6-cyano-7-nitroquinoxaline-2-3-dione and pyrrolidine-2-4-dicarboxylic-acid

The properties of functional kainate receptor-mediated EPSCs were studied in acute slices from 19-35-day-old rats. EPSCs elicited in pyramidal and fast-spiking cells in layers 2/3 and 5 of the rat motor cortex by extracellular single shock stimulus in the presence of GYKI 53655 and D-2-amino-5-phosphopentanoic resulted in a residual current. This current was not enhanced by cyclothiazide but was blocked by 6-cyano-7-nitroquinoxalin-2,3-dione and is thought to be mediated by kainate receptors. These kainate receptor-mediated currents displayed a wide range of time courses depending on which pre-synaptic fibres were activated. With paired recordings, unitary EPSCs elicited in pyramidal cells were almost totally blocked by GYKI 53655 and D-2-amino-5-phosphopentanoic. However, when L-transpyrrolidine-2,4-dicarboxylate (PDC), a glutamate uptake blocker, was introduced in the bath, the amplitude of kainate receptor-mediated currents, which is resistant to GYKI 53655 and D-2-amino-5-phosphopentanoic, was revealed. The rise and decay time constants of the kainate receptor-mediated currents were identical to control EPSCs. PDC was not required to reveal the kainate receptor-mediated currents elicited in fast-spiking cells which also displayed similar rise and decay time constants to the control EPSCs. Excitatory input onto pyramidal and fast-spiking cells in the neocortex mediated by kainate receptors contributed between 14 and 40% of the total control unitary EPSCs which displayed identical time courses to the AMPA receptor-mediated component of the EPSCs. Post-synaptic kainate receptors at connected pyramidal cell synapses may be located extra-synaptically.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Antihypertensive Agents; Benzodiazepines; Benzothiadiazines; Dicarboxylic Acids; Drug Interactions; Electric Conductivity; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Membrane Potentials; Neocortex; Neural Pathways; Neurotransmitter Uptake Inhibitors; Patch-Clamp Techniques; Pyramidal Cells; Pyrrolidines; Rats; Rats, Wistar; Receptors, Kainic Acid; Synaptic Transmission; Time Factors; Valine

Early visual experience is essential in the refinement of developing neural connections. In vivo whole-cell recording from the tectum of Xenopus tadpoles showed that repetitive dimming-light stimulation applied to the contralateral eye resulted in persistent enhancement of glutamatergic inputs, but not GABAergic or glycinergic inputs, on tectal neurons. This enhancement can be attributed to potentiation of retinotectal synapses. It required spiking of postsynaptic tectal cells as well as activation of NMDA receptors, and effectively occluded long-term potentiation (LTP) of retinotectal synapses induced by direct electrical stimulation of retinal ganglion cells. Thus, LTP-like synaptic modification can be induced by natural visual inputs and may be part of the underlying mechanism for the activity-dependent refinement of developing connections.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Dicarboxylic Acids; Light; Long-Term Potentiation; Neurons; Neurotransmitter Uptake Inhibitors; Photic Stimulation; Pyrrolidines; Retina; Superior Colliculi; Synapses; Visual Pathways; Visual Perception; Xenopus

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca2+ concentration ([Ca2+]i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen-glucose deprivation) elicited a gradual increase in [Ca2+]i in normal Krebs solution. At high extracellular sodium concentrations ([Na+]o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na+]o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na+]o was reversed by pre-incubation of the slice at a low [Na+]o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na+]o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na+-Ca2+ exchanger only slightly influenced the effects of high [Na+]o. These observations suggest that disturbance of the transmembrane gradient of Na+ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na+-dependent glutamate transporter rather than an exocytotic process.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acid Transport System X-AG; Animals; ATP-Binding Cassette Transporters; Bepridil; Biological Transport; Calcium; Calcium Channel Blockers; Chelating Agents; Dicarboxylic Acids; Excitatory Amino Acid Antagonists; Fura-2; Glutamic Acid; Hippocampus; Hypoxia; Neurotransmitter Uptake Inhibitors; Organ Culture Techniques; Pyrrolidines; Rats; Rats, Wistar; Sodium; Sodium-Calcium Exchanger

