AMPA Receptor Desensitization Research Articles

Mechanism and impact of allosteric AMPA receptor modulation by the Ampakine TM CX546

Glutamate release at central synapses is transduced into a characteristic fast postsynaptic response by AMPA receptor gating and agonist affinity. The effect of two classes of modulators of AMPA receptor desensitization, the benzothiadiazides (cyclothiazide and IDRA 21) and the benzoylpiperidines (CX516 and CX546), were studied on gating kinetics of recombinant, native AMPA receptors and on synaptic currents. CX546 reduced the degree of desensitization more potently than CX516 or IDRA 21, but not as efficiently as cyclothiazide. In presence of CX516/CX546, desensitization of GluR2 flip receptors was inhibited more than of GluR1 flip, whereas they had no effect upon response shape or conductance. CX546 increased agonist affinity threefold on nondesensitizing AMPA receptors by slowing agonist unbinding. Analysis of modulatory action suggests that, in contrast to cyclothiazide or IDRA 21, the Ampakine TM CX546 binds specifically to the agonist bound nondesensitized receptor, most likely acting by destabilizing the desensitized receptor conformation. All modulators tested showed higher efficiency on native receptors as compared to homomeric receptors. At the glutamatergic synapse, evoked synaptic amplitudes were weakly potentiated, while EPSC decay was slowed by nearly a factor of three in the presence of CX546 or cyclothiazide. In the presence of CX546, the current induced by short pulses of glutamate from recombinant GluR2 receptors decayed with a time course that was approximately twentyfold faster than EPSCs. The unique properties of CX546 may be beneficial for therapeutical use.

Subunit Interactions and AMPA Receptor Desensitization

Most AMPA-type glutamate receptors (GluRs) exhibit rapid and virtually complete desensitization when activated by glutamate, and at some central synapses it is largely desensitization that determines the decay of EPSCs. However, the mechanisms underlying the conformation change that results in desensitization are not fully understood. AMPA receptor subunits that contain a single amino acid substitution have been shown to form homomeric channels that show markedly reduced desensitization. We show here that the coexpression of wild-type GluR1 with one such mutant, GluR1(L497Y), results in heteromeric channels that show desensitization behavior that is intermediate between wild-type and mutant homomers. The relative amplitudes of the multiple exponential components present in the decay of glutamate-evoked currents depended on the relative abundance of wild-type and mutant subunits and were described by the combinatorial distribution of the two types of subunits into tetrameric, but not pentameric, assemblies. Our results are consistent with recent structural data suggesting that AMPA receptors are tetrameric assemblies composed of two dimers.

Gadolinium reduces AMPA receptor desensitization and deactivation in hippocampal neurons.

The actions of the trivalent cation Gd(3+) on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd(3+) (0.1-10 microM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 microM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd(3+) also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd(3+) reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd(3+) inhibited both peak and steady-state currents, indicating that Gd(3+) both reduces desensitization and inhibits these currents. Gd(3+) reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd(3+). The Gd(3+)-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd(3+) reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd(3+) potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.

Blocking AMPA receptor desensitization prolongs spontaneous EPSC decay times and depolarizes H1 horizontal cells in carp retinal slices.

Desensitization of H1 horizontal cell (H1 HC) glutamate receptors was investigated in carp retinal slices using cyclothiazide (CTZ), an inhibitor of AMPA receptor desensitization. 100 microM CTZ depolarized H1 HCs and increased the amplitude of light responses, without any prominent changes in their kinetics. Spontaneous EPSCs (sEPSCs) in H1 HCs were observed in the presence of 2.5 mM heptanol, an uncoupling agent of gap junctions. 20 microM GYKI52466 (an AMPA receptor antagonist) blocked the sEPSCs, consistent with the sEPSCs being mediated by AMPA receptors. 100 microM cobalt suppressed the frequency of sEPSCs without changing their mean peak amplitude, suggesting that calcium-dependent transmitter release from cones was not affected by heptanol. CTZ increased the total inward charge transferred per sEPSC by increasing the sEPSC decay time constant twofold, without any significant change in their frequency and mean peak amplitude. This suggests that the depolarizing effect of CTZ on H1 HCs was due to blocking desensitization of AMPA receptors, increasing the inward current induced by glutamate released from cone synaptic terminals. The desensitization of glutamate receptors may function to extend the dynamic range of H1 HC light responses.

