6-cyano-7-nitroquinoxaline-2-3-dione and (alpha-carboxycyclopropyl)glycine

Transporters for L-glutamate (excitatory amino acid transporters; EAATs), localized to astrocytes, are involved intimately in intermediary metabolism within the brain. Because (2S,4R)-4-methylglutamate (4MG) has affinity for glial EAATs, we employed [(3)H]4MG to define the characteristics of EAATs in cultured murine astrocytes and describe new approaches to analyze EAAT function. Specific binding of [(3)H]4MG in astrocytic membranes at 4 degrees C represented 90% of total binding. Binding was rapid (apparent t(1/2) approximately 7 min) and saturable. Saturation and Scatchard analyses indicated a single binding site (n(H) = 0.8) with a K(d) of 6.0 +/- 1.5 microM and B(max) = 9.7 +/- 2.9 pmol/mg protein. Binding of [(3)H]4MG to astrocytic homogenates was Na(+)-dependent and inhibited by K(+). Compounds acting at EAATs, such as L-glutamate (Glu), D-aspartate (D-Asp), L-(2S,3S,4R)-2-(carboxycyclopropyl)glycine and L-trans-pyrrolidine-2,4-dicarboxylate displaced binding to nonspecific levels. L-Serine-O-sulphate, an EAAT1-preferring ligand, fully displaced binding of [(3)H]4MG. In contrast, inhibitors having preferential affinity for EAAT2, L-threo-3-methylglutamate, dihydrokainate, and kainate, were relatively ineffective binding displacers. Agonists and antagonists for Glu receptors failed to significantly inhibit [(3)H]4MG binding. Studies with [(3)H]D-Asp reinforced evidence that [(3)H]4MG was binding to EAATs. These data were consistent with Western blot analyses, which indicated abundant expression of EAAT1 but not EAAT2. [(3)H]4MG was also accumulated rapidly (apparent t(1/2) approximately 4 min) into whole astrocytes by a sodium- and temperature-sensitive process (K(m) of 146 +/- 24 microM, V(max) = 336 +/- 27 nmol/mg protein/min), which possessed an EAAT1-like pharmacologic profile. These findings confirm that 4MG is a substrate for EAAT1 and that the binding assay developed using [(3)H]4MG can be utilized in various preparations including cultured astrocytes.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acid Transport System X-AG; Amino Acids, Dicarboxylic; Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Binding, Competitive; Blotting, Western; Cell Membrane; Cells, Cultured; D-Aspartic Acid; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Amino Acid Transporter 1; Glutamates; Kainic Acid; Ligands; Mice; Potassium; Serine; Sodium; Temperature; Tritium

It has been suggested recently that presynaptic kainate receptors (KARs) are involved in short-term and long-term synaptic plasticity at hippocampal mossy fiber synapses. Using genetic deletion and pharmacology, we here assess the role of GLU(K5) and GLU(K6) in synaptic plasticity at hippocampal mossy fiber synapses. We found that the kainate-induced facilitation was completely abolished in the GLU(K6)-/- mice, whereas it was unaffected in the GLU(K5)-/-. Consistent with this finding, synaptic facilitation was reduced in the GLU(K6)(-/-) and was normal in the GLU(K5)-/-. In agreement with these results and ruling out any compensatory effects in the genetic deletion models, application of the GLU(K5)-specific antagonist LY382884 [(3S,4aR,6S,8aR)-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid] did not affect short-term and long-term synaptic plasticity at the hippocampal mossy fiber synapses. We therefore conclude that the facilitatory effects of kainate on mossy fiber synaptic transmission are mediated by GLU(K6)-containing KARs.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids, Dicarboxylic; Animals; Benzodiazepines; Cyclopropanes; Excitatory Postsynaptic Potentials; Gene Deletion; GluK2 Kainate Receptor; Glycine; Isoquinolines; Isoxazoles; Kainic Acid; Mice; Mice, Knockout; Mossy Fibers, Hippocampal; Neuronal Plasticity; Patch-Clamp Techniques; Potassium; Propionates; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Kainic Acid; Synaptic Transmission

Patch pipettes were used to record currents in whole-cell configuration to study the effects of group II metabotropic glutamate receptor (mGluR) stimulation on synaptic transmission in slices of rat subthalamic nucleus. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were reversibly reduced by the selective group II mGluR agonist (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) in a concentration-dependent manner, with an IC50 of 0.19 +/- 0.05 microM. DCG IV (1 microM) had no effect on inhibitory postsynaptic currents mediated by GABA. DCG IV-induced inhibition of EPSCs was reversed by the selective group II mGluR antagonist LY 341495 (100 nM) and mimicked by another selective group II agonist (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Inhibition of EPSC amplitude by DCG IV and L-CCG-I was associated with an increase in the paired-pulse ratio of EPSCs. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (2 microM) reduced the inhibitory effect of DCG IV on EPSCs. However, the response to DCG IV was not affected by the protein kinase A (PKA) activator forskolin (20 microM), by the adenylyl cyclase inhibitor MDL 12230A (20 microM), or by the phosphodiesterase inhibitor Ro 20-1724 (50 microM). DCG IV-induced inhibition of EPSCs was reduced by the non-selective protein kinase inhibitors H-7 (100 microM), H-8 (50 microM) and HA-1004 (100 microM). These results suggest that group II mGluR stimulation acts presynaptically to inhibit glutamate release by a PKC-dependent mechanism in the subthalamic nucleus.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 2-Amino-5-phosphonovalerate; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenylyl Cyclase Inhibitors; Amino Acids; Amino Acids, Dicarboxylic; Animals; Colforsin; Cyclopropanes; Electric Stimulation; Electrophysiology; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Glycine; Imines; In Vitro Techniques; Isoquinolines; Male; Picrotoxin; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Subthalamic Nucleus; Sulfonamides; Synaptic Transmission; Tetradecanoylphorbol Acetate; Xanthenes

G-protein-coupled metabotropic glutamate receptors (mGluRs) are being implicated in various forms of neuroplasticity and CNS disorders. This study examined whether the sensitivities of mGluR agonists are modulated in a distinct fashion in different models of synaptic plasticity, specifically, kindling and chronic cocaine treatment. The influence of kindling and chronic cocaine exposure in vivo was examined in vitro on the modulation of synaptic transmission by group II and III metabotropic glutamate receptors using whole cell voltage-clamp recordings of central amygdala (CeA) neurons. Synaptic transmission was evoked by electrical stimulation of the basolateral amygdala (BLA) and ventral amygdaloid pathway (VAP) afferents in brain slices from control rats and from rats treated with cocaine or exposed to three to five stage-five kindled seizures. This study shows that after chemical stimulation with chronic cocaine exposure or after electrical stimulation with kindling the receptor sensitivities for mGluR agonists are altered in opposite ways. In slices from control rats, group II agonists, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG1) and (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), depressed neurotransmission more potently at the BLA-CeA than at the VAP-CeA synapse while group III agonist, L(+)-2-amino-4-phosphonobutyrate (LAP4), depressed neurotransmission more potently at the VAP-CeA synapse than at the BLA-CeA. These agonist actions were not seen (were absent) in amygdala neurons from chronic cocaine-treated animals. In contrast, after kindling, concentration response relationships for LCCG1 and LAP4 were shifted to the left, suggesting that sensitivity to these agonists is increased. Except at high concentrations, LCCG1, LY354740, and LAP4 neither induced membrane currents nor changed current-voltage relationships. Loss of mGluR inhibition with chronic cocaine treatment may contribute to counter-adaptive changes including anxiety and depression in cocaine withdrawal. Drugs that restore the inhibitory effects of group II and III mGluRs may be novel tools in the treatment of cocaine dependence. The enhanced sensitivity to group II and III mGluR agonists in kindling is similar to that recorded at the lateral to BLA synapse in the amygdala where they reduce epileptiform bursting. These findings suggest that drugs modifying mGluRs may prove useful in the treatment of cocaine withdrawal or epilepsy.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids, Dicarboxylic; Aminobutyrates; Amygdala; Animals; Bridged Bicyclo Compounds; Cell Membrane; Chromones; Cocaine; Cocaine-Related Disorders; Dopamine Uptake Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Kindling, Neurologic; Male; Patch-Clamp Techniques; Phosphinic Acids; Propanolamines; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Synapses; Synaptic Transmission

Intracellular recordings were obtained from neocortical brain slices of adult rats maintained in vitro. The effect of metabotropic glutamate receptor activation on spike frequency adaptation in regular spiking layer II and III neurons was determined. Putative metabotropic glutamate receptor agonists and antagonists, as well as inhibitors of intracellular signaling systems, were tested. Activation of metabotropic glutamate receptors by bath applied (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD; 50-200 microM) reduced the first interspike interval and increased action potential frequency at all current intensities. This effect was not blocked by ionotropic glutamate receptor antagonists. Under these recording conditions, quisqualate (1-10 microM) similarly reduced spike frequency adaptation. Neither 1R,3S-ACPD, L-