1. Whole-cell recordings were obtained from Bergmann glial cells in rat cerebellar slices. 2. The cells had low input resistances (70 +/- 38 M omega; n = 13) and a mean resting potential of -82 +/- 6 mV (n = 12) with a potassium-based internal solution. Electrical and dye coupling between Bergmann glia were confirmed. 3. Stimulation of parallel fibres induced a complex, mostly inward current which could be decomposed pharmacologically. 4. The ionotropic glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), but not DL-2-amino-5-phosphonopentanoic acid (DL-APV; 100 microM) consistently blocked an early inward current component that may reflect synaptic activation of AMPA/kainate receptors in Bergmann glia. 5. Addition of cadmium ions (100 microM) to inhibit transmitter release blocked most of the CNQX-APV-insensitive current. This component probably reflects electrogenic uptake of the synaptically released glutamate. 6. Tetrodotoxin (TTX; 1 microM) blocked the remaining inward current: a slow component, possibly produced by the potassium ion efflux during action potential propagation in parallel fibres. An initial triphasic component of the response was also TTX sensitive and reflected passage of the parallel fibre action potential volley. 7. The putative glutamate uptake current was further characterized; it was blocked by the competitive uptake blockers D-aspartate (0.5 mM) and L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC; 0.5 mM), and by replacement of sodium with lithium. Monitoring the triphasic TTX-sensitive component showed that this inhibition did not result from changes of action potential excitation and propagation. 8. Intracellular nitrate ions increased the putative uptake current, consistent with the effect of this anion on glutamate transporters. 9. The putative uptake current was reduced by depolarization, consistent with the voltage dependence of glutamate uptake. 10. It is concluded that a large fraction of the current induced by parallel fibre stimulation reflects the uptake of synaptically released glutamate. The uptake current activated rapidly, with a 20-80% rise time of 2.3 +/- 0.7 ms (n = 10), and decayed with a principal time constant of 25 +/- 6 ms (n = 10).

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Aspartic Acid; Cadmium; Cerebellum; Dicarboxylic Acids; Electric Stimulation; Evoked Potentials; Glutamic Acid; In Vitro Techniques; Male; Nerve Fibers; Neuroglia; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Rats; Rats, Wistar; Synapses; Tetrodotoxin

L-trans-Pyrrolidine-2,4-dicarboxylate (L-t-PDC) and L-threo-3- hydroxyaspartate (L-t-3OHA), compounds known to interact strongly with the Na(+)-dependent high affinity uptake of excitatory amino acids in central nervous tissue, were tested as potential inhibitors of binding to glutamate receptors and transport sites in frozen sections of rat brain. [3H] alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionate (AMPA), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and [3H] kainate were used as ligands for the binding sites on the "non-NMDA" classes of glutamate receptors and [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653) was used to label NMDA receptor binding sites. The Na(+)-dependent glutamate-uptake site was marked by [3H]L-aspartate. The autoradiograms, obtained by exposing 3H-sensitive film to sections of rat forebrain preincubated with 3H-labelled ligands, were scanned by laser beam and quantified. Distribution patterns of the receptor and transporter sites visualized by the 3H-labelled ligands were compatible with previously published results. [3H]CNQX binding, however, was found to be significantly decreased by Na+.L-t-3OHA was about an order of magnitude stronger than L-t-PDC as an inhibitor of [3H]L-aspartate binding. Neither of the compounds had any important effect at the "non-NMDA" receptor binding sites but L-t-3OHA was a weak inhibitor of [3H]CGP 39653 binding (< 40% at 100 microM). The results suggest that, at low nanomolar concentrations, both compounds are likely to be selective for Na(+)-dependent high affinity glutamate transporter sites. Moreover, L-t-3OHA seems to have a sufficiently high affinity for the site to be almost certainly useful, if available in a 3H-labelled form, as a ligand in autoradiographic studies.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Aspartic Acid; Autoradiography; Dicarboxylic Acids; Excitatory Amino Acid Antagonists; Kainic Acid; Neurotransmitter Uptake Inhibitors; Prosencephalon; Pyrrolidines; Rats; Receptors, N-Methyl-D-Aspartate; Tritium

