNA levels, consistent with the association of this subunit and binding site in vitro. There were more modest effects on AMPA and kainate receptor expression, even by direct antagonists. Together, these data suggest that ionotropic glutamate receptors interact at the level of expression. These data also suggest that drug regimens targeting one ionotropic glutamate receptor subtype may indirectly affect other subtypes, potentially producing unwanted side effects.     

Transient Ca2+-permeable AMPA receptors in postnatal rat primary auditory neurons

Fast excitatory transmission in the nervous system is mostly mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+-permeable before hearing onset as evidenced using agonist-induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100 microM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 microM) and blocked by GYKI 52466 (80 microM) and Joro spider toxin (1 microM). It was unaffected by D-AP5 (50 microM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 microM glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynaptically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.     

Comparison of anticonvulsive and acute neuroprotective activity of three 2,3-benzodiazepine compounds, GYKI 52466, GYKI 53405, and GYKI 53655

GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine], a non-competitive AMPA [alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate] and kainate receptor antagonist and its two analogues, GYKI 53405 [1-(4-aminophenyl)-3-acetyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine] and GYKI 53655 [1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine] were investigated in two seizure models and in MgCl2 induced global cerebral ischaemia, as an acute neuroprotective model. The ED(50) values of GYKI 52466 for suppression of the tonic and clonic phases of sound-induced seizures were 3.6 and 4.3 mg/kg, respectively. The corresponding data for GYKI 53405 were 1.1 and 3.1 mg/kg, while ED(50) values of GYKI 53655 were 1.3 and 2.0 mg/kg, respectively. The inhibition of seizure evoked by maximal electroshock was also found to be remarkable: the ED(50) values of GYKI 52466 and its two analogues were 6.9, 2.6, and 2.2 mg/kg, respectively. All compounds prolonged the survival times in MgCl2 induced global cerebral ischaemia test in a dose-dependent fashion, with PD(50) (dose of 50% prolongation) values of 24.1, 8.3, and 8.2 mg/kg intraperitoneal, respectively. In audiogenic seizure model the duration of anticonvulsant action of 10 mg/kg GYKI 52466 and 5 mg/kg GYKI 53405, GYKI 53655 were examined, too. The effect of GYKI 52466 decreased to 50% after 2 h, while the analogues showed more than 80% seizure suppression 3 h after treatment. After 6 h the effect of GYKI 53655 decreased to zero, while the effect of GYKI 52466, remained on the 50% level.     

Low-affinity kainate receptor agonists induce insult-dependent apoptosis and necrosis in cultured murine cortical neurons

Overstimulation of ionotropic glutamate receptors leads to excitotoxic neuronal death, which has been implicated in the neurodegeneration of neurological diseases. The present study examined the role of putative low-affinity kainate receptor subtype (GluR5-7) agonists in excitotoxicity in cultured murine cortical neurons. The concentration-dependent decrease in cell viability induced by the agonists kainate (1-1,000 microM) and (RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA; 1-1,000 microM) was only attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466; 20 microM). (S)-5-iodowillardiine (1-1,000 microM)-induced toxicity was attenuated by CNQX (20 microM), GYKI 52466 (20 microM) and MK-801 (10 microM); however, (2S, 4R)-4-methylglutamate (1-120 microM)-induced toxicity was not attenuated by the antagonists. None of the agonists possessed selective actions at GluR5-7. Morphological observations (phase-contrast and fluorescence microscopy) revealed that the agonists induced two distinct patterns of neuronal injury. After 24 hr of treatment, low concentrations of agonists (1-30 microM) produced cellular shrinkage and nuclear granulation consistent with slow, apoptotic-like neuronal death. Pyknotic labeling with the DNA binding dye Sytox green confirmed these apoptotic characteristics, which significantly decreased with increasing concentrations. After 4 hr, increasing concentrations of agonists (100-1,000 microM) induced cellular swelling, with subsequent extracellular debris; labeling with propidium iodide revealed isolated nuclei consistent with the increased involvement of rapid necrosis. Thus, all putative GluR5-7 agonists produced excitotoxicity across a necrotic-apoptotic continuum in murine cortical neuron cultures.     