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 EC 50 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 EC 50 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.

Binding of an AMPA receptor potentiator ([ 3H]LY395153) to native and recombinant AMPA receptors

LY395153 is a member of a newly described class of arylpropylsulfonamide AMPA receptor potentiators. Here, we characterize and compare [ 3H]LY395153 binding to native AMPA receptors from rat cerebral cortex and recombinant human GluR4 flip receptors expressed in HEK293 cells. l-Glutamate and AMPA increased [ 3H]LY395153 binding to both native and recombinant AMPA receptors in a concentration dependent and stereoselective manner; this effect of AMPA receptor agonists reflects an apparent increase in ligand affinity. In the presence of l-glutamate (500 μM), [ 3H]LY395153 binding is saturable; the affinity of this radioligand is slightly, albeit statistically significantly higher at human GluR4 flip ( K d =55.6±5.3 nM ) than rat cortical receptors ( K d =110±15.1 nM ). NBQX competitively inhibited l-glutamate-induced increases in [ 3H]LY395153 binding in both native and recombinant receptors, whilst LY303070 (the active isomer of GYKI53655) noncompetitively inhibited this effect in native, but not recombinant receptors. The prototypic AMPA receptor potentiator cyclothiazide competitively inhibited [ 3H]LY395153 binding with a potency ( K i ∼7 μM ) comparable to EC 50 values reported in electrophysiological studies. In contrast, the structurally unrelated AMPA receptor potentiator CX 516 did not inhibit [ 3H]LY395153 binding at concentrations of up to 600 μM. Further, at concentrations reported to facilitate AMPA receptor desensitization, thiocyanate acts as a competitive inhibitor of [ 3H]LY395153 binding. [ 3H]LY395153 binding was unaffected by a variety of structurally (and mechanistically) diverse compounds tested at a concentration of 10 μM. These data indicate [ 3H]LY395153 is a useful probe for labeling a unique modulatory site on both native and recombinant AMPA receptors.

A computational model of synaptic transmission at the calyx of Held

The calyx of Held is a giant glutamatergic synapse in the mammalian auditory pathway designed to ensure faithful transmission of high frequency action potential trains. Pre- and postsynaptic recordings from this synapse reveal several forms of facilitation and depression. A computational model of synaptic transmission has been developed to investigate the mechanisms underlying modulation at the calyx of Held. The model includes paired-pulse facilitation and depression due to vesicle depletion and postsynaptic AMPA receptor desensitization. Accurately matching the experimental time course and magnitude of the depression requires both slow and fast replenishment of vesicles at the presynaptic release sites. The model demonstrates that the temporal accuracy of postsynaptic spike generation depends on the amplitude of the EPSC and so accuracy decreases as the EPSCs depress.

Characteristics of AMPA receptor-mediated responses of cultured cortical and spinal cord neurones and their correlation to the expression of glutamate receptor subunits, GluR1-4.

