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Gary Lynch - One of the best experts on this subject based on the ideXlab platform.
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Interactions between recording technique and AMPA receptor modulators.
Brain research, 2002Co-Authors: Bin Lin, Laura Lee Colgin, Fernando A. Brucher, Amy Christine Arai, Gary LynchAbstract:Whole cell recording (EPSCs) and extracellular recording (field EPSPs) were compared in hippocampal field CA1 with regard to the effects of experimental treatments that increase AMPA receptor gated currents. Cyclothiazide, which maintains AMPA receptors in the sensitized state, caused a rapid and pronounced increase in EPSCs but only minor changes in field EPSPs. This difference was evident in recordings carried out at 22 and 32 degrees C and with different solutions in the clamp pipette. The larger effect of Cyclothiazide on EPSCs was unaffected by blockade of GABA and NMDA receptors. Two-dimensional current source density analyses derived from 64 recording sites were used to provide extracellular estimates of AMPA receptor mediated synaptic currents. With this method, Cyclothiazide again had much smaller effects than were obtained with whole cell clamp. Differences between whole cell and extracellular recordings were present, although not as pronounced, for the ampakines, a class of drugs that slow both deactivation and desensitization of AMPA receptors. Additionally, increases in synaptic responses produced by frequency facilitation, a manipulation that enhances the number of bound receptors, were not qualitatively different between recording techniques. These results support the conclusion that the whole cell clamp technique may alter AMPA receptors in such a way as to increase the relative importance of desensitization.
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Interactions between recording technique and AMPA receptor modulators.
Brain Research, 2002Co-Authors: Bin Lin, Amy Arai, Laura Lee Colgin, Fernando A. Brucher, Gary LynchAbstract:Whole cell recording (EPSCs) and extracellular recording (field EPSPs) were compared in hippocampal field CA1 with regard to the effects of experimental treatments that increase AMPA receptor gated currents. Cyclothiazide, which maintains AMPA receptors in the sensitized state, caused a rapid and pronounced increase in EPSCs but only minor changes in field EPSPs. This difference was evident in recordings carried out at 22 and 32 °C and with different solutions in the clamp pipette. The larger effect of Cyclothiazide on EPSCs was unaffected by blockade of GABA and NMDA receptors. Two-dimensional current source density analyses derived from 64 recording sites were used to provide extracellular estimates of AMPA receptor mediated synaptic currents. With this method, Cyclothiazide again had much smaller effects than were obtained with whole cell clamp. Differences between whole cell and extracellular recordings were present, although not as pronounced, for the ampakines, a class of drugs that slow both deactivation and desensitization of AMPA receptors. Additionally, increases in synaptic responses produced by frequency facilitation, a manipulation that enhances the number of bound receptors, were not qualitatively different between recording techniques. These results support the conclusion that the whole cell clamp technique may alter AMPA receptors in such a way as to increase the relative importance of desensitization.
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Effects of the potent ampakine CX614 on hippocampal and recombinant AMPA receptors: interactions with Cyclothiazide and GYKI 52466.
Molecular pharmacology, 2000Co-Authors: Amy Arai, Markus Kessler, Gary Rogers, Gary LynchAbstract:R,S-a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor up-modulators of the benzamide type (“ampakines”) have previously been shown to enhance excitatory synaptic transmission in vivo and in vitro and AMPA receptor currents in excised patches. The present study analyzed the effects of an ampakine (CX614; 2H,3H,6aH-pyrrolidino[20,1039,29]1,3-oxazino[69,59-5,4]benzo[e]1,4-dioxan-10-one) that belongs to a benzoxazine subgroup characterized by greater structural rigidity and higher potency. CX614 enhanced the size (amplitude and duration) of field excitatory postsynaptic potentials in hippocampal slices and autaptically evoked excitatory postsynaptic currents in neuronal cultures with EC50 values of 20 to 40 mM. The compound blocked desensitization (EC50 5 44 mM) and slowed deactivation of responses to glutamate by a factor of 8.4 in excised patches. Currents through homomeric, recombinant AMPA receptors were enhanced with EC50 values that did not differ greatly across GluR1‐3 flop subunits (19 ‐37 mM) but revealed slightly lower potency at corresponding flip variants. Competition experiments using modulation of [ 3 H]fluorowillardiine binding suggested that CX614 and Cyclothiazide share a common binding site but Cyclothiazide seems to bind to an additional site not recognized by the ampakine. CX614 did not reverse the effect of GYKI 52466 on responses to brief glutamate pulses, which indicates that they act through separate sites, a conclusion that was confirmed in binding experiments. In sum, these results extend prior evidence that ampakines are effective in enhancing synaptic responses, most likely by slowing deactivation, and that their effects are exerted through sites that are only in part shared with other modulators.
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AMPA receptor desensitization modulates synaptic responses induced by repetitive afferent stimulation in hippocampal slices.