Kindled seizure-evoked somatostatin release in the hippocampus: inhibition by MK-801

The aim of this study was to evaluate the contribution of ionotropic glutamate receptors to kindled seizure-evoked somatostatin release in the hippocampus, using a microdialysis approach. Basal and amygdala stimulation-evoked somatostatin-like immunoreactivity (-LI) release was significantly greater in kindled compared to naive rats. In naive rats, neither hippocampal perfusion with the selective AMPA/kainate receptor antagonist GYKI 52466 nor with the selective NMDA receptor antagonist MK-801 affected behavior, EEG, or somatostatin-LI release. In kindled rats, GYKI 52466 was still devoid of any effect, while MK-801 significantly decreased stimulus-evoked (but not basal) somatostatin-LI efflux. MK-801 produced identical effects when injected i.p. This study provides the first direct evidence that kindled seizure-evoked somatostatin release in the hippocampus is partly NMDA receptor dependent.     

Treatment of early and late kainic acid-induced status epilepticus with the noncompetitive AMPA receptor antagonist GYKI 52466

Purpose:  Benzodiazepines such as diazepam may fail to effectively treat status epilepticus because benzodiazepine-sensitive GABA_A receptors are progressively internalized with continued seizure activity. Ionotropic glutamate receptors, including AMPA receptors, are externalized, so that AMPA receptor antagonists, which are broad-spectrum anticonvulsants, could be more effective treatments for status epilepticus. We assessed the ability of the noncompetitive AMPA receptor antagonist GYKI 52466 to protect against kainic acid–induced status epilepticus in mice.
Methods:  Groups of animals treated with kainic acid received GYKI 52466 (50 mg/kg followed in 15 min by 50 mg/kg) or diazepam (25 mg/kg followed in 20 min by 12.5 mg/kg) beginning at 5 min of continuous seizure activity or 25 min later. The duration of seizure activity was determined by EEG recording from epidural cortical electrodes.
Results:  Both GYKI 52466 and diazepam rapidly terminated electrographic and behavioral seizures when administered early. However, diazepam-treated animals exhibited more seizure recurrences. With late administration, GYKI 52466 also rapidly terminated seizures and they seldom recurred, whereas diazepam was slow to produce seizure control and recurrences were common. Although both treatments caused sedation, GYKI 52466-treated animals retained neurological responsiveness whereas diazepam-treated animals did not. GYKI 52466 did not affect blood pressure whereas diazepam caused a sustained drop in mean arterial pressure.
Discussion:  Noncompetitive AMPA receptor antagonists represent a promising approach for early treatment of status epilepticus; they may also be effective at later times when there is refractoriness to benzodiazepines.     

Effect of a glutamate receptor antagonist (GYKI 52466) on 4-aminopyridine-induced seizure activity developed in rat cortical slices

In the present experiments we have tested the effect of the noncompetitive AMPA antagonist GYKI 52466 (20-80 microM) on spontaneous epileptic discharges developed as the consequence of 4-aminopyridine application in neocortex slices of adult rats. Parallel to the changes of spontaneous activity, the field potentials, evoked by electrical stimulation of the corpus callosum, were also analyzed. Glass microcapillary extracellular recording electrode was positioned in the third layer of the somatosensory cortex slice, while the stimulating electrode was placed at the border of the white and gray matter. 4-aminopyridine and GYKI 52466 were bath-applied. The application of 40 microM GYKI 52466 caused about 40% decrease in the frequency and the amplitude of spontaneous seizures as well as the duration of each discharges developed in 4-amino-pyridine. Pre-incubation with the AMPA antagonist effectively inhibited both the development of seizure activity and the maintenance of the discharges. GYKI 52466 also decreased the duration and amplitude of field responses evoked by stimulation of the corpus callosum. This inhibitory effect was dose-dependent. Our data in the in vitro cortex slice epilepsy model suggest that the non-competitive AMPA antagonist GYKI 52466 is a potent anticonvulsant and neuroprotective compound because it reduced the fully developed epileptic discharges or prevented their development.     

Agonist- and subunit-dependent potentiation of glutamate receptors by a nootropic drug aniracetam.

GluR1 and GluR2 cDNAs encoding non-NMDA subtypes of glutamate receptor were isolated from a rat brain cDNA library by Boulter et al. (Science, 249 (1990) 1033-1037). Functional receptors activated by kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and glutamate were expressed in Xenopus oocytes injected with GluR1, GluR2 or a mixture of GluR1 and GluR2 RNAs. In GluR1-expressed oocytes, 1 mM aniracetam potentiated AMPA-induced currents by 99 +/- 10% (mean +/- S.E.M., n = 5) and glutamate-induced currents by 140 +/- 8% (n = 4), but little affected kainate-induced currents. Aniracetam was effective from a concentration of 0.1 mM, and it exhibited more conspicuous effects with the increase of the dose. In oocytes injected with GluR1 plus GluR2 RNAs, aniracetam more markedly potentiated current responses to AMPA and glutamate than those in oocytes injected with GluR1 RNA alone. For example, 1 mM aniracetam potentiated AMPA-induced currents by 396 +/- 76% (n = 4) and glutamate-induced currents by 970 +/- 65% (n = 5) in oocytes injected with 10% GluR1 and 90% GluR2 RNAs. In these oocytes, however, the potentiation of kainate-induced currents by 1 mM aniracetam was only 8 +/- 5% (n = 4). Thus, we conclude that the potentiation of the AMPA/kainate receptor by aniracetam depends on both species of agonists and subunit composition of the receptor.     