Electrophysiological recordings have been used to characterize responses mediated by AMPA receptors expressed by cultured rat cortical and spinal cord neurones. The EC(50) values for AMPA were 17 and 11 microM, respectively. Responses of cortical neurones to AMPA were inhibited competitively by NBQX (pK(i)=6.6). Lower concentrations of NBQX (< or =1 microM) also potentiated the plateau responses of spinal cord neurones to AMPA, which could be attributed to a depression of desensitization to AMPA. GYKI 52466 inhibited responses of spinal cord neurones to AMPA to about twice the extent of responses of cortical neurones. Blockade of AMPA receptor desensitization by cyclothiazide (CTZ) potentiated responses of spinal cord neurones (6.8 fold) significantly more than responses of cortical neurones (4.8 fold). Responses of cortical neurones to KA were potentiated 3.5 fold by CTZ, while responses of spinal cord neurones were unaffected. Ultra-fast applications of AMPA to outside-out patches showed responses of spinal cord neurones desensitized by 97.5% and exhibit marked inward rectification, whereas cortical neurones desensitized by 91% and exhibited slight outward rectification. The time constants of deactivation and desensitization were about twice as fast in spinal cord than cortical neurones. In cortical neurones, single-cell RT - PCR showed GluR2 and GluR1 accounted for 91% of all subunits and were expressed together in 67% of neurones, predominantly as the flip variants (78%). GluR2 was detected alone in 24% of neurones. GluR3 and GluR4 were present in only 14 and 29% of neurones, respectively. For spinal cord neurones, GluR4(o) was detected in 81% of neurones, whereas predominantly flop versions of GluR1, 2 and 3 were detected in 38, 13 and 13% of neurones, respectively. These expression patterns are related to the respective pharmacological and mechanistic properties.

Minimizing Synaptic Depression by Control of Release Probability

Transmission at the end-bulb synapse formed by auditory nerve terminals onto the soma of neurons in the avian nucleus magnocellularis is characterized by high transmitter release probability and strong synaptic depression. Activation of presynaptic GABA(B) receptors minimizes depression at this synapse and significantly enhances synaptic strength during high-frequency activity. Here we investigate synaptic mechanisms underlying this phenomenon. EPSC amplitudes evoked by 200 Hz trains increased more than twofold when release probability was reduced with Cd(2+) or baclofen. This effect was not exhibited by a transmitter depletion model of presynaptic depression, which predicts that EPSC amplitudes reach a common steady-state amplitude during high-frequency trains, despite alterations of initial release probability. However, an additional source of postsynaptic depression was sufficient to explain our findings. Aniracetam, a modulator of AMPA receptors that reduces desensitization, decreased the amount of synaptic depression during trains, indicating that desensitization occurred during trains of stimuli. However, this effect of aniracetam was absent when release probability was lowered with baclofen or Cd(2+). No effect of aniracetam on the NMDA component of the EPSC was seen, confirming a postsynaptic site of action of aniracetam. When desensitization was reduced with aniracetam, steady-state EPSC amplitudes during trains were found to converge over a wide range of release probabilities, as predicted by the depletion model. Additional evidence of AMPA receptor desensitization was provided by direct measurement of quantal amplitudes immediately after stimulus trains. Thus, presynaptic modulation by GABA(B) receptors regulates the extent of AMPA receptor desensitization and controls synaptic strength, thereby modulating the flow of information at an auditory synapse.

Domain Interactions Regulating AMPA Receptor Desensitization

Desensitization is a common property of glutamate and other ligand-gated ion channels, yet its molecular mechanism is unknown. For glutamate receptors, agonist binding involves interactions with identified amino acids from two lobes and may result in stabilizing the lobes in a closed "clamshell" conformation. The present studies demonstrate that two structures, beta-strands 7 and 8 and alpha-helices J and K, functionally interact with each other and likely form hinges between the two lobes, influencing the coupling between agonist binding and desensitization. Two amino acids identified within these regions form a solvent-exposed interface with a third amino acid, a mutation of which was shown previously to block receptor desensitization (L(507) in glutamate receptor 3). This interface may regulate a concerted conformational shift of the AMPA subtype of glutamate receptor subunits to the desensitized state.