Brain research, 1998Co-Authors: Amy Arai, Gary LynchAbstract:Abstract In patches excised from CA1 pyramidal cells, peak amplitudes of currents evoked by brief glutamate pulses grew progressively smaller over a series of high-frequency pulses. This decline was eliminated by Cyclothiazide, a drug previously shown to block AMPA receptor desensitization. In hippocampal slices, synaptically evoked bursts exhibited an increase from the first to the second response, presumably due to facilitation of transmitter release, but the subsequent responses gradually declined in amplitude. Cyclothiazide attenuated or reversed this decline; after normalization to the first response, the amplitudes of the later responses to a 50 Hz series of afferent stimulation were increased by 20–25% in regular recording medium and by as much as 40% when transmitter release was enhanced in a high-calcium medium. The effect of Cyclothiazide was greatly diminished when the stimulation frequency was reduced to 33 or 25 Hz. Comparable results were obtained in slices in which NMDA, GABA A , and GABA B receptors were blocked. The ampakine drug CX516 which has only a minor influence on desensitization kinetics did not differentially facilitate the later responses to high-frequency afferent stimulation. These results suggest that the desensitization of AMPA receptors contributes importantly to synaptic activity when afferents are repetitively activated at high-frequency.
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Effects of Cyclothiazide on synaptic responses in slices of adult and neonatal rat hippocampus.
Neuroreport, 1994Co-Authors: John Larson, Randy A. Hall, Gary LynchAbstract:The effects of Cyclothiazide, a drug that blocks AMPA receptor desensitization, on synaptic responses were studied in field CA1 of hippocampal slices from adult and neonatal rats. Cyclothiazide (100 microM) reliably increased AMPA receptor-mediated field EPSP decay times in adult slices but only after prolonged (60-120 min) applications. In neonatal slices, 30 min applications of Cyclothiazide were sufficient to produce large and long-lasting increases in response decay times. Since the effects of Cyclothiazide on AMPA receptor binding properties were similar in adult and neonatal forebrain membranes, the data indicate that the slow action of the drug in adult slices is probably due to diffusion barriers. The prolongation of EPSP decay by Cyclothiazide suggests that AMPA receptor-mediated synaptic responses in hippocampus are terminated by receptor desensitization.
Mark L Mayer - One of the best experts on this subject based on the ideXlab platform.
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ampa receptor flip flop mutants affecting deactivation desensitization and modulation by Cyclothiazide aniracetam and thiocyanate
The Journal of Neuroscience, 1996Co-Authors: Kathryn M Partin, Mark W Fleck, Mark L MayerAbstract:AMPA receptor GluRA subunits with mutations at position 750, a residue shown previously to control allosteric regulation by Cyclothiazide, were analyzed for modulation of deactivation and desensitization by Cyclothiazide, aniracetam, and thiocyanate. Point mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val, and Tyr were constructed in GluRAflip. The last four of these mutants were not functional; S750D was active only in the presence of Cyclothiazide, and the remaining mutants exhibited altered rates of deactivation and desensitization for control responses to glutamate, and showed differential modulation by Cyclothiazide and aniracetam. Results from kinetic analysis are consistent with aniracetam and Cyclothiazide acting via distinct mechanisms. Our experiments demonstrate for the first time the functional importance of residue 750 in regulating intrinsic channel-gating kinetics and emphasize the biological significance of alternative splicing in the M3–M4 extracellular loop.
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AMPA Receptor Flip/Flop Mutants Affecting Deactivation, Desensitization, and Modulation by Cyclothiazide, Aniracetam, and Thiocyanate
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1996Co-Authors: Kathryn M Partin, Mark W Fleck, Mark L MayerAbstract:AMPA receptor GluRA subunits with mutations at position 750, a residue shown previously to control allosteric regulation by Cyclothiazide, were analyzed for modulation of deactivation and desensitization by Cyclothiazide, aniracetam, and thiocyanate. Point mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val, and Tyr were constructed in GluRAflip. The last four of these mutants were not functional; S750D was active only in the presence of Cyclothiazide, and the remaining mutants exhibited altered rates of deactivation and desensitization for control responses to glutamate, and showed differential modulation by Cyclothiazide and aniracetam. Results from kinetic analysis are consistent with aniracetam and Cyclothiazide acting via distinct mechanisms. Our experiments demonstrate for the first time the functional importance of residue 750 in regulating intrinsic channel-gating kinetics and emphasize the biological significance of alternative splicing in the M3–M4 extracellular loop.
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AMPA receptor heterogeneity in rat hippocampal neurons revealed by differential sensitivity to Cyclothiazide.
