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

Topics: Animals; Apoptosis; Cells, Cultured; Dizocilpine Maleate; Dopaminergic Neurons; Kainic Acid; Membrane Potential, Mitochondrial; Mesencephalon; Mice; Neuromuscular Depolarizing Agents; Neurotoxins; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

The increasing occurrence of poisoning accidents in marine animals caused by the amnesic shellfish toxin, domoic acid (DOM), necessitates a better understanding of the factors contributing to DOM neurotoxicity. Here we evaluated the contribution and temporal involvement of NMDA, non-NMDA- and metabotropic-type glutamate receptors (GluRs) in DOM-induced neuronal death using rat primary mixed cortical cultures. Co-application of antagonists for AMPA/kainate- (NBQX) and NMDA-type GluRs (D-AP5) but not for metabotropic GluRs reduced DOM toxicity induced by either of three EC50 dose/duration exposure paradigms. Maximal protection offered by D-AP5 and NBQX either extended or not to the 30- to 60-min period after DOM exposure, respectively. Antagonists were ineffective if applied with a 2-h delay, indicating the presence of a critical time window for neuronal protection after DOM exposure. Early effects correlated with neuronal swelling was seen as early as 10 min post-DOM, which has been linked to non-NMDAR-mediated depolarization and release of endogenous glutamate. That DOM toxicity is dictated by iGluRs is supported by the finding that increased efficacy and potency of DOM with in vitro neuronal maturation are positively correlated with elevated protein levels of iGluR subunits, including NR1, GluR1, GluR2/3, GluR5, and GluR6/7. We determined the time course of DOM excitotoxicity. At >10 microM maximal neuronal death occurs within 2 h, while doses < or = 10 microM continue to produce death during the subsequent 22-h washout period, indicating a quicker progression of the neuronal death cascade with high DOM concentrations. Accordingly, NBQX applied 30 min post-DOM afforded better protection against low dose/prolonged duration (3 microM/24 h) than against high dose/brief duration exposure (50 microM/10 min). Interestingly, prior exposure to subthreshold DOM dose-dependently aggravated toxicity produced by a subsequent exposure to DOM. These findings provide greater insight into the complex properties underlying DOM toxicity, including the sequential involvement of multiple GluRs, greater potency with increasing neuronal maturation and protein levels of iGluRs, varying efficacy depending on dose, duration, and prior history of DOM exposure.

Topics: Animals; Blotting, Western; Cell Survival; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Combinations; Excitatory Amino Acid Antagonists; Fetus; Immunochemistry; Kainic Acid; Marine Toxins; Neurons; Neurotoxins; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate

The neurotoxicity of domoic acid was studied in 2-3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1-100 microM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC(50) values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC(50) value of 6 microM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 microM NBQX against 3 and 10 microM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 microM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 microM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD.

Topics: Animals; Culture Techniques; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Hippocampus; Indicators and Reagents; Kainic Acid; Microtubule-Associated Proteins; Neurons; Neurotoxins; Propidium; Quinoxalines; Rats; Rats, Wistar

Kainate receptors (KARs) are abundantly expressed in the basal ganglia, but their function in synaptic transmission has not been established. In the present study, we show that the GluR6 subunit of KARs is expressed in both substance P- and enkephalin-containing GABAergic projection neurons of the mouse striatum. Using whole-cell voltage-clamp recordings in brain slices, we demonstrate the presence of functional KARs in the dorsal striatum activated by low concentrations of the AMPA/KAR agonist domoate in wild-type but not GluR6-deficient mice. Despite the abundance of KARs, we found no evidence for synaptic activation of these receptors after single or repetitive stimulation of glutamatergic afferents. Domoate induces a transient increase in the frequency of spontaneous IPSCs of small amplitude and a sustained depression of large IPSCs evoked by minimal electrical stimulation within the striatum in wild-type mice but not in GluR6-deficient mice. This depressant effect is inhibited in presence of adenosine A(2A) receptor antagonists, ZM-241385 and SCH-58261. These data strongly suggest that, in striatal neurons, KARs depress GABAergic synaptic transmission indirectly via release of adenosine acting on A(2A) receptors.

Topics: Animals; Benzodiazepines; Corpus Striatum; Down-Regulation; Evoked Potentials; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Gene Expression; GluK2 Kainate Receptor; Kainic Acid; Mice; Mice, Inbred C57BL; Neuromuscular Depolarizing Agents; Neurons; Quinoxalines; Receptors, Adrenergic, alpha-2; Receptors, Kainic Acid; RNA, Messenger; Synaptic Transmission

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-type glutamate receptors mediate most excitatory postsynaptic currents (EPSCs) at central synapses, and their conductance determines in part the size of EPSCs. The conductance of a recombinant AMPA receptor depends on the number of agonist molecules bound to the channel. Here we tested whether native AMPA and kainate receptors show this behavior in outside-out patches from neurons in situ by measuring conductance levels of single channels over a wide range of agonist concentrations. We found that the conductance of AMPA, but not kainate, receptors depended strongly on agonist concentration. Our results suggest that alterations in the glutamate concentration in the synaptic cleft may change the apparent unitary conductance of postsynaptic AMPA receptors.