Cortical and Striatal Neuronal Cultures of the Same Embryonic Origin Show Intrinsic Differences in Glutamate Receptor Expression and Vulnerability to Excitotoxicity

Cortical and striatal cultures were prepared from the same embryonic rat brains and maintained in identical culture conditions. In this way, the intrinsic, genetically imprinted differences determine the responses of cortical and striatal neurons in comparative studies. Cortical and striatal neurons differed in their sensitivity to glutamate receptor-mediated neurotoxicity as measured by the MTT cell viability assay. On the 8th day in vitro, striatal cultures were less sensitive to N-methyl-d-aspartate (NMDA)-induced toxicity than cortical, although both cultures were equally vulnerable to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)- or kainate-induced toxicity. The AMPA receptor-mediated cell death in cortical cultures, however, was much more dependent on preventing AMPA receptor desensitization than in striatal cultures. Furthermore, glutamate-induced neurotoxicity was primarily mediated by NMDA receptors in cortical cultures, while blockade of either NMDA or AMPA receptors gave almost complete protection against glutamate in striatal cultures. To elucidate the molecular mechanisms responsible for the observed differences, we analyzed the expression of NMDA receptor subunits (NR1, NR2A-C) at the mRNA and the protein level in cortical and striatal cultures as well as in standard cerebellar granule cell cultures. The lowest expression level of NMDA receptor subunits was found in striatal cultures, thereby providing a possible explanation for their lower sensitivity to NMDA. Remarkable differences were found between the relative rates of mRNA and protein expression for NR1 and NR2B in the three cultures, indicative of intrinsic differences in the posttranscriptional regulation of NMDA receptor subunit expression in cultures from various brain regions.

Presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated stimulation of glutamate and GABA release in the rat striatum in vivo: a dual-label microdialysis study

The existence of presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate autoreceptors on glutamate nerve terminals in vitro has recently been demonstrated using synaptosomal and brain slice preparations. In the present study we have used a modification of a rapid dual-label intracerebral microdialysis method, previously developed by Young and co-workers 80,81 for the study of presynaptic mechanisms of neurotransmitter release, to investigate whether presynaptic AMPA receptors also play a role in the control of striatal glutamate release in vivo. For comparative purposes, the action of locally applied AMPA on striatal GABA release in vivo was also monitored. Local application of AMPA (0.01–100 μM), by reverse dialysis, into the striatum resulted in concentration-dependent increases in the Ca 2+-dependent efflux of both [ 3H] l-glutamate and [ 14C]GABA. Maximum responses reached 142.0±6.5% and 166.8±7.7% of basal efflux for [ 3H] l-glutamate and [ 14C]GABA, respectively. No marked behavioural changes were observed at any dose of the agonist. Unexpectedly, the AMPA-evoked responses were not potentiated by the AMPA receptor desensitization inhibitors cyclothiazide (10–100 μM) or aniracetam (1 mM). Consistent with this finding, AMPA-stimulated [ 3H] l-glutamate and [ 14C]GABA efflux were significantly attenuated by co-perfusion with the selective, competitive AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo(F)quinoxaline-2,3-dione (100 μM) but not 1-(aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (100 μM), a non-competitive AMPA receptor antagonist known to interact with the cyclothiazide site to control AMPA receptor function. The broad spectrum ionotropic glutamate receptor antagonist, kynurenic acid (100–1000 μM) also markedly inhibited the AMPA-evoked responses in the striatum in vivo. None of the antagonists, when given alone, influenced basal efflux of [ 3H] l-glutamate suggesting a lack of tonic regulatory control of glutamate release via presynaptic AMPA-type autoreceptors in the rat striatum. These results demonstrate the presence of presynaptic AMPA receptors, of a novel cyclothiazide- and aniracetam-insensitive subtype, on presynaptic nerve terminals in the rat striatum in vivo, acting to enhance glutamate and GABA release. Our data support the concept of AMPA receptor heterogeneity in vivo, a finding which may facilitate the development of novel, more selective drugs for the treatment of a range of neurological disorders associated with abnormal cerebral glutamate release. The pharmacological profile of these novel presynaptic receptors is currently under investigation.

Modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization by extracellular protons.