Journal of neurophysiology, 1996Co-Authors: M. W. Fleck, R. Bahring, D. K. Patneau, Mark L MayerAbstract:1. The kinetics of onset of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization by glutamate, and the extent of attenuation of AMPA receptor desensitization by Cyclothiazide, showed pronounced cell-to-cell variation in cultures of rat hippocampal neurons. Cultures prepared from area CA1 stratum radiatum tended to show weaker modulation by Cyclothiazide than cultures prepared from the whole hippocampus. 2. Kinetic analysis of concentration jump responses to glutamate revealed multiple populations of receptors with fast (approximately 400 ms), intermediate (approximately 2-4 s), and slow (> 20 s) time constants for recovery from modulation by Cyclothiazide. The amplitudes of these components varied widely between cells, suggesting the existence of at least three populations of AMPA receptor subtypes, the relative density of which varied from cell to cell. 3. The complex patterns of sensitivity to Cyclothiazide seen in hippocampal neurons could be reconstituted by assembly of recombinant AMPA receptor subunits generated from cDNAs encoding the flip (i) and flop (o) splice variants of the GluR-A and GluR-B subunits. Recovery from modulation by Cyclothiazide was slower for GluR-AiBi and GluR-AoBi than for GluR-AiBo and GluR-AoBo. 4. Coexpression of the flip and flop splice variants of GluR-A, in the absence of GluR-B, revealed that heteromeric AMPA receptors with intermediate sensitivity to Cyclothiazide, similar to responses observed for the combinations GluR-AoBi or GluR-AiBo, could be generated independently of the presence of the GluR-B subunit. However, recovery from modulation by Cyclothiazide was twofold slower for GluR-AiBi than for homomeric GluR-Ai, indicating that the GluR-A and GluR-B subunits are not functionally equivalent in controlling sensitivity to Cyclothiazide. 5. These results demonstrate that AMPA receptors expressed in hippocampal neurons are assembled in a variety of subunit and splice variant combinations that might serve as a mechanism to fine-tune the kinetics of synaptic transmission.
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Negative allosteric modulation of wild-type and mutant AMPA receptors by GYKI 53655.
Molecular pharmacology, 1996Co-Authors: Kathryn M Partin, Mark L MayerAbstract:Benzothiadiazides such as Cyclothiazide potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor responses, whereas 2,3-benzodiazepines such as 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4 -dihydro- 5H-2,3-benzodiazepine (GYKI 53655) act as noncompetitive antagonists; both drugs act through allosteric modulation. Controversy exists as to whether Cyclothiazide and GYKI 53655 act at a common site. Recent mutational analysis has led to the identification of a serine residue in flip splice variants that is critical for directing the interaction of Cyclothiazide with AMPA receptors. We tested whether the mutation of this residue to glutamine, which abolishes potentiation by Cyclothiazide, can in addition block antagonism by 2,3-benzodiazepines, as would be predicted for action at a common site. We found that the S to Q mutation does not alter antagonism by 2,3-benzodiazepines, suggesting that the molecular determinants directing the interaction between GYKI 53655 and AMPA receptors are not identical to those controlling sensitivity to Cyclothiazide. Additional support for this was obtained from analysis of the responses of AMPA receptor flip/flop splice variants, which, despite differences in equilibrium desensitization and sensitivity to Cyclothiazide, show only small differences in sensitivity to 2,3-benzodiazepines. Furthermore, introduction of the flip exon from GluRA into GluR6, conferred sensitivity to Cyclothiazide but did not increase sensitivity to 2,3-benzodiazepines. Of interest, experiments with native AMPA receptors generated from hippocampal and forebrain poly(A)+ mRNA revealed greater sensitivity to 2,3-benzodiazepines than receptors generated by expression of recombinant AMPA receptors, possibly indicating the existence of an unidentified accessory protein or novel receptor subunit.
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Structural determinants of allosteric regulation in alternatively spliced AMPA receptors
Neuron, 1995Co-Authors: Kathryn M Partin, Derek Bowie, Mark L MayerAbstract:Abstract The flip and flop splice variants of AMPA receptors show strikingly different sensitivity to allosteric regulation by Cyclothiazide; heteromers assembled from GluR-A and GIuR-B also exhibit splice variant-dependent differences in efficacy for activation by glutamate and kainate. The sensitivity for attenuation of desensitization by Cyclothiazide for homomeric GIuR-A was solely dependent upon exchange of Ser-750 (flip) and Asn-750 (flop), and was unaffected by mutagenesis of other divergent residues. In contrast, substantial alteration of the relative efficacy of glutamate versus kainate required mutation of multiple residues in the flip/flop region. Modulation by Cyclothiazide was abolished by mutation of Ser-750 to Gin, the residue found at the homologous site in kainate-preferring subunits, whereas introduction of Ser at this site in GIuR6 imparted sensitivity to Cyclothiazide.
Kathryn M Partin - One of the best experts on this subject based on the ideXlab platform.