Topics: Animals; Animals, Newborn; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Ion Channels; Kainic Acid; Mice; Mice, Inbred C57BL; Neuromuscular Depolarizing Agents; Organ Culture Techniques; Quinoxalines; Receptors, AMPA; Receptors, Kainic Acid; Synaptic Membranes

The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N-methyl-D-aspartate (NMDA) receptors, (2) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine (MK-801), an NMDA receptor antagonist, was able to block part of the toxicity. The activation of NMDA receptors is most likely a secondary effect resulting from glutamate release upon kainate or domoate stimulation. 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-ethyle nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization of kainate receptors led to an increased neurotoxicity upon stimulation with kainate or domoate. In neurons cultured for 12 days in vitro a small but significant neurotoxic effect was observed when stimulated with agonist in the presence of MK-801 and GYKI 53655. This indicates that the toxicity is produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures.

Topics: Animals; Benzodiazepines; Blotting, Western; Cell Survival; Cells, Cultured; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Kainic Acid; Mice; Neocortex; Neuromuscular Depolarizing Agents; Neurotoxins; Quinoxalines; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate

The participation of NMDA and non-NMDA receptors in domoic acid-induced neurotoxicity was investigated in cultured rat cerebellar granule cells (CGCs). Neurons were exposed to 300 microM L-glutamate or 10 microM domoate for 2 h in physiologic buffer at 22 degrees C followed by a 22-h incubation in 37 degrees C conditioned growth media. Excitotoxic injury was monitored as a function of time by measurement of lactate dehydrogenase (LDH) activity in both the exposure buffer and the conditioned media. Glutamate and domoate evoked, respectively, 50 and 65% of the total 24-h increment in LDH efflux after 2 h. Hyperosmolar conditions prevented this early response but did not significantly alter the extent of neuronal injury observed at 24 h. The competitive NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid and the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) reduced glutamate-induced LDH efflux totals by 73 and 27%, respectively, whereas, together, these glutamate receptor antagonists completely prevented neuronal injury. Domoate toxicity was reduced 65-77% when CGCs were treated with competitive and noncompetitive NMDA receptor antagonists. Unlike the effect on glutamate toxicity, NBQX completely prevented domoate-mediated injury. HPLC analysis of the exposure buffer revealed that domoate stimulates the release of excitatory amino acids (EAAs) and adenosine from neurons. Domoate-stimulated EAA release occurred almost exclusively through mechanisms related to cell swelling and reversal of the glutamate transporter. Thus, whereas glutamate-induced injury is mediated primarily through NMDA receptors, the full extent of neurodegeneration is produced by the coactivation of both NMDA and non-NMDA receptors. Domoate-induced neuronal injury is also mediated primarily through NMDA receptors, which are activated secondarily as a consequence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor-mediated stimulation of EAA efflux.

Topics: Animals; Cells, Cultured; Cerebellum; Culture Media, Conditioned; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Amino Acids; Glutamic Acid; Kainic Acid; Kinetics; L-Lactate Dehydrogenase; Neurons; Neurotoxins; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin

The binding of [3H]NS 257 (1,2,3,6,7,8-hexahydro-3-(hydroxyimino)-N,N-[3H]dimethyl-7-methyl- 2- oxobenzo[2,1-b:3,4-c']dipyrrole-5-sulfonamide) to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [3H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 mM. In the presence of thiocyanate [3H]NS 257 bound to a single population of binding sites with an affinity of 225 +/- 8 nM and a binding site density of 0.61 +/- 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [3H]NS 257 for both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and L-glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > L-glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [3H]NS 257 binding sites in rat brain was similar to that of [3H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Autoradiography; Binding, Competitive; Cell Membrane; Cerebral Cortex; Hydrogen-Ion Concentration; Indoles; Kainic Acid; Male; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Sulfonamides; Thiocyanates; Tritium

5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime (NS-102), a new competitive glutamate receptor antagonist displaced binding to non-N-methyl-D-aspartate (non-NMDA) binding sites with no activity at the NMDA and strychnine-insensitive glycine binding sites. Under experimental conditions in which both high- and low-affinity sites were labelled, NS-102 only partially inhibited the binding of [3H]kainate. Studies of NS-102 displacement of high-affinity versus low-affinity [3H]kainate binding showed a high selectivity of NS-102 for the low-affinity [3H]kainate binding site (Ki = 0.6 microM) compared to the high-affinity [3H]kainate binding site (Ki > 10 microM). NS-102 was a relatively weak inhibitor of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) binding (IC50 = 7.2 microM). NS-102 and related compounds with similar pharmacological profiles may become valuable tools in the characterization of the functional importance of the low-affinity [3H]kainate binding site.

Topics: Animals; Binding, Competitive; Calcium; Calcium Chloride; Cerebral Cortex; Excitatory Amino Acid Antagonists; Indoles; Kainic Acid; Male; Membranes; Molecular Structure; Neuromuscular Depolarizing Agents; Oximes; Quinoxalines; Radioligand Assay; Rats; Rats, Wistar; Tritium