The interstitial milieu of the brain is buffered to an average pH of 7.3, but synaptic activation produces a temporal sequence of events that can affect pH in the synaptic cleft. Furthermore, pathophysiological processes such as ischemia and seizures produce global and prolonged acidification of interstitial pH. Changes in pH, in turn, can affect neuronal excitability by modulating receptors and channels. Patch-clamp recordings were made from cultured rat hippocampal neurons to determine whether physiologically relevant changes in interstitial pH (6.5-7.8) significantly affect AMPA receptor function. Acidic pH, such as that typically associated with ischemia (pH 6.5), significantly inhibited AMPA receptor-mediated responses in neurons. The effect of pH was agonist-dependent, with 2-fold greater inhibition of responses evoked by the strongly desensitizing agonists glutamate and quisqualate than the weakly desensitizing agonist kainate. Additional experiments tested the hypothesis that protons modulate AMPA receptor desensitization. In the presence of drugs that block AMPA receptor desensitization, pH 6. 5 had no effect on glutamate-evoked responses. In neuronal macropatches, protons increased equilibrium desensitization without affecting macroscopic desensitization or deactivation kinetics. The mechanisms and molecular determinants of pH-mediated effects were further investigated using human embryonic kidney 293 cells expressing recombinant AMPA receptors. Inhibition of kainate-evoked responses varied with subunit and isoform composition, ranging from 10% to >40%. Flop isoforms, which exhibit faster and more extensive desensitization, were most strongly inhibited. These findings suggest that interstitial acidification can modulate AMPA receptor-mediated synaptic transmission and that differences in receptor sensitivity to proton modulation may underlie the selective vulnerability of certain neuronal populations to ischemia.

The lethal expression of the GluR2flip/GluR4flip AMPA receptor in HEK293 cells.

alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) -type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1-GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+-permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2-lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip-mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor-mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.

AMPA Receptor Current Density, Not Desensitization, Predicts Selective Motoneuron Vulnerability

Spinal motoneurons are more susceptible to AMPA receptor-mediated injury than are other spinal neurons, a property that has been implicated in their selective degeneration in amyotrophic lateral sclerosis (ALS). The aim of this study was to determine whether this difference in vulnerability between motoneurons and other spinal neurons can be attributed to a difference in AMPA receptor desensitization and/or to a difference in density of functional AMPA receptors. Spinal motoneurons and dorsal horn neurons were isolated from embryonic rats and cultured on spinal astrocytes. Single-cell RT-PCR quantification of the relative abundance of the flip and flop isoforms of the AMPA receptor subunits, which are known to affect receptor desensitization, did not reveal any difference between the two cell populations. Examination of AMPA receptor desensitization by patch-clamp electrophysiological measurements on nucleated and outside-out patches and in the whole-cell mode also yielded similar results for the two cell groups. However, AMPA receptor current density was two- to threefold higher in motoneurons than in dorsal horn neurons, suggesting a higher density of functional AMPA receptors in motoneuron membranes. Pharmacological reduction of AMPA receptor current density in motoneurons to the level found in dorsal horn neurons eliminated selective motoneuron vulnerability to AMPA receptor activation. These results suggest that the greater AMPA receptor current density of spinal motoneurons may be sufficient to account for their selective vulnerability to AMPA receptor agonists in vitro.

Ca2+-dependent desensitization of AMPA receptors.

The effect of changes in the external concentrations (0.4-10 mM) of Ca2+ ions on AMPA receptors (AMPARs) of different subunit composition was studied on freshly isolated rat brain neurones. Ca2+ produces rapid and reversible voltage-independent inhibition of AMPARs. Ca2+-permeable and Ca2+-impermeable AMPARs are equally sensitive to external Ca2+ suggesting that the effect is not addressed to the ion channel. The inhibition of responses evoked by AMPA is significantly larger than those evoked by kainate or glutamate. Cyclothiazide and aniracetam, which are known to prevent AMPAR desensitization, both greatly diminish inhibition of AMPARs by Ca2+. Cyclothiazide is more potent than aniracetam in both preventing of AMPAR desensitization and protecting against the Ca2+ inhibitory effect on hippocampal pyramidal cells. On giant cholinergic interneurones of striatum, aniracetam but not cyclothiazide significantly prevents inhibition by Ca2+. This agrees with available data on relative abundance of flip and flop splice variants in these cell types. The results suggest that Ca2+ may allosterically increase AMPA receptor desensitization independently on subunit composition and splice variants.