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Structural and Functional Analysis of Two New Positive Allosteric Modulators of GluA2 Desensitization and Deactivation
Molecular pharmacology, 2011Co-Authors: David E. Timm, Morris Benveniste, Autumn M. Weeks, Eric S. Nisenbaum, Kathryn M PartinAbstract:At the dimer interface of the extracellular ligand-binding domain of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors a hydrophilic pocket is formed that is known to interact with two classes of positive allosteric modulators, represented by Cyclothiazide and the ampakine 2H,3H,6aH-pyrrolidino(2,1–3′,2′)1,3-oxazino(6′,5′-5,4)benzo(e)1,4-dioxan-10-one (CX614). Here, we present structural and functional data on two new positive allosteric modulators of AMPA receptors, phenyl-1,4-bis-alkylsulfonamide (CMPDA) and phenyl-1,4-bis-carboxythiophene (CMPDB). Crystallographic data show that these compounds bind within the modulator-binding pocket and that substituents of each compound overlap with distinct moieties of Cyclothiazide and CX614. The goals of the present study were to determine 1) the degree of modulation by CMPDA and CMPDB of AMPA receptor deactivation and desensitization; 2) whether these compounds are splice isoform-selective; and 3) whether predictions of mechanism of action could be inferred by comparing molecular interactions between the ligand-binding domain and each compound with those of Cyclothiazide and CX614. CMPDB was found to be more isoform-selective than would be predicted from initial binding assays. It is noteworthy that these new compounds are both more potent and more effective and may be more clinically relevant than the AMPA receptor modulators described previously.
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The contributions of GluR2 to allosteric modulation of AMPA receptors.
Neuropharmacology, 2000Co-Authors: Julie L. S. Cotton, Kathryn M PartinAbstract:Abstract Native AMPA receptor complexes in the CNS are composed of hetero-oligomers of the GluR1-4 subunits, and generally contain the GluR2 subunit. To determine the contributions of GluR2 to pharmacological properties of receptor complexes, the effect of hetero-oligomerization with GluR2 on allosteric modulation of recombinant AMPA receptors was studied. The study of homo-oligomeric GluR2 was facilitated with a site-directed mutant of the pore, GluR2(R607Q), which allowed robust currents from this normally low-conducting subunit. The efficacy of the allosteric modulators was tested on homo-oligomeric GluR1-4, and then compared with hetero-oligomeric GluR1/GluR2, GluR3/GluR2 and GluR4/GluR2. Two selective allosteric modulators were tested, a positive modulator, Cyclothiazide, and a negative modulator, LY300164. The results show that the pharmacological properties of homo-oligomeric GluR2 are not significantly different from those of GluR1, GluR3 or GluR4. The apparent affinity of Cyclothiazide is not significantly changed upon hetero-oligomerization. However, the extent of potentiation of kainate responses by Cyclothiazide is significantly decreased upon hetero-oligomerization. Hetero-oligomerization increases the apparent affinity of LY300164, a (−) isomer of the 2,3-benzodiazepine LY293606. These data indicate that although GluR2 has a dominant effect on the permeation properties, this subunit does not have a similarly dominant effect on pharmacological properties of native receptors. However, the state of hetero-oligomerization can alter the pharmacological properties of AMPA receptors.
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ampa receptor flip flop mutants affecting deactivation desensitization and modulation by Cyclothiazide aniracetam and thiocyanate
The Journal of Neuroscience, 1996Co-Authors: Kathryn M Partin, Mark W Fleck, Mark L MayerAbstract:AMPA receptor GluRA subunits with mutations at position 750, a residue shown previously to control allosteric regulation by Cyclothiazide, were analyzed for modulation of deactivation and desensitization by Cyclothiazide, aniracetam, and thiocyanate. Point mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val, and Tyr were constructed in GluRAflip. The last four of these mutants were not functional; S750D was active only in the presence of Cyclothiazide, and the remaining mutants exhibited altered rates of deactivation and desensitization for control responses to glutamate, and showed differential modulation by Cyclothiazide and aniracetam. Results from kinetic analysis are consistent with aniracetam and Cyclothiazide acting via distinct mechanisms. Our experiments demonstrate for the first time the functional importance of residue 750 in regulating intrinsic channel-gating kinetics and emphasize the biological significance of alternative splicing in the M3–M4 extracellular loop.
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AMPA Receptor Flip/Flop Mutants Affecting Deactivation, Desensitization, and Modulation by Cyclothiazide, Aniracetam, and Thiocyanate
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1996Co-Authors: Kathryn M Partin, Mark W Fleck, Mark L MayerAbstract:AMPA receptor GluRA subunits with mutations at position 750, a residue shown previously to control allosteric regulation by Cyclothiazide, were analyzed for modulation of deactivation and desensitization by Cyclothiazide, aniracetam, and thiocyanate. Point mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val, and Tyr were constructed in GluRAflip. The last four of these mutants were not functional; S750D was active only in the presence of Cyclothiazide, and the remaining mutants exhibited altered rates of deactivation and desensitization for control responses to glutamate, and showed differential modulation by Cyclothiazide and aniracetam. Results from kinetic analysis are consistent with aniracetam and Cyclothiazide acting via distinct mechanisms. Our experiments demonstrate for the first time the functional importance of residue 750 in regulating intrinsic channel-gating kinetics and emphasize the biological significance of alternative splicing in the M3–M4 extracellular loop.
