2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and cyclothiazide

In the vestibular periphery neurotransmission between hair cells and primary afferent nerves occurs via specialized ribbon synapses. Type I vestibular hair cells (HCIs) make synaptic contacts with calyx terminals, which enclose most of the HCI basolateral surface. To probe synaptic transmission, whole cell patch-clamp recordings were made from calyx afferent terminals isolated together with their mature HCIs from gerbil crista. Neurotransmitter release was measured as excitatory postsynaptic currents (EPSCs) in voltage clamp. Spontaneous EPSCs were classified as simple or complex. Simple events exhibited a rapid rise time and a fast monoexponential decay (time constant < 1 ms). The remaining events, constituting ~40% of EPSCs, showed more complex characteristics. Extracellular Sr

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Benzothiadiazines; Calcium Channel Blockers; Calcium Channels, L-Type; Cells, Cultured; Dipeptides; Excitatory Postsynaptic Potentials; Female; Gerbillinae; Hair Cells, Vestibular; Male; Nifedipine; Quinoxalines; Receptors, AMPA; Strontium; Synapses

Several lines of evidence indicate that ketamine has a rapid antidepressant-like effect in rodents and humans, but underlying mechanisms are unclear. In the present study, we investigated the effect of ketamine on serotonin (5-HT) release in the rat prefrontal cortex by in vivo microdialysis. A subcutaneous administration of ketamine (5 and 25 mg/kg) significantly increased the prefrontal 5-HT level in a dose-dependent manner, which was attenuated by local injection of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonists into the dorsal raphe nucleus (DRN). Direct stimulation of AMPARs in the DRN significantly increased prefrontal 5-HT level, while intra-DRN injection of ketamine (36.5 nmol) had no effect. Furthermore, intra-DRN injection of an α 4 β 2-nicotinic acetylcholine receptor (nAChR) antagonist, dihydro-β-erythroidine (10 nmol), significantly attenuated the subcutaneous ketamine-induced increase in prefrontal 5-HT levels. These results suggest that AMPARs and α 4 β 2-nAChRs in the DRN play a key role in the ketamine-induced 5-HT release in the prefrontal cortex.

Topics: Animals; Antidepressive Agents; Benzothiadiazines; Dihydro-beta-Erythroidine; Dorsal Raphe Nucleus; Dose-Response Relationship, Drug; Injections, Subcutaneous; Ketamine; Male; Microdialysis; Microinjections; Nicotinic Antagonists; Prefrontal Cortex; Quinoxalines; Rats; Receptors, AMPA; Receptors, Nicotinic; Serotonin; Spermine

In mammalian brains, d-serine has been shown to be required for the regulation of glutamate neurotransmission as an endogenous co-agonist for the N-methyl-d-aspartate type glutamate receptor that is essential for the expression of higher-order brain functions. The exact control mechanisms for the extracellular d-serine dynamics, however, await further elucidation. To obtain an insight into this issue, we have characterized the effects of agents acting at the α-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid (AMPA) type glutamate receptor on the extracellular d-serine contents in the medial prefrontal cortex of freely moving rats by an in vivo microdialysis technique in combination with high-performance liquid chromatography with fluorometric detection. In vivo experiments are needed in terms of a crucial role of d-serine in the neuron-glia communications despite the previous in vitro studies on AMPA receptor-d-serine interactions using the separated preparations of neurons or glial cells. Here, we show that the intra-cortical infusion of (S)-AMPA, an active enantiomer at the AMPA receptor, causes a significant and concentration-dependent reduction in the prefrontal extracellular contents of d-serine, which is reversed by an AMPA/kainate receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt, and a calcium permeable AMPA receptor antagonist, 1-naphthyl acetyl spermine. The d-serine reducing effects of (S)-AMPA are augmented by co-infusion of cyclothiazide that prevents AMPA receptor desensitization. Our data support the view that a calcium permeable AMPA receptor subtype may exert a phasic inhibitory control on the extracellular d-serine release in the mammalian prefrontal cortex in vivo.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Area Under Curve; Benzothiadiazines; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Excitatory Amino Acid Agents; Extracellular Fluid; Fluorometry; Gas Chromatography-Mass Spectrometry; Male; Microdialysis; Prefrontal Cortex; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Serine; Spermine; Time Factors

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2(flop) receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.

Topics: Animals; Benzothiadiazines; Glutamic Acid; Ion Channel Gating; Lipid Bilayers; Lipids; Liposomes; Membrane Potentials; Microscopy, Atomic Force; Protein Conformation; Quinoxalines; Rats; Receptors, AMPA

The endocannabinoid (eCB) system and the cannabinoid CB1 receptor (CB1R) play key roles in the modulation of brain functions. Although actions of eCBs and CB1Rs are well described at the synaptic level, little is known of their modulation of neural activity at the network level. Using microelectrode arrays, we have examined the role of CB1R activation in the modulation of the electrical activity of rat and mice cortical neural networks in vitro. We find that exogenous activation of CB1Rs expressed on glutamatergic neurons decreases the spontaneous activity of cortical neural networks. Moreover, we observe that the net effect of the CB1R antagonist AM251 inversely correlates with the initial level of activity in the network: blocking CB1Rs increases network activity when basal network activity is low, whereas it depresses spontaneous activity when its initial level is high. Our results reveal a complex role of CB1Rs in shaping spontaneous network activity, and suggest that the outcome of endogenous neuromodulation on network function might be state dependent.

Topics: 4-Aminopyridine; Action Potentials; Animals; Animals, Newborn; Benzothiadiazines; Brain; Cannabinoid Receptor Modulators; Cells, Cultured; Electric Stimulation; Endocannabinoids; Excitatory Amino Acid Antagonists; GABA Antagonists; In Vitro Techniques; Mice; Mice, Transgenic; Nerve Net; Neural Inhibition; Neurons; Organophosphorus Compounds; Picrotoxin; Piperidines; Potassium Channel Blockers; Pyrazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Sodium Channel Blockers; Tetrodotoxin; Valine

Cochlear inner hair cells (IHCs) convert sounds into receptor potentials and via their ribbon synapses into firing rates in auditory nerve fibers. Multivesicular release at individual IHC ribbon synapses activates AMPA-mediated EPSCs with widely ranging amplitudes. The underlying mechanisms and specific role for multivesicular release in encoding sound are not well understood. Here we characterize the waveforms of individual EPSCs recorded from afferent boutons contacting IHCs and compare their characteristics in immature rats (postnatal days 8-11) and hearing rats (postnatal days 19-21). Two types of EPSC waveforms were found in every recording: monophasic EPSCs, with sharp rising phases and monoexponential decays, and multiphasic EPSCs, exhibiting inflections on rising and decaying phases. Multiphasic EPSCs exhibited slower rise times and smaller amplitudes than monophasic EPSCs. Both types of EPSCs had comparable charge transfers, suggesting that they were activated by the release of similar numbers of vesicles, which for multiphasic EPSCs occurred in a less coordinated manner. On average, a higher proportion of larger, monophasic EPSCs was found in hearing compared to immature rats. In addition, EPSCs became significantly faster with age. The developmental increase in size and speed could improve auditory signaling acuity. Multiphasic EPSCs persisted in hearing animals, in some fibers constituting half of the EPSCs. The proportion of monophasic versus multiphasic EPSCs varied widely across fibers, resulting in marked heterogeneity of amplitude distributions. We propose that the relative contribution of two modes of multivesicular release, generating monophasic and multiphasic EPSCs, may underlie fundamental characteristics of auditory nerve fibers.

Topics: Animals; Animals, Newborn; Benzothiadiazines; Biophysics; Cochlea; Cochlear Nerve; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hair Cells, Auditory, Inner; Hearing; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Sprague-Dawley; Synapses

To further understand the excitatory effects of motoneurons on spinal network function, we investigated the entrainment of disinhibited rhythms by ventral root (VR) stimulation in the neonatal mouse spinal cord. A brief train of stimuli applied to a VR triggered bursting reliably in 31/32 experiments. The same roots that entrained disinhibited bursting could also produce locomotor-like activity with a similar probability when the network was not disinhibited. The ability of VR stimulation to entrain the rhythm persisted in nicotinic and muscarinic cholinergic antagonists but was blocked by the AMPAR antagonist NBQX. Bath application of the type I mGluR1 receptor antagonist CPCCOEt reduced the ability of both dorsal root and VR stimulation to entrain the disinhibited rhythm and abolished the ability of either type of stimulation to evoke locomotor-like activity. Calcium imaging through the lateral aspect of the cord revealed that VR stimulation and spontaneously occurring bursts were accompanied by a wave of activity that originated ventrally and propagated dorsally. Imaging the cut transverse face of L(5) revealed that the earliest VR-evoked optical activity began ventrolaterally. The optical activity accompanying spontaneous bursts could originate ventrolaterally, ventromedially, or throughout the mediolateral extent of the ventral horn or very occasionally dorsally. Collectively, our data indicate that VR stimulation can entrain disinhibited spinal network activity and trigger locomotor-like activity through a mechanism dependent on activation of both ionotropic and metabotropic glutamate receptors. The effects of entrainment appear to be mediated by a ventrolaterally located network that is also active during spontaneously occurring bursts.

Topics: Action Potentials; Animals; Animals, Newborn; Benzothiadiazines; Bicuculline; Biophysics; Carbodiimides; Chromones; Electric Stimulation; Electroporation; Excitatory Amino Acid Antagonists; Functional Laterality; GABA Antagonists; Glycine Agents; In Vitro Techniques; Mice; Motor Neurons; Neural Inhibition; Neural Pathways; Organic Chemicals; Quinoxalines; Reaction Time; Spectrum Analysis; Spinal Cord; Spinal Nerve Roots; Strychnine; Time Factors

At central glutamatergic synapses, neurotransmitter often saturates postsynaptic AMPA receptors (AMPARs), thereby restricting the dynamic range of synaptic efficacy. Here, using simultaneous pre- and postsynaptic whole-cell recordings, at the calyx of Held synapse of immature rats, we have investigated the mechanism by which transmitter glutamate saturates postsynaptic AMPARs. When we loaded L-glutamate (1-100 mM) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration-dependent manner. At physiological temperature (36-37 degrees C), the qEPSC amplitude increased when intraterminal L-glutamate concentration was elevated from 1 mM to 10 mM, but it reached a plateau at 10 mM. This plateau persisted after bath-application of the low affinity AMPAR antagonist kynurenate, suggesting that it was caused by saturation of vesicular filling with glutamate rather than by saturation of postsynaptic AMPARs. In contrast to qEPSCs, action potential-evoked EPSCs remained unchanged by increasing intraterminal L-glutamate from 1 mM to 100 mM , even at room temperature, indicating that multi-quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 microM). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 nM; this was far below the concentration required for AMPAR desensitization. We conclude that rapid AMPAR desensitization, caused by glutamate released from multiple vesicles during synaptic transmission, underlies postsynaptic AMPAR saturation at this immature calyceal synapse before the onset of hearing.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Animals; Aspartic Acid; Benzothiadiazines; Brain Stem; Excitatory Postsynaptic Potentials; Glutamic Acid; Kynurenic Acid; Patch-Clamp Techniques; Presynaptic Terminals; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptic Vesicles; Temperature; Tetrodotoxin

The neurosteroid pregnenolone sulfate (PREGS), which is synthesized in glial cells, plays a significant role in learning and memory performance. The aim of this study was to investigate the regulation of expression of the steroid sulfotransferase SULT2B1a, which catalyzes the conversion of pregnenolone to PREGS, using the rat C6 glioma cell line. Rat C6 glioma cells expressed the SULT2B1a isoform, which sulfonates pregnenolone, but, neither the SULT2B1b isoform, which catalyzes cholesterol, nor the prototypical steroid sulfotransferase SULT2A1 were expressed in these cells. Increasing concentrations of l-glutamic acid in the presence of cyclothiazide, which prevents AMPA receptor desensitization, attenuated SULT2B1a mRNA expression; however, neither NMDA nor kainic acid had a significant effect. Exposure to the synthetic glutamate analogue alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the presence of cyclothiazide also inhibited SULT2B1a expression. Attenuation of SULT2B1a expression by L-glutamic acid was reversed by the selective AMPA/kainate receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), and partially reversed by the specific neuronal nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI). Induction of inducible NOS by TNF-alpha in combination with lipopolysaccharide (LPS) dramatically attenuated SULT2B1a expression; this was partially reversed by the specific inducible NOS inhibitor N(6)-(1-iminoethyl)-L-lysine hydrochloride (L-NIL). Furthermore, exposure to exogenous NO donors inhibited SULT2B1a mRNA expression, and exposure to sodium nitroprusside, LPS/TNF-alpha and L-glutamic acid in combination with cyclothiazide increased the production of nitrite, a stable degradation product of NO. These findings suggest that expression of SULT2B1a, which catalyzes PREGS production, is inhibited by activation of excitatory amino acid receptors of the AMPA subtype, via facilitation of intracellular NO signaling.

Topics: Animals; Benzothiadiazines; Cell Line, Tumor; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glioma; Glutamic Acid; Isoenzymes; Neuroglia; Nitric Oxide; Pregnenolone; Quinoxalines; Rats; Receptors, AMPA; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sulfotransferases

We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.

Topics: Aniline Compounds; Benzothiadiazines; Calcium; Cell Line; Combinatorial Chemistry Techniques; Concanavalin A; Drug Evaluation, Preclinical; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescence; Glutamic Acid; Humans; Kainic Acid; Quinoxalines; Receptors, AMPA; Receptors, Glutamate; Signal Transduction; Xanthenes

Rod signals traverse several synapses en route to cone bipolar cells. In one pathway, rods communicate directly with cones via gap junctions. In a second pathway, signals flow rods-rod bipolars-AII amacrines-cone bipolars. The relative contribution of each pathway to retinal function is not well understood. Here we have examined this question from the perspective of the AII amacrine. AIIs form bidirectional electrical synapses with on cone bipolars. Consequently, as on cone bipolars are activated by outer plexiform inputs, they too should contribute to the AII response. Rod bipolar inputs to AIIs were blocked by AMPA receptor antagonists, revealing a smaller, non-AMPA component of the light response. This small residual response did not reverse between -70 and +70 mV and was blocked by carbenoxolone, suggesting that the current arose in on cone bipolars and was transmitted to AIIs via gap junctions. The residual component was evident for stimuli 2 log units below cone threshold and was prolonged for bright stimuli, demonstrating that it was rod driven. Because the rod bipolar-AII pathway was blocked, the rod-driven residual current likely was generated via the rod-cone pathway activation of on cone bipolars. Thus for a large range of intensities, rod signals reach the inner retina by both rod bipolar-AII and rod-cone coupling pathways.

Topics: Amacrine Cells; Animals; Benzodiazepines; Benzothiadiazines; Diagnostic Imaging; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Fluorescein; GABA Antagonists; Glycine Agents; In Vitro Techniques; Light; Male; Membrane Potentials; Models, Biological; Neurons; Patch-Clamp Techniques; Picrotoxin; Quinoxalines; Rabbits; Retina; Retinal Rod Photoreceptor Cells; Strychnine; Synapses; Synaptic Transmission; Visual Pathways

Synaptic transmission from cones is faster than transmission from rods. Using paired simultaneous recordings from photoreceptors and second-order neurones in the salamander retina, we studied the contributions of rod-cone differences in glutamate receptor properties and synaptic release rates to shaping postsynaptic responses. Depolarizing steps evoked sustained calcium currents in rods and cones that in turn produced transient excitatory postsynaptic currents (EPSCs) in horizontal and OFF bipolar cells. Cone-driven EPSCs rose and decayed faster than rod-driven EPSCs, even when comparing inputs from a rod and cone onto the same postsynaptic neurone. Thus, rod-cone differences in EPSCs reflect properties of individual rod and cone synapses. Experiments with selective AMPA and KA agonists and antagonists showed that rods and cones both contact pharmacologically similar AMPA receptors. Spontaneous miniature EPSCs (mEPSCs) exhibited unimodal distributions of amplitude and half-amplitude time width and there were no rod-cone differences in mEPSC properties. To examine how release kinetics shape the EPSC, we convolved mEPSC waveforms with empirically determined release rate functions for rods and cones. The predicted EPSC waveform closely matched the actual EPSC evoked by cones, supporting a quantal release model at the photoreceptor synapse. Convolution with the rod release function also produced a good match in rod-driven cells, although the actual EPSC was often somewhat slower than the predicted EPSC, a discrepancy partly explained by rod-rod coupling. Rod-cone differences in the rates of exocytosis are thus a major factor in producing faster cone-driven responses in second-order retinal neurones.

Topics: Animals; Benzodiazepines; Benzothiadiazines; Calcium; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Kinetics; Models, Biological; Neurotransmitter Agents; Quinoxalines; Receptors, AMPA; Receptors, Glutamate; Retinal Bipolar Cells; Retinal Cone Photoreceptor Cells; Retinal Horizontal Cells; Retinal Rod Photoreceptor Cells; Synaptic Transmission; Synaptic Vesicles; Urodela

We investigated the role of nitric oxide (NO) on mitochondrial complexes activity, following short-term non-desensitizing activation of AMPA receptors with kainate (KA) plus cyclothiazide (CTZ), in cultured rat hippocampal neurons. In these conditions, we observed a decrease in the activity of mitochondrial complexes I, II/III, and IV. A selective neuronal nitric oxide synthase inhibitor, 7-Nitroindazole, prevented the decrease in the activity of mitochondrial complex I, but not for the other complexes. Exposure to KA plus CTZ also increased cyclic GMP levels significantly, and led to increased levels of 3-nitrotyrosine, a biomarker for peroxynitrite production. Taken together, our results suggest that non-desensitizing activation of AMPA receptors causes inhibition of mitochondrial complex I via peroxynitrite.

Topics: Analysis of Variance; Animals; Antihypertensive Agents; Benzothiadiazines; Cells, Cultured; Cyclic GMP; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; Immunohistochemistry; Kainic Acid; Microtubule-Associated Proteins; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peroxynitrous Acid; Proton Pumps; Quinoxalines; Rats; Receptors, AMPA; Tyrosine

Two main features make microelectrode arrays (MEAs) a valuable tool for electrophysiological measurements under the perspective of pharmacological applications, namely: (i) they are non-invasive and permit, under appropriate conditions, to monitor the electrophysiological activity of neurons for a long period of time (i.e. from several hours up to months); (ii) they allow a multi-site recording (up to tens of channels). Thus, they should allow a high-throughput screening while reducing the need for animal experiments. In this paper, by taking advantages of these features, we analyze the changes in activity pattern induced by the treatment with specific substances, applied on dissociated neurons coming from the chick-embryo spinal cord. Following pioneering works by Gross and co-workers (see e.g. Gross and Kowalski, 1991. Neural Networks, Concepts, Application and Implementation, vol. 4. Prentice Hall, NJ, pp. 47-110; Gross et al., 1992. Sensors Actuators, 6, 1-8.), in this paper analysis of the drugs' effects (e.g. NBQX, CTZ, MK801) to the collective electrophysiological behavior of the neuronal network in terms of burst activity, will be presented. Data are simultaneously recorded from eight electrodes and besides variations induced by the drugs also the correlation between different channels (i.e. different area in the neural network) with respect to the chemical stimuli will be introduced (Bove et al., 1997. IEEE Trans. Biomed. Eng., 44, 964-977.). Cultured spinal neurons from the chick embryo were chosen as a neurobiological system for their relative simplicity and for their reproducible spontaneous electrophysiological behavior. It is well known that neuronal networks in the developing spinal cord are spontaneously active and that the presence of a significant and reproducible bursting activity is essential for the proper formation of muscles and joints (Chub and O'Donovan, 1998. J. Neurosci., 1, 294-306.). This fact, beside a natural variability among different biological preparations, allows a comparison also among different experimental session giving reliable results and envisaging a definition of a bioelectronic 'neuronal sensory system'.

Topics: Algorithms; Animals; Benzothiadiazines; Biosensing Techniques; Cells, Cultured; Cells, Immobilized; Chick Embryo; Chickens; Dizocilpine Maleate; Microelectrodes; Nerve Net; Quinoxalines; Signal Processing, Computer-Assisted

Immunocytochemical and Co(2+) uptake studies revealed that in primary cultures of rat cortical neurones, the majority of neurones are gamma-aminobutyric acid (GABA) immunopositive and can express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. By fura-2 microfluorimetry, it was shown that the stimulation with the selective agonist (S)-AMPA (0.3-300 microM) induced a concentration-dependent but cell-variable increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (EC(50) value 7.4 microM) in more than 80% of the medium-sized multipolar neurones studied. The AMPA-induced rise in [Ca(2+)](i) seems to be due to Ca(2+) entry through AMPA receptor channels, because the response was abolished in Ca(2+)-free solution and by AMPA receptor selective antagonists, but was not significantly influenced by cyclopiazonic acid, an inhibitor of the endoplasmatic Ca(2+)-ATPase, by selective N-methyl-D-aspartic acid (NMDA) receptor antagonists, as well as the Na(+) channel blocker tetrodotoxin and the majority of tested Ca(2+) channel blockers. In conclusion, the results indicate that the cerebral cortical neurones in culture represent mostly GABAergic interneurone-like cells and the majority of them possess Ca(2+)-permeable AMPA receptors, important for intracellular signal transduction and neuronal plasticity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzodiazepines; Benzothiadiazines; Calcium; Cells, Cultured; Cerebral Cortex; Cobalt; Diazoxide; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Fluorescence; Fluorometry; Fura-2; gamma-Aminobutyric Acid; Immunohistochemistry; N-Methylaspartate; Neurons; Piperazines; Quinoxalines; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

The existence of presynaptic alpha-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 microM), 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 (10-100microM) or aniracetam (1mM). 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 (100microM) but not 1-(aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (100microM), 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 (100-1000microM) 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

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Anti-Anxiety Agents; Antihypertensive Agents; Benzodiazepines; Benzothiadiazines; Calcium; Carbon Radioisotopes; Corpus Striatum; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glutamic Acid; Kynurenic Acid; Male; Microdialysis; Nootropic Agents; Potassium; Presynaptic Terminals; Pyrrolidinones; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Tritium

Cerebellar long-term synaptic depression (LTD) is a model system of neuronal information storage that is expressed postsynaptically as a functional down-regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. What properties of postsynaptic AMPA receptors are changed? Several lines of evidence argue against changes in AMPA-receptor kinetics. Neither LTD evoked in cultured granule-cell Purkinje cell (PC) pairs nor an LTD-like phenomenon evoked by phorbol ester application was associated with alterations in evoked AMPA receptor-mediated excitatory post-synaptic current (EPSC) or mEPSC kinetics. LTD produced by pairing glutamate pulses with depolarization was not altered by prior application of the desensitization-reducing compound cyclothiazide. Finally, rapid application of glutamate to lifted PCs revealed no significant alterations in AMPA-receptor kinetic properties after LTD induction. When this system was used to apply varying concentrations of glutamate, no alteration in AMPA-receptor glutamate affinity was seen after LTD induction. Finally, peak-scaled nonstationary fluctuation analysis was applied to estimate AMPA-receptor unitary conductance before and after LTD induction in a cultured cell pair, and this analysis too revealed no significant change. These results suggest that cerebellar LTD may be expressed solely as a reduction in the number of functional AMPA receptors in the postsynaptic density [Wang, Y.-T. & Linden, D. J. (2000) Neuron 25, 635-664].

Topics: 2-Amino-5-phosphonovalerate; Animals; Benzothiadiazines; Cells, Cultured; Cerebellum; Electrophysiology; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Long-Term Potentiation; Mice; Phorbol Esters; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

High affinity glutamate transport plays an important role in maintaining a low extracellular glutamate concentration in the CNS. Excitotoxicity due to a loss of glutamate transporter function has been implicated in disease processes such as stroke and amyotrophic lateral sclerosis (ALS). We studied the effects of glutamate transport inhibitors on thalamocortical synapses at developing (postnatal day 3-8) layer IV neurons in the barrel cortex using the thalamocortical slice preparation and whole-cell recordings. Inhibition of glutamate transport by D,L-threo-beta-hydroxyaspartate (THA), a combination of THA and dihydrokainate (DHK), or by L-trans-pyrrolidine-2,4-dicarboxylate (tPDC), caused a reversible blockade of AMPA and kainate receptor-mediated dual component excitatory postsynaptic currents (AMPA/KA EPSCs). This effect was not blocked by cyclothiazide (CTZ) indicating that is was not due to desensitisation of AMPARs. Under conditions in which NMDA receptors were unblocked the transport inhibitors caused the massive activation of NMDA receptors leading to the rapid loss of recordings. Previous studies using these transport inhibitors on brain slices from older animals reported no or only modest effects on synaptic transmission. Therefore the data in the present study suggest that neurons in the developing neocortex are particularly sensitive to glutamate transporter function. Furthermore the effects of transport inhibition are dependent upon whether neurons are sufficiently depolarised to relieve the voltage-dependent block of NMDA receptors.

Topics: Amino Acid Transport System X-AG; Animals; Aspartic Acid; ATP-Binding Cassette Transporters; Benzothiadiazines; Biological Transport; Dicarboxylic Acids; Diuretics; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glutamic Acid; In Vitro Techniques; Kainic Acid; Neurotransmitter Uptake Inhibitors; Picrotoxin; Pyrrolidines; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Sodium Chloride Symporter Inhibitors; Somatosensory Cortex; Synaptic Transmission; Thalamus

Despite its importance in the cerebellum, the functions of the orphan glutamate receptor delta2 are unknown. We examined a mutant delta2 receptor channel in lurcher mice that was constitutively active in the absence of ligand. Because this mutation was within a highly conserved motif (YTANLAAF), we tested its effect on several glutamate receptors. Mutant delta2 receptors showed distinct channel properties, including double rectification of the current-voltage relationship, sensitivity to a polyamine antagonist and moderate Ca 2+ permeability, whereas other constitutively active mutant glutamate channels resembled wild-type channels in these respects. Moreover, the kinetics of ligand-activated currents were strikingly altered. We conclude that the delta2 receptor has a functional ion channel pore similar to that of glutamate receptors. The motif may have a role in the channel gating of glutamate receptors.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Anti-Anxiety Agents; Antihypertensive Agents; Benzodiazepines; Benzothiadiazines; Cell Line; Conserved Sequence; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Humans; Ion Channel Gating; Kainic Acid; Kidney; Mice; Mice, Neurologic Mutants; Molecular Sequence Data; Mutagenesis; Neuromuscular Depolarizing Agents; Patch-Clamp Techniques; Purkinje Cells; Quinoxalines; Receptors, Glutamate; Transfection

AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well.

Topics: Alternative Splicing; Animals; Animals, Newborn; Benzodiazepines; Benzothiadiazines; Excitatory Amino Acid Antagonists; Female; Hippocampus; In Vitro Techniques; Kinetics; Patch-Clamp Techniques; Pyramidal Cells; Quinoxalines; Rats; Receptors, AMPA; Receptors, Kainic Acid

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), alpha-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.

Topics: Acoustic Stimulation; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Suckling; Benzothiadiazines; Dizocilpine Maleate; Drug Administration Routes; Excitatory Amino Acid Antagonists; Female; Hypothyroidism; Inferior Colliculi; Injections, Intraventricular; Male; Maternal Exposure; N-Methylaspartate; Propylthiouracil; Proto-Oncogene Proteins c-fos; Quinoxalines; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Seizures

The present study describes the effect of (S)-2,3-dihydro-[3, 4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986-1), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with cognitive-enhancing effects, on (S)-AMPA-induced [3H]noradrenaline release in rat hippocampal and frontal cortex slices. (S)-AMPA significantly increased [3H]noradrenaline release in rat hippocampus and frontal cortex slices, whereas S18986-1 (3-1000 microM) alone, was inactive. However, S18986-1 between 30 and 1000 microM potently enhanced (+200%) (S)-AMPA-mediated [3H]noradrenaline release in both hippocampal and frontal cortex slices. The capacity of S18986-1 to potentiate [3H]noradrenaline release was specific for AMPA receptors as S18986-1 failed to potentiate either kainate and N-methyl-D-aspartate (NMDA)-mediated release of [3H]noradrenaline in rat hippocampal slices. Moreover, 1, 2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) and 1-(4-aminophenyl)-3-methylcarbamoyl-4-methyl-3, 4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-53655) but not (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ((+)-MK-801), inhibited (S)-AMPA and S18986-induced stimulation of (S)-AMPA-mediated [3H]noradrenaline release. In addition, S18986-1-induced stimulation of (S)-AMPA-evoked [3H]noradrenaline release was markedly attenuated in the presence of tetrodotoxin (1 microM) and in Ca(2+)-free buffer. S18986-1 enhanced (S)-AMPA-mediated [3H]noradrenaline release to a greater extent than its corresponding (R)-enantiomer S19024-1 and racemic mixture S17951-1. However, positive allosteric modulators of AMPA receptors such as aniracetam failed to potentiate AMPA-mediated noradrenaline release in hippocampal slices, whereas cyclothiazide potently enhanced (S)-AMPA-mediated [3H]noradrenaline release. These results suggest that the capacity of S18986-1 to enhance AMPA receptor-mediated release of noradrenaline in rat hippocampus and frontal cortex, could contribute to the cognition enhancing mechanisms of S18986-1.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzodiazepines; Benzothiadiazines; Calcium; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Synergism; Excitatory Amino Acid Antagonists; Frontal Lobe; Hippocampus; In Vitro Techniques; Male; Norepinephrine; Pyrrolidinones; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Stereoisomerism; Tetrodotoxin; Tritium

alpha-Amino-3-hydroxy-5-methyl-4-isoxazle propionic acid (AMPA) receptors are ubiquitously expressed; however, their subtypes and abundance vary from region to region. We classified the neurons in various forebrain regions (hippocampus, striatum, amygdala, piriform cortex and somatosensory cortex) into six types: [R1+/R2+], [R1-/R2+], [R1+/R2-], [R1-/R2-], [R1++/R2+] and [R1++/R2-], and analysed the expression of Ca2+-binding proteins, such as parvalbumin and calbindin-D28k, using a triple-staining method. The neurons showing a high GluR1 : GluR2 expression ratio, [R1+/R2-], [R1++/R2+] and [R1++/R2-] neurons, comprised 13-30% of the total neuronal population. In addition, the expression of Ca2+-binding proteins was mainly observed in these three types of neurons. The results suggest that Ca2+-binding protein-positive neurons express Ca2+-permeable AMPA receptors, because the Ca2+-permeability of AMPA receptors is enhanced by the relative scarcity of the GluR2 subunit. To directly test the possibility that Ca2+-binding protein-positive neurons express Ca2+-permeable AMPA receptors, we performed Ca2+-imaging experiments in cultured cortical neurons. Ca2+ influx through AMPA receptors was measured selectively by addition of AMPA together with cyclothiazide in the presence of blockers of other Ca2+ influx routes. More than half of the calbindin-D28k-positive neurons showed a large increase in the intracellular Ca2+ concentration ([Ca2+]i), whilst most of the calbindin-D28k-undetectable neurons exhibited only a slight rise in [Ca2+]i after AMPA addition. These results suggest that the expression of calbindin-D28k is related to the expression of Ca2+-permeable AMPA receptors.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Antihypertensive Agents; Benzothiadiazines; Biological Transport; Calbindin 1; Calbindins; Calcium; Calcium Channel Blockers; Cells, Cultured; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression; In Situ Hybridization; Neurons; Parvalbumins; Prosencephalon; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; RNA, Messenger; S100 Calcium Binding Protein G

Aniracetam, 1-(1,3-benzodioxol-5-yl-carbonyl)piperidine (1-BCP) and cyclothiazide, three compounds considered to enhance cognition through modulation of alpha-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 microM) antagonism of the [3H]NA release elicited by 100 microM NMDA with high potency (EC50< or =0.1 microM). 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-sulphamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) added at 5 microM, 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 microM cyclothiazide and reversed to an enhancement of AMPA-evoked [3H]NA release by 10 microM 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.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Antihypertensive Agents; Benzothiadiazines; Dioxoles; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Heterocyclic Compounds; Hippocampus; In Vitro Techniques; Kynurenic Acid; Male; N-Methylaspartate; Nootropic Agents; Norepinephrine; Piperidines; Pyrrolidinones; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Tritium

AMPA receptors and glutamate transporters expressed by cerebellar Bergmann glial cells are activated by neurotransmitter released from climbing fibers (). Based on anatomical evidence, this is most likely the result of glutamate diffusing out of the climbing fiber-Purkinje cell synaptic clefts (). We used the change in the EC50 of the Bergmann glia AMPA receptors produced by cyclothiazide (CTZ) to estimate the concentration of glutamate reached at the glial membrane. The decrease of the EC50 gives rise to a concentration-dependent potentiation of the AMPA receptor-mediated responses (). By comparing the increase in amplitude of the AMPA receptor response in the Bergmann glia (840 +/- 240%; n = 8) with the shift in the glutamate dose-response curve measured in excised patches (EC50, 1810 microM in control vs 304 microM in CTZ), we estimate that the extrasynaptic transmitter concentration reaches 160-190 microM. This contrasts with the concentration in the synaptic cleft, thought to rapidly rise above 1 mM, but is still high enough to activate glutamate receptors. These results indicate that the sphere of influence of synaptically released glutamate can extend beyond the synaptic cleft.

Topics: Amino Acid Transport System X-AG; Animals; ATP-Binding Cassette Transporters; Benzothiadiazines; Cell Membrane; Dose-Response Relationship, Drug; Glutamic Acid; In Vitro Techniques; Kinetics; Models, Biological; Neuroglia; Patch-Clamp Techniques; Probability; Purkinje Cells; Quinoxalines; Rats; Receptors, AMPA; Synapses; Temperature

We have cloned the human ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR3 flip splice variant (hGluR3i) and developed a stable cell line expressing this receptor in HEK293 cells. Electrophysiological recordings demonstrated that glutamate-evoked currents desensitize rapidly, with a mean desensitization time constant of 5.4 ms. Robust glutamate-evoked increases in intracellular Ca++ ([Ca++]i) were observed in the presence of cyclothiazide, which attenuated receptor desensitization. [Ca++]i measurements were used to perform a detailed pharmacological characterization of hGluR3i with reference agonists and antagonists. The results of these studies showed that kainate and domoate were not fully efficacious agonists relative to glutamate. The binding affinities of agonists and competitive antagonists were determined in a [3H]AMPA competition binding assay. There was a good correlation between the functional data and the binding affinities obtained for competitive antagonists. However, the binding affinities of the agonists did not correlate with their functional EC50 values from [Ca++]i data, possibly because the binding assay predominantly measures the desensitized high-affinity state of the receptor. [3H]AMPA binding also was performed on membranes prepared from rat forebrain, and comparison of the data from HEK293 cells expressing hGluR3i and rat forebrain suggest that nearly all of the reference compounds show similar binding activities between the two membrane preparations, with the exception of fluoro-willardiine, kainate and 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2-3-dione (NBQX). These data suggest that cells stably expressing recombinant hGluR3i represent pharmacologically valid experimental systems to study human AMPA receptors.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzothiadiazines; Binding, Competitive; Brain; Cells, Cultured; Diuretics; Electrophysiology; Excitatory Amino Acid Antagonists; Hippocampus; Humans; Kidney; Quinoxalines; Rats; Receptors, AMPA; Sodium Chloride Symporter Inhibitors

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

Topics: Animals; Benzothiadiazines; Cell Survival; Cells, Cultured; Dioxoles; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Neurons; Neurotoxins; Piperidines; Quinoxalines; Rats; Receptors, AMPA

N-methyl-D-aspartate (NMDA) stimulated release of [3H]noradrenaline (NA) from prelabelled rat spinal cord slices. The release was partially insensitive to tetrodotoxin (TTX) and was inhibited by the NMDA antagonist MK-801. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) also evoked release of [3H]NA, which was enhanced by blocking AMPA receptor desensitization with cyclothiazide. AMPA-evoked release was inhibited by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX) but was not affected by TTX. NMDA and AMPA showed synergistic effects, indicating co-existence of NMDA and AMPA receptors on noradrenergic terminals. Kainate evoked [3H]NA release only at high concentrations and the release was not potentiated by blocking kainate receptor desensitization with concanavalin A. Thus, the results indicate that there are stimulatory presynaptic NMDA and AMPA receptors on noradrenergic axon terminals in the spinal cord and that they interact synergistically to evoke release of [3H]NA.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzothiadiazines; Concanavalin A; Dizocilpine Maleate; In Vitro Techniques; Kainic Acid; Male; N-Methylaspartate; Norepinephrine; Presynaptic Terminals; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Tetrodotoxin

The diazoxide derivative 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-S,S-dioxide (IDRA21) enhances memory and learning in rodents, most likely by potentiating AMPAergic synaptic activity. We examined IDRA21's effect upon AMPAergic synaptic currents and whole-cell glutamate currents in cultured rat hippocampal neurons to determine whether IDRA21 was a partial modulator of AMPA receptor desensitization and deactivation. Comparable to cyclothiazide, IDRA21 prolonged AMPAergic autaptic currents (5.6 times control, EC50 150 microM) and slowed the rate of AMPA deactivation (3 times control) following 1-ms applications of 1 mM glutamate to excised, outside-out membrane patches. IDRA21 also augmented autaptic GABA currents by 27 +/- 8.1%, although it had two opposing effects, reducing the peak amplitude versus prolonging autaptic GABA currents. IDRA21 (200 microM) inhibited whole-cell GABA currents elicited by exogenously applied 1 mM GABA by 41 +/- 11%. At sufficient concentrations, IDRA21 reduced AMPA receptor desensitization and slowed the rate of deactivation, most consistent with full agonist activity with lower potency compared to cyclothiazide. IDRA21 slightly augments GABAergic synaptic currents.

Topics: Animals; Animals, Newborn; Antihypertensive Agents; Benzothiadiazines; Cells, Cultured; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Neural Inhibition; Neurons; Neurotoxins; Patch-Clamp Techniques; Quinoxalines; Rats; Receptors, AMPA; Receptors, GABA; Synapses; Synaptic Transmission

Rats neonatally treated with 0.02% propylthiouracil (PTU) through mother's milk showed a high incidence of audiogenic seizures after maturation. These audiogenic seizures were differently modified by MK-801 and NBQX; while intraperitoneal MK-801 equally inhibited running fit (RF) and generalized tonic-clonic seizure (GTCS), NBQX administered into cisterna ambiens significantly inhibited RF but not GTCS. The possible involvement of glutamate receptors in the inferior colliculus was further investigated using naive Sprague-Dawley rats injected with NMDA, AMPA or cyclothiazide, known as an inhibitor of desensitization of AMPA action. All drugs tested successfully induced RF followed by GTCS, resembling audiogenic seizures in PTU-treated rats. However, sound stimulation could augment AMPA-induced, but not NMDA-induced GTCS. Systemic administration with MK-801 potently blocked GTCS induced by AMPA/cyclothiazide, but the same drug failed to block RF after intracisternal injection with AMPA/cyclothiazide. Furthermore, intracisternal administration with NBQX significantly inhibited only RF induced by AMPA/cyclothiazide. The present study suggests that: 1) glutamate receptors in the brainstem, possible in the inferior colliculus, play a crucial role in audiogenic seizures, namely the initiation of RF and propagation into GTCS; and 2) the initiation mechanism is regulated by both NMDA and AMPA receptors, whereas propagation is mainly controlled by NMDA receptors.

Topics: Acoustic Stimulation; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Anticonvulsants; Benzothiadiazines; Brain Stem; Dizocilpine Maleate; Epilepsy, Tonic-Clonic; Excitatory Amino Acid Agonists; Inferior Colliculi; N-Methylaspartate; Neuroprotective Agents; Propylthiouracil; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Seizures

Effects of the glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), on the activator protein-1 (AP-1) DNA binding activity were studied in primary cultures of rat cerebellar granule cells. Application of NMDA as well as of AMPA produced a concentration-dependent enhancement of AP-1 binding. Further examination revealed that only a brief exposure (10 min) to NMDA or AMPA was required for the initiation of a significant, four- to sixfold enhancement of AP-1 DNA binding activity. Blockade of the desensitization of AMPA receptors by cyclothiazide further reduced the exposure time needed to activate the AP-1 complex. The time needed to achieve a maximal increase of AP-1 binding activity varied depending on the glutamate receptor agonist used. NMDA gave maximal AP-1 stimulation after 60 min exposure, whereas stimulation with AMPA alone reached a maximum after 240 min exposure. When AMPA was applied together with cyclothiazide the maximal enhancement of AP-1 binding was reached much faster, within 120 min. Supershift analysis with specific antibodies against the members of Fos and Jun protein families (c-Fos, Fos B, c-Jun, Jun B, Jun D) revealed that the NMDA-induced AP-1 complex was composed predominantly of Jun D and c-Fos. The composition of the AP-1 complex activated by AMPA alone was similar to that produced by NMDA, but with an additional contribution of Fos B. In contrast, application of AMPA plus cyclothiazide induced an AP-1 transcription with contribution of Jun D, c-Fos, Fos B, c-Jun and Jun B proteins. These findings indicate that glutamate is able to enhance AP-1 DNA binding activity in cerebellar granule cells through both NMDA and AMPA glutamate receptors.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzothiadiazines; Cells, Cultured; Cerebellum; Dizocilpine Maleate; DNA-Binding Proteins; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Glycine; Kinetics; N-Methylaspartate; Neurons; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Transcription Factor AP-1

L-Glutamate, N-methyl-D-aspartate, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate increased the release of neuropeptide Y-like immunoreactivity from primary cultures of rat hippocampal neurons incubated in Mg2+(1.2 mM)-containing medium. The neuropeptide Y-like immunoreactivity released by 100 microM glutamate was mainly accounted for by neuropeptide Y (1-36), but consisted in part (about 20%) of peptide YY. The effect of 100 microM glutamate on neuropeptide Y-like immunoreactivity release was largely (about 70%) prevented by the N-methyl-D-aspartate receptor antagonist dizocilpine maleate (10 microM), while the remainder (about 30%) was sensitive to the AMPA/ kainate receptor antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2-3-dione (10 microM). The AMPA(100 microM)-evoked release of neuropeptide Y-like immunoreactivity was strongly antagonized by 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2-3-dione and by 1-aminophenyl-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine, but it was in part (15-20%) sensitive to dizocilpine. The releases of neuropeptide Y-like immunoreactivity elicited by glutamate, N-methyl-D-aspartate, AMPA and kainate were all strongly Ca(2+)-dependent. Tetrodotoxin (1 microM) abrogated the N-methyl-D-aspartate-evoked release and partly inhibited the release caused by glutamate, but did not modify significantly AMPA- or kainate-evoked release. Removal of Mg2+ from the medium caused increase of neuropeptide Y-like immunoreactivity release, an effect prevented by dizocilpine maleate or 7-Cl-kynurenate. Cyclothiazide (10 microM), a drug known to prevent AMPA receptor desensitization, enhanced the neuropeptide Y-like immunoreactivity release elicited by 100 microM AMPA, but not that caused by 100 microM kainate. However, when used at a lower concentration (50 microM), kainate elicited a response that was potentiated significantly by cyclothiazide. It is concluded that glutamate can stimulate Ca(2+)-dependent release of neuropeptide Y from hippocampal neurons mainly through N-methyl-D-aspartate receptors and, less so, by activating cyclothiazide-sensitive receptors of the AMPA-preferring type.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Anti-Anxiety Agents; Antibody Specificity; Antihypertensive Agents; Benzodiazepines; Benzothiadiazines; Calcium; Cells, Cultured; Chromatography, High Pressure Liquid; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Kainic Acid; Magnesium; Neurons; Neuropeptide Y; Quinoxalines; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin

Monitoring of extracellular cGMP during intracerebral microdialysis in freely moving rats permits the study of the functional changes occurring in the glutamate receptor/nitric oxide (NO) synthase/guanylyl cyclase pathway and the relationship of these changes to animal behaviour. When infused into the rat hippocampus in Mg2+-free medium, cyclothiazide, a blocker of desensitization of the AMPA-preferring receptor, increased cGMP levels. The effect of cyclothiazide (300 microM) was abolished by the NO synthase inhibitor L-NARG (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (100 microM). During cyclothiazide infusion the animals displayed a pre-convulsive behaviour characterized by frequent "wet dog shakes" (WDS). Neither L-NARG nor ODQ decreased the WDS episodes. Both cGMP and WDS responses elicited by cyclothiazide were prevented by blocking NMDA receptor function with the glutamate site antagonist CGS 19755 (100 microM), the channel antagonist MK-801 (30 microM) or Mg2+ ions (1 mM). The AMPA/kainate receptor antagonists DNQX (100 microM) and NBQX (100 microM) abolished the WDS episodes but could not inhibit the cyclothiazide-evoked cGMP response. DNQX or NBQX (but not MK-801) elevated, on their own, extracellular cGMP levels. The cGMP response elicited by the antagonists appears to be due to prevention of a glutamate-dependent inhibitory GABAergic tone, since infusion of bicuculline (50 microM) caused a strong cGMP response. The results suggest that (a) AMPA/kainate receptors linked to the NO/cGMP pathway in the hippocampus (but not NMDA receptors) are tonically activated and kept in a desensitized state by endogenous glutamate; (b) blockade of AMPA/kainate receptor desensitization by cyclothiazide leads to endogenous activation of NMDA receptors; (c) the hippocampal NO/cGMP system is under a GABAergic inhibitory tone driven by non-NMDA ionotropic receptors; (d) the pre-convulsive episodes observed depend on hippocampal NMDA receptor activation but not on NO and cGMP production.

Topics: Animals; Behavior, Animal; Benzothiadiazines; Cyclic GMP; Diuretics; Drug Interactions; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Hippocampus; Male; Nitric Oxide; Nitric Oxide Synthase; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Sodium Chloride Symporter Inhibitors

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

Topics: Allosteric Regulation; Animals; Anti-Anxiety Agents; Antihypertensive Agents; Benzodiazepines; Benzothiadiazines; Drug Interactions; Electrophysiology; Excitatory Amino Acid Antagonists; Hippocampus; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Kainic Acid

While it is widely accepted that parasympathetic activity plays a significant role in cardiovascular, bronchomotor, and gastrointestinal function, little is known about the synaptic control of parasympathetic vagal neurons. In this study, we identified the neurotransmitter(s) and postsynaptic responses in dorsal motor nucleus of the vagus (DMNX) neurons upon stimulation of the nucleus of the solitary tract (NTS). Neurons were visualized in rat brain stem slices, and perforated patch-clamp techniques were used to record postsynaptic currents. NTS stimulation activated glutamatergic currents in DMNX that were separated into N-methyl-D-aspartate (NMDA) and non-NMDA components using D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. The non-NMDA component was further characterized using cyclothiazide and concanavalin A to block desensitization of DL-alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) and kainate receptors, respectively. Cyclothiazide increased the postsynaptic amplitude, whereas concanavalin A augmented duration, suggesting kainate, but not AMPA, currents are curtailed by desensitization. High frequency stimulations did not alter synaptic efficacy. In conclusion, this study demonstrates the existence of a monosynaptic glutamatergic pathway from NTS that activates NMDA, kainate, and AMPA postsynaptic receptors in DMNX neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Benzothiadiazines; Brain; Concanavalin A; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Antagonists; Neurons; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Solitary Nucleus; Synapses; Vagus Nerve

Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or 45Ca2+ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45Ca2+ accumulation. 45Ca2+ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall 45Ca2+ influx. These included Ca2+ uptake through NMDA-receptor channels, voltage-sensitive Ca2+ channels, and via Na+/Ca2+ exchange. However, nearly one-fifth of the total 45Ca2+ influx remained unaccounted for and this estimate was similar to 45Ca2+ influx observed under Na(+)-free conditions. This observation suggested that a significant proportion of the Ca2+ flux passes through the AMPA-receptor channel proper, a view supported by Co2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Anti-Anxiety Agents; Benzodiazepines; Benzothiadiazines; Calcium; Calcium Radioisotopes; Cell Survival; Cells, Cultured; Cerebellum; Cobalt; Concanavalin A; Excitatory Amino Acids; Kainic Acid; N-Methylaspartate; Quinoxalines; Rats; Receptors, AMPA; Wheat Germ Agglutinins

Since the lifetime of synaptically released glutamate is thought to be very brief, reflecting diffusion and glutamate uptake, the decay of synaptic currents is thought to represent the average elementary lifetime of a receptor channel bound only once by transmitter molecules. We show here that the decay of evoked non-NMDA synaptic currents can reflect presynaptic factors, in particular, the prolonged action of transmitter at postsynaptic receptors under conditions of enhanced transmitter release. We show that diffusion, high-affinity glutamate uptake, and non-NMDA receptor desensitization are insufficiently rapid to limit the decays of evoked synaptic currents to those of miniature synaptic currents in microcultures of rat hippocampal cells. Our results are consistent with recent studies suggesting that during evoked release, multiple glutamate quanta can interact with overlapping postsynaptic receptor domains.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Anti-Anxiety Agents; Benzodiazepines; Benzothiadiazines; Calcium; Cells, Cultured; Evoked Potentials; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Kinetics; Magnesium; Neurons; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synapses; Time Factors

On rat cortical slices, cyclothiazide, 1-100 microM, (ED50 = 7.1 +/- 1.1 microM) enhanced the depolarizing action of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) but not that of N-methyl-D-aspartate (NMDA). Cyclothiazide 10 microM also reversed the action of a 2,3-benzodiazepine, GYKI 53655, which is a non-competitive AMPA receptor antagonist, but not that of the quinoxalinedione, NBQX, which is a competitive AMPA receptor antagonist.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzodiazepines; Benzothiadiazines; Cerebral Cortex; Diuretics; Excitatory Amino Acid Antagonists; Ibotenic Acid; In Vitro Techniques; Kainic Acid; N-Methylaspartate; Quinoxalines; Rats; Receptors, AMPA; Sodium Chloride Symporter Inhibitors