Characterization of Audiogenic-Like Seizures in Naive Rats Evoked by Activation of AMPA and NMDA Receptors in the Inferior Colliculus

The role of glutamate receptors in the inferior colliculus (IC) in audiogenic and audiogenic-like seizures was investigated in adult rats with transient neonatal hypothyroidism by 0.02% propylthiouracil (PTU) treatment through mother's milk (PTU rats) and in naive rats treated intracisternally with N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA), or cyclothiazide, an inhibitor of rapid AMPA receptor desensitization. All rats showed audiogenic or audiogenic-like seizures characterized by running fit (RF) and generalized tonic–clonic seizures (GTCS). While systemically administered MK-801 inhibited GTCS, intracisternally administered NBQX inhibited RF and GTCS in both audiogenic and audiogenic-like seizures. Auditory stimulation shortened the latency to GTCS induced by AMPA, but not NMDA, at a subclinical dose and further elongated the shortened duration of RF, but not GTCS, induced by MK-801 pretreatment. Furthermore, Northern blot analysis was used to evaluate the expression of the immediate-early gene c-fos in the IC following induction of audiogenic or audiogenic-like seizures. The significant induction of c-fos mRNA by audiogenic seizures in PTU rats or by AMPA- or cyclothiazide-induced seizures in naive rats was prominent in the IC. MK-801 suppressed c-fos mRNA expression in the IC induced by audiogenic seizures in PTU rats or by AMPA-induced seizures in naive rats. NBQX suppressed the expression of c-fos mRNA in the IC induced by AMPA-induced seizures but did not suppress c-fos mRNA in PTU rats or rats with cyclothiazide-induced seizures. Auditory stimuli failed to affect c-fos mRNA induction by AMPA. The present study suggests that audiogenic-like seizures can be reproduced by glutamate receptor agonists in which AMPA receptors are primarily linked to the initiation of audiogenic seizures (RF) while NMDA receptors presumably located within the IC are involved in the propagation of GTCS in audiogenic seizures.

Dietary restriction and 2-deoxyglucose enhance synaptic glucose and glutamate transport and protect against synaptic dysfunction in experimental models of Alzheimer's disease

Cultured neocortical neurons, which predominantly consist of GABAergic neurons exhibit a pronounced stimulus-coupled GABA release. Since the cultures may contain a small population of glutamatergic neurons and the GABAergic neurons have a high content of glutamate it was of interest to examine if glutamate in addition to γ-aminobutyric acid (GABA) could be released from these cultures. The neurons were preloaded with [3H]d-aspartate and subsequently its release was followed during depolarization induced by a high potassium concentration or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonists, AMPA and kainate. Depolarization of the neurons with 55 mM potassium increased the release of [3H]d-aspartate by more than 10-fold. When the non-specific calcium-channel blockers cobalt or lanthanum were included in the stimulation buffer with potassium, the release of [3H]d-aspartate was decreased by about 40%. These results indicated that some of the released [3H]d-aspartate might originate from a vesicular pool. When AMPA was applied to the neurons, the release of [3H]d-aspartate was increased 2-fold and could not be prevented or decreased by addition of cobalt. Since AMPA has a rapid desensitizing effect on AMPA receptors, it was examined whether AMPA under non-desensitizing conditions was able to induce an increased release of [3H]d-aspartate as compared to the conditions of applying AMPA alone. The desensitization of AMPA receptors was blocked by 6-chloro-3,4-dihydro-3-(2-norbornen-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide). Under the non-desensitizing conditions, the AMPA-induced release of [3H]d-aspartate was highly enhanced showing about a 10-fold increase over basal release. Addition of cobalt or lanthanum did not decrease the amount of [3H]d-aspartate released, indicating that the release originated from a cytoplasmic pool. Kainate, which induces an almost non-desensitizing effect on AMPA receptors, showed similar results as observed for AMPA under non-desensitizing conditions. The NMDA receptor antagonist (5R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had only minor effects on the [3H]d-aspartate release induced by AMPA and kainate. Thus, the depolarization-induced release of [3H]d-aspartate from cultured GABAergic neurons appears to be caused mainly by reversal of the glutamate transporters.

Kainate receptor-mediated activation of the AP-1 transcription factor complex in cultured rat cerebellar granule cells

The sequence-specific DNA-binding activity of the AP-1 transcription factor complex was measured in cultured rat cerebellar granule cells by electrophoretic mobility shift assay. A low concentration of kainate (KA; 10 μM), but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 10 μM), enhanced DNA-binding of the AP-1 transcription factor in cultures pretreated with Concanavalin A (Con A), to prevent KA receptor desensitization. In the presence of cyclothiazide (an inhibitor of AMPA receptor desensitization), KA (10 μM) caused only a slight increase of AP-1 DNA-binding, in contrast to the threefold enhancement produced by AMPA (10 or 30 μM) or by a higher concentration of KA (30 μM), suggesting that the effect of KA, in the presence of Con A, is mediated by activation of putative KA receptors. To confirm this, the effects of the AMPA receptor-selective, non-competitive antagonist, 1-(4-aminophenyl)-3-methylcarbamoyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 53655; 50 μM), the mixed AMPA/KA receptor competitive antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM), and the AMPA and GluR5 KA receptor competitive antagonist, (-)(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid monohydrate (LY 326325; 100 μM), were examined on AMPA- and KA-induced AP-1 activation, respectively. Our results suggest that stimulation of native KA receptors is responsible for the observed KA-specific activation of the AP-1 transcription factor complex.

Depolarization-induced release of [3H]d-aspartate from GABAergic neurons caused by reversal of glutamate transporters

Cultured neocortical neurons, which predominantly consist of GABAergic neurons exhibit a pronounced stimulus-coupled GABA release. Since the cultures may contain a small population of glutamatergic neurons and the GABAergic neurons have a high content of glutamate it was of interest to examine if glutamate in addition to γ-aminobutyric acid (GABA) could be released from these cultures. The neurons were preloaded with [3H]d-aspartate and subsequently its release was followed during depolarization induced by a high potassium concentration or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonists, AMPA and kainate. Depolarization of the neurons with 55 mM potassium increased the release of [3H]d-aspartate by more than 10-fold. When the non-specific calcium-channel blockers cobalt or lanthanum were included in the stimulation buffer with potassium, the release of [3H]d-aspartate was decreased by about 40%. These results indicated that some of the released [3H]d-aspartate might originate from a vesicular pool. When AMPA was applied to the neurons, the release of [3H]d-aspartate was increased 2-fold and could not be prevented or decreased by addition of cobalt. Since AMPA has a rapid desensitizing effect on AMPA receptors, it was examined whether AMPA under non-desensitizing conditions was able to induce an increased release of [3H]d-aspartate as compared to the conditions of applying AMPA alone. The desensitization of AMPA receptors was blocked by 6-chloro-3,4-dihydro-3-(2-norbornen-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide). Under the non-desensitizing conditions, the AMPA-induced release of [3H]d-aspartate was highly enhanced showing about a 10-fold increase over basal release. Addition of cobalt or lanthanum did not decrease the amount of [3H]d-aspartate released, indicating that the release originated from a cytoplasmic pool. Kainate, which induces an almost non-desensitizing effect on AMPA receptors, showed similar results as observed for AMPA under non-desensitizing conditions. The NMDA receptor antagonist (5R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had only minor effects on the [3H]d-aspartate release induced by AMPA and kainate. Thus, the depolarization-induced release of [3H]d-aspartate from cultured GABAergic neurons appears to be caused mainly by reversal of the glutamate transporters.