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Negative allosteric modulation of wild-type and mutant AMPA receptors by GYKI 53655.
Molecular pharmacology, 1996Co-Authors: Kathryn M Partin, Mark L MayerAbstract:Benzothiadiazides such as Cyclothiazide potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor responses, whereas 2,3-benzodiazepines such as 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4 -dihydro- 5H-2,3-benzodiazepine (GYKI 53655) act as noncompetitive antagonists; both drugs act through allosteric modulation. Controversy exists as to whether Cyclothiazide and GYKI 53655 act at a common site. Recent mutational analysis has led to the identification of a serine residue in flip splice variants that is critical for directing the interaction of Cyclothiazide with AMPA receptors. We tested whether the mutation of this residue to glutamine, which abolishes potentiation by Cyclothiazide, can in addition block antagonism by 2,3-benzodiazepines, as would be predicted for action at a common site. We found that the S to Q mutation does not alter antagonism by 2,3-benzodiazepines, suggesting that the molecular determinants directing the interaction between GYKI 53655 and AMPA receptors are not identical to those controlling sensitivity to Cyclothiazide. Additional support for this was obtained from analysis of the responses of AMPA receptor flip/flop splice variants, which, despite differences in equilibrium desensitization and sensitivity to Cyclothiazide, show only small differences in sensitivity to 2,3-benzodiazepines. Furthermore, introduction of the flip exon from GluRA into GluR6, conferred sensitivity to Cyclothiazide but did not increase sensitivity to 2,3-benzodiazepines. Of interest, experiments with native AMPA receptors generated from hippocampal and forebrain poly(A)+ mRNA revealed greater sensitivity to 2,3-benzodiazepines than receptors generated by expression of recombinant AMPA receptors, possibly indicating the existence of an unidentified accessory protein or novel receptor subunit.
Maurizio Raiteri - One of the best experts on this subject based on the ideXlab platform.
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Extracellular protons differentially potentiate the responses of native AMPA receptor subtypes regulating neurotransmitter release.
British journal of pharmacology, 2005Co-Authors: Anna Pittaluga, Daniela Segantini, Marco Feligioni, Maurizio RaiteriAbstract:The effects of pH changes on the basal and evoked release of [3H]noradrenaline ([3H]NA) and [3H]5-hydrohytryptamine ([3H]5-HT) from hippocampal synaptosomes and of [3H]dopamine ([3H]DA) and [3H]acetylcholine ([3H]ACh) from striatal and cortical synaptosomes were investigated in rat brain. Changing pH between 6.4 and 8.0 did not affect the spontaneous release of the four [3H]neurotransmitters; alkalinization to pH 8.8 significantly enhanced release. Acidification to pH 6.4 augmented the AMPA-evoked overflows of [3H]NA, [3H]5-HT and [3H]DA, but not that of [3H]ACh. In contrast, lowering pH to 6.4 decreased the K+-evoked overflows of [3H]NA, [3H]5-HT, [3H]DA and [3H]ACh. AMPA released transmitters in a Ca2+-dependent, exocytotic manner since its effects, at pH 7.4 or 6.4, were abolished by omitting external Ca2+ or by depleting vesicular transmitter stores with bafilomycin A1. AMPA did not evoke carrier-mediated release because the uptake blockers nisoxetine, 6-nitroquipazine, GBR12909 and hemicholinium-3 could not inhibit the AMPA-induced release of [3H]NA, [3H]5-HT, [3H]DA and [3H]ACh. Extraterminal acidification to pH 6.4 prevented the potentiating effect of Cyclothiazide on the AMPA-evoked release of [3H]NA, [3H]DA and [3H]5-HT, whereas the proton-insensitive AMPA-evoked release of [3H]ACh, previously found to be Cyclothiazide-insensitive at pH 7.4 was Cyclothiazide-resistant also at pH 6.4. To conclude, the Cyclothiazide-sensitive AMPA receptors mediating release of NA, 5-HT and DA, but not the Cyclothiazide-insensitive AMPA receptors mediating the release of ACh, become more responsive when external pH is lowered to pathophysiologically relevant values. The results with Cyclothiazide suggest that H+ ions may prevent desensitization of some AMPA receptor subtypes. Keywords: pH, AMPA receptor subtypes, noradrenaline release, dopamine release, 5-HT release, acetylcholine release, receptor desensitization Introduction AMPA/kainate receptors mediate fast excitatory synaptic transmission exerting relevant roles in development, neuroplasticity and excitotoxicity (for reviews see Hollmann & Heinemann, 1994; Bettler & Mulle, 1995; Bleakman & Lodge, 1998, Dingledine et al., 1999). Evidence has been provided that rat hippocampal noradrenergic and serotonergic terminals, as well as striatal dopaminergic and cortical cholinergic nerve endings, are endowed with AMPA/kainate receptors whose activation causes Ca2+-dependent exocytotic-like neurotransmitter release. The pharmacological characterization of these receptors, obtained with a number of receptor antagonists, suggests that they all belong to the AMPA-preferring type, but appear to represent four pharmacologically distinct subtypes (Ghersi et al., 2003). It is well known that AMPA receptors undergo desensitization, which can be prevented by drugs like Cyclothiazide (Partin et al., 1993; Bettler & Mulle, 1995; Bleakman & Lodge, 1998); the existence of endogenous factors mimicked by these Cyclothiazide-like compounds is unknown. Glutamate receptors of the NMDA type exhibit decreased function when extracellular pH is lowered to pathophysiologically relevant values (Traynelis & Cull-Candy, 1990; Traynelis et al., 1995; Pittaluga et al., 2001). In contrast, the relationships between acidification and function of AMPA receptors are controversial. These receptors have been reported to be unaffected by protons at pH 6.5 (see Traynelis, 1998). On the other hand, at pH values lower than 6.5, which are of more biochemical than pathophysiological interest, the responses to AMPA were found to be inhibited (Christensen & Hida, 1990; Traynelis & Cull-Candy, 1991; Traynelis et al., 1995; Lei et al., 2001). Recent results, obtained in primary cell cultures from murine brain, show that extracellular acidification could inhibit AMPA receptor-mediated responses (Ihle & Patneau, 2000), but potentiated AMPA receptor-mediated excitotoxicity (McDonald et al., 1998). The aim of the present work was to investigate whether the function of native AMPA receptors mediating enhancement of noradrenaline (NA), dopamine (DA), 5-HT and ACh release in nerve endings isolated from adult rat brain is sensitive to extracellular pH changes. We here find that protons potentiate the release evoked by AMPA from the vesicular pool of NA, DA and 5-HT, leaving unaffected the AMPA-evoked release of ACh. Moreover, our results show that H+ ions strengthen the function of release-regulating AMPA receptors previously found to be Cyclothiazide-sensitive, but not that of receptors found to be insensitive to the drug (Pittaluga et al., 1997).
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Aniracetam, 1-BCP and Cyclothiazide differentially modulate the function of NMDA and AMPA receptors mediating enhancement of noradrenaline release in rat hippocampal slices.
Naunyn-Schmiedeberg's archives of pharmacology, 1999Co-Authors: Anna Pittaluga, Andrea Bonfanti, Daniela Arvigo, Maurizio RaiteriAbstract:Aniracetam, 1-(1,3-benzodioxol-5-yl-carbo-nyl)piperidine (1-BCP) and Cyclothiazide, three compounds considered to enhance cognition through modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, were evaluated in the ‘kynurenate test’, a biochemical assay in which some nootropics have been shown to prevent the antagonism by kynurenic acid of the N-methyl-d-aspartate (NMDA)-evoked [3H]noradrenaline ([3H]NA) release from rat hippocampal slices. Aniracetam attenuated the kynurenate (100 μM) antagonism of the [3H]NA release elicited by 100 μM NMDA with high potency (EC50≤0.1 μM). Cyclothiazide and 1-BCP were about 10 and 100 times less potent than aniracetam, respectively. The effect of aniracetam persisted in the presence of the AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) added at 5 μM, a concentration that did not affect NMDA receptors; in contrast, NBQX reduced the effect of 1-BCP and abolished that of Cyclothiazide. The AMPA-evoked release of [3H]NA from hippocampal slices or synaptosomes was enhanced by Cyclothiazide, less potently by 1-BCP and weakly by aniracetam. High concentrations of kynurenate (1 mM) antagonized the AMPA-evoked [3H]NA release in slices; this antagonism was attenuated by 1 μM cyclo-thiazide and reversed to an enhancement of AMPA-evoked [3H]NA release by 10 μM of the drug, but was insensitive to 1-BCP or aniracetam. It is concluded that aniracetam exerts a dual effect on glutamatergic transmission: modulation of NMDA receptor function at nanomolar concentrations, and modulation of AMPA receptors at high micromolar concentrations. As to Cyclothiazide and 1-BCP, our data concur with the idea that both compounds largely act through AMPA receptors, although an NMDA component may be involved in the effect of 1-BCP.
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Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function
Naunyn-Schmiedeberg's archives of pharmacology, 1997Co-Authors: Anna Pittaluga, Andrea Bonfanti, Maurizio RaiteriAbstract:The release of tritium from rat hippocampal synaptosomes prelabeled with [3H]noradrenaline ([3H]NA) or [3H]5-hydroxytryptamine ([3H]5-HT) and from rat neocortex synaptosomes prelabeled with [3H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 µM AMPA were, in all cases, almost totally dependent on external Ca2+ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When Cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 µM) concomitantly with 100 µM AMPA or kainate, the EAA-evoked release of [3H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 µM) on the release of [3H]NA evoked by AMPA or kainate. The effect of Cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, Cyclothiazide potentiated the release of [3H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither Cyclothiazide (1 or 10 µM) nor concanavalin A (3 or 10 µM) could affect the release of [3H]ACh or endogenous GABA provoked by 100 µM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with Cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 µM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 µM) pulse. When hippocampal synaptosomes prelabeled with [3H]NA were subjected to three subsequent AMPA (100 µM) stimuli (S1, S2 and S3), the release of [3H]NA decreased dramatically from S1 to S3 (S3/S1 = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 µM Cyclothiazide (S3/S1 = 0.36 ± 0.06). This value did not differ from the S3/S1 ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K+. The release evoked by high-K+ was insensitive to Cyclothiazide. Finally, the effect of AMPA on the release of [3H]ACh did not respond to Cyclothiazide also during three subsequent stimuli with 100 µM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies.
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Desensitization of AMPA receptors and AMPA‐NMDA receptor interaction: an in vivo cyclic GMP microdialysis study in rat cerebellum
British journal of pharmacology, 1996Co-Authors: Ernesto Fedele, Maurizio RaiteriAbstract:1. Desensitization is an important characteristic of glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type. 2. Stimulation of N-methyl-D-aspartate (NMDA) or AMPA receptors in cerebellum results in increased production of cyclic GMP. We have investigated AMPA receptor desensitization in vivo by monitoring extracellular cyclic GMP during intracerebellar microdialysis in conscious unrestrained adult rats. 3. Local infusion of AMPA (10 to 100 microM) caused dose-related elevations of cyclic GMP levels. The effect of AMPA was prevented by the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NOARG). 4. In the absence of AMPA, DNQX lowered the basal levels of cyclic GMP whereas the NMDA receptor channel antagonist dizocilpine (MK-801) was ineffective. 5. Cyclothiazide, a blocker of AMPA receptor desensitization, potentiated the cyclic GMP response to exogenous AMPA. Moreover, Cyclothiazide (100-300 microM) produced on its own dose-dependent elevations of extracellular cyclic GMP. The Cyclothiazide-induced response was prevented not only by DNQX but also by MK-801. 6. While the cyclic GMP response elicited by AMPA was totally insensitive to MK-801, the response produced by AMPA (10 microM) plus Cyclothiazide (30 microM) was strongly attenuated by the NMDA receptor antagonist (30 microM). 7. The results suggest that (a) AMPA receptors linked to the NO-cyclic GMP pathway in the cerebellum can undergo desensitization in vivo during exposure to exogenous AMPA; Cyclothiazide inhibits such desensitization; (b) AMPA receptors (but not NMDA receptors) are 'tonically' activated and kept in a partly desensitized state by endogenous glutamate; (c) if Cyclothiazide is present, activation of AMPA receptors may permit endogenous activation of NMDA receptors.
Amy Arai - One of the best experts on this subject based on the ideXlab platform.
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Interactions between recording technique and AMPA receptor modulators.
Brain Research, 2002Co-Authors: Bin Lin, Amy Arai, Laura Lee Colgin, Fernando A. Brucher, Gary LynchAbstract:Whole cell recording (EPSCs) and extracellular recording (field EPSPs) were compared in hippocampal field CA1 with regard to the effects of experimental treatments that increase AMPA receptor gated currents. Cyclothiazide, which maintains AMPA receptors in the sensitized state, caused a rapid and pronounced increase in EPSCs but only minor changes in field EPSPs. This difference was evident in recordings carried out at 22 and 32 °C and with different solutions in the clamp pipette. The larger effect of Cyclothiazide on EPSCs was unaffected by blockade of GABA and NMDA receptors. Two-dimensional current source density analyses derived from 64 recording sites were used to provide extracellular estimates of AMPA receptor mediated synaptic currents. With this method, Cyclothiazide again had much smaller effects than were obtained with whole cell clamp. Differences between whole cell and extracellular recordings were present, although not as pronounced, for the ampakines, a class of drugs that slow both deactivation and desensitization of AMPA receptors. Additionally, increases in synaptic responses produced by frequency facilitation, a manipulation that enhances the number of bound receptors, were not qualitatively different between recording techniques. These results support the conclusion that the whole cell clamp technique may alter AMPA receptors in such a way as to increase the relative importance of desensitization.
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Effects of the potent ampakine CX614 on hippocampal and recombinant AMPA receptors: interactions with Cyclothiazide and GYKI 52466.
Molecular pharmacology, 2000Co-Authors: Amy Arai, Markus Kessler, Gary Rogers, Gary LynchAbstract:R,S-a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor up-modulators of the benzamide type (“ampakines”) have previously been shown to enhance excitatory synaptic transmission in vivo and in vitro and AMPA receptor currents in excised patches. The present study analyzed the effects of an ampakine (CX614; 2H,3H,6aH-pyrrolidino[20,1039,29]1,3-oxazino[69,59-5,4]benzo[e]1,4-dioxan-10-one) that belongs to a benzoxazine subgroup characterized by greater structural rigidity and higher potency. CX614 enhanced the size (amplitude and duration) of field excitatory postsynaptic potentials in hippocampal slices and autaptically evoked excitatory postsynaptic currents in neuronal cultures with EC50 values of 20 to 40 mM. The compound blocked desensitization (EC50 5 44 mM) and slowed deactivation of responses to glutamate by a factor of 8.4 in excised patches. Currents through homomeric, recombinant AMPA receptors were enhanced with EC50 values that did not differ greatly across GluR1‐3 flop subunits (19 ‐37 mM) but revealed slightly lower potency at corresponding flip variants. Competition experiments using modulation of [ 3 H]fluorowillardiine binding suggested that CX614 and Cyclothiazide share a common binding site but Cyclothiazide seems to bind to an additional site not recognized by the ampakine. CX614 did not reverse the effect of GYKI 52466 on responses to brief glutamate pulses, which indicates that they act through separate sites, a conclusion that was confirmed in binding experiments. In sum, these results extend prior evidence that ampakines are effective in enhancing synaptic responses, most likely by slowing deactivation, and that their effects are exerted through sites that are only in part shared with other modulators.
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The norbornenyl moiety of Cyclothiazide determines the preference for flip-flop variants of AMPA receptor subunits.
Neuroscience letters, 2000Co-Authors: Markus Kessler, Gary Rogers, Amy AraiAbstract:Cyclothiazide and two analogs in which the norbornenyl part was replaced with a cyclohexyl or a cyclohexenyl moiety were examined with regard to their preference for flop vs. flip splice variants of the (±)-αamino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits GluR2, 3 and 4. The studies were carried out by measuring the effects of the drugs on the binding of [3H]AMPA or [3H]fluorowillardiine to membranes from HEK293 cells that stably express the AMPA receptor subunits. Cyclothiazide had four to nine times lower EC50 values at flip than at flop receptors, as previously reported. In contrast, the two analogs showed little discrimination for GluR3 or GluR4 splice variants and a clear preference for the flop variant in the case of GluR2. These results indicate that it is the norbornenyl component of Cyclothiazide which confers the selectivity vis-a-vis flip-flop variants.
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AMPA receptor desensitization modulates synaptic responses induced by repetitive afferent stimulation in hippocampal slices.
Brain research, 1998Co-Authors: Amy Arai, Gary LynchAbstract:Abstract In patches excised from CA1 pyramidal cells, peak amplitudes of currents evoked by brief glutamate pulses grew progressively smaller over a series of high-frequency pulses. This decline was eliminated by Cyclothiazide, a drug previously shown to block AMPA receptor desensitization. In hippocampal slices, synaptically evoked bursts exhibited an increase from the first to the second response, presumably due to facilitation of transmitter release, but the subsequent responses gradually declined in amplitude. Cyclothiazide attenuated or reversed this decline; after normalization to the first response, the amplitudes of the later responses to a 50 Hz series of afferent stimulation were increased by 20–25% in regular recording medium and by as much as 40% when transmitter release was enhanced in a high-calcium medium. The effect of Cyclothiazide was greatly diminished when the stimulation frequency was reduced to 33 or 25 Hz. Comparable results were obtained in slices in which NMDA, GABA A , and GABA B receptors were blocked. The ampakine drug CX516 which has only a minor influence on desensitization kinetics did not differentially facilitate the later responses to high-frequency afferent stimulation. These results suggest that the desensitization of AMPA receptors contributes importantly to synaptic activity when afferents are repetitively activated at high-frequency.
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Effect of Cyclothiazide on binding properties of AMPA-type glutamate receptors: lack of competition between Cyclothiazide and GYKI 52466.
Molecular pharmacology, 1996Co-Authors: Markus Kessler, Amy Arai, Alex Quan, G LynchAbstract:The effects of Cyclothiazide on the properties of (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors were studied using equilibrium binding techniques and interactions with other compounds known to modulate the receptors. Cyclothiazide caused a reduction in [3H]AMPA binding in assays carried out in the presence of thiocyanate, a chaotropic ion that markedly increases the affinity of AMPA receptors and accelerates their desensitization. In the absence of thiocyanate, however, Cyclothiazide had no reliable effect on the binding of [3H]AMPA or on the affinity for this agonist assessed from the displacement of [3H]CNQX. The interaction of Cyclothiazide with the receptor appears not to be changed by the presence of thiocyanate. Analysis of the results with a kinetic model of the AMPA receptor suggests that Cyclothiazide does not block receptor desensitization by making the desensitized state inaccessible but rather by stabilizing the active state, i.e., by increasing the affinity of the latter to a point where it becomes energetically more favorable than the desensitized state. GYKI 52466, an atypical benzodiazepine that blocks AMPA receptor-gated currents, did not reverse the changes in binding affinity produced by Cyclothiazide in the presence of thiocyanate. Physiological experiments conducted in excised patches collected from hippocampal pyramidal cells indicated that thiocyanate does not block access of GYKI 52466 to AMPA receptors. These results point to the conclusion that Cyclothiazide acts at a site on the AMPA receptor different from that for GYKI 52466.