Modulation of AMPA receptors in spinal motor neurons by the neuroprotective agent riluzole

We investigated the interaction of riluzole, a therapeutic agent used in amyotrophic lateral sclerosis (ALS), with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels in mouse spinal motor neurons in culture using whole-cell patch-clamp recording techniques. Kainate elicited concentration-dependent (EC(50) = 35 microM) inward currents in all the patched cells. These responses were mediated primarily through the activation of AMPA receptors with a negligible contribution from kainate receptors, because bath application of 100 microM GYKI53655, a potent noncompetitive AMPA receptor antagonist, completely blocked the kainate-induced currents. Riluzole (0.5-100 microM) reduced in a dose-dependent manner the kainate-induced currents with an IC(50) of 1.54 microM in all tested neurons (n = 25) and this effect was found to be reversible. The response to kainate decreased in the presence of 1 microM riluzole in all spinal motor neurons tested, without changing its EC(50), indicating a noncompetitive mechanism of inhibition. The amplitude of the responses induced by kainate under control condition and during riluzole was a linear function of the membrane potential. The reversal potential of the current was not significantly different in the two experimental conditions, whereas the total conductance of the motor neurons for the currents induced by 100 microM kainate was reduced significantly in the presence of 1 microM riluzole (P < 0.05). These results reveal an interaction of riluzole with glutamatergic neurotransmission in spinal cord motor neurons and can contribute to explain its beneficial effect in the ALS treatment.     

Comparative Antagonism of Kainate-activated Kainate and AMPA Receptors in Hippocampal Neurons

Native kainate receptors expressed by cultured hippocampal cells were studied in the whole-cell configuration of the patch-clamp technique by using a fast perfusion system. About 80% of the neurons expressed kainate receptors independently of the time in culture (0–4 days), which coincided with the number of cells immunoreactive for a monoclonal antibody against the GluR5/6/7 subunits. Three types of cells were considered: neurons in which the rapid application of kainate induced a rapidly desensitizing current, cells in which kainate induced a more slowly rising, non-desensitizing, response and those in which a mixture of both responses was apparent. Steady responses induced by 300 μM kainate were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in a dose-dependent manner (IC₅₀= 0.92 μM). CNQX was less potent in blocking transient kainite-induced responses (IC₅₀= 6.1 μM). Responses to kainate, whether steady or transient, were also inhibited by NS102, showing poor selectivity for the transient response (IC₅₀= 4.1 and 2.2 μM respectively). The new α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor antagonist NS394 was very potent in inhibiting steady kainate-induced currents (IC₅₀= 0.45 μM), but was even more effective in preventing peak responses (IC₅₀= 0.13 μM). In contrast, cyclothiazide did not affect transient kainate-induced responses but did potentiate current induced by activation of AMPA receptors by AMPA or kainate. These results demonstrate the lack of complete selectivity amongst some available competitive antagonists for AMPA and kainate receptors, and indicate that kainate receptors expressed by hippocampal cells lack the cyclothiazide modulatory site present at AMPA receptors. In addition, the present data support the idea that low-affinity kainate binding sites in the brain correspond to receptor channels selectively activated by kainate.     

Excitotoxic profile of LY339434, a GluR5 agonist, in cultured murine cortical neurons

The neurotoxic profile of (2S,4R, 6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434), a low-affinity kainate receptor subtype 5 (GluR5) agonist at recombinant human glutamate receptors, was evaluated to investigate the involvement of GluR5 in excitotoxic neuronal death. Murine cortical neurons were exposed to treatments for 24 h and assessed by a cell viability assay and phase-contrast microscopy. LY339434 (1-1000 microM) caused a concentration-dependent decrease in cell viability (EC(50)=11.4+/-1.2 microM) that was only attenuated by (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine (MK-801, 10 microM), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, 20 microM). Labeling with nucleic acid binding dyes revealed that LY339434 induced few apoptotic-like characteristics. These findings indicate that in cultured murine cortical neurons, LY339434 acts predominantly through N-methyl-D-aspartate (NMDA) receptors rather than GluR5 to effect neuronal death that is rapid and involves predominantly necrosis rather than morphological apoptosis.