The purposes of our study were to determine the type of respiratory changes that would occur when either an excitatory amino acid receptor agonist or an uptake inhibitor was administered into the caudal subretrofacial area. This was done by microinjecting either L-glutamate or L-pyrrolidine-2,4-dicarboxylate (L-trans-2,4-PDC) into the caudal subretrofacial area while monitoring tidal volume, respiratory rate, mean arterial blood pressure and heart rate. Bilateral microinjection of 2.5 nmol of L-glutamate into the caudal subretrofacial area produced apnea in eight of eight animals tested, and the duration of apnea was 27 +/- 2 s. To determine the type of L-glutamate receptor responsible for mediating the apneic response, antagonists of the N-methyl-D-aspartate (NMDA) and non-NMDA receptor, namely, 3-[(RS)-carboxypiperazin-4-yl]-propyl-phosphonic acid (CPP), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, were tested. Neither antagonist in doses that blocked NMDA (in the case of CPP) and amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) (in the case of CNQX) blocked apnea elicited by L-glutamate. In addition, kynurenic acid, an antagonist of NMDA and non-NMDA ionotropic receptors, failed to block the effect of L-glutamate. Microinjection of the metabotropic receptor agonist drug, trans-L-1-amino-1,3-cyclopentone-dicarboxylic acid (L-trans-ACPD), into the caudal subretrofacial area failed to have any effect on respiratory activity. Because of the inability to block the effect of L-glutamate in the caudal subretrofacial area, and the lack of effect of L-trans-ACPD, the data suggest that the apneic response produced by L-glutamate is mediated by an as yet undefined receptor. Microinjection of the L-glutamate uptake inhibitor, L-trans-2,4-PDC, was found to produce apnea. Using the dose of 0.5 nmol of L-trans-2,4-PDC, we examined the type of excitatory amino acid receptor that mediated the response. Neither pretreatment with the NMDA receptor antagonist, CPP, nor the non-NMDA receptor antagonist, CNQX, affected L-trans-2,4-PDC-induced apnea. However, combined use of these two antagonists prevented L-trans-2,4-PDC-induced apnea. These data suggest that the effect of synaptically released exitatory amino acid at the caudal subretrofacial area on breathing is apnea, and that this effect is mediated by simultaneous activation of both NMDA and non-NMDA ionotropic receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Apnea; Blood Pressure; Cats; Cycloleucine; Dicarboxylic Acids; Excitatory Amino Acid Antagonists; Female; Glutamic Acid; Heart Rate; Kynurenic Acid; Male; Neurotoxins; Neurotransmitter Uptake Inhibitors; Piperazines; Pyrrolidines; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Respiration; Respiratory Function Tests; Tidal Volume; Trigeminal Caudal Nucleus

Binding of [3H]L-aspartate to thaw-mounted coronal sections of frozen rat forebrain was strong in grey regions of telencephalon (neocortex, hippocampus and neostriatum), but it was weaker and unevenly distributed in diencephalon. At low nanomolar concentrations of ligand used in the present studies, [3H]L-aspartate binding was strongly inhibited by L-threo-3-hydroxyaspartate and L-trans-pyrrolidine-2,4-dicarboxylate, compounds known to be substrate/inhibitors of the high affinity uptake of L-glutamate and L-aspartate. None of the typical ligands for the glutamate and aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-methyl-D-aspartate and kainate, produced a strong enough inhibition (only CNQX at 100 microM weakly inhibited) of the Na(+)-dependent [3H]L-aspartate binding to suggest that [3H]L-aspartate was bound to the receptor binding sites. Furthermore, the binding was absolutely dependent on the presence of Na+ in the incubation medium. It is concluded that [3H]L-aspartate is a ligand suitable for autoradiographic studies of the distribution of Na(+)-dependent, high affinity uptake of acidic amino acids in the central nervous system (CNS). However, feasibility of using [3H]L-aspartate as a specific marker of glutamatergic and/or aspartergic synapses in the CNS requires further investigation.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Aspartic Acid; Autoradiography; Dicarboxylic Acids; Female; Glutamates; Glutamic Acid; In Vitro Techniques; Kinetics; Male; Neurotransmitter Uptake Inhibitors; Presynaptic Terminals; Prosencephalon; Pyrrolidines; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Amino Acid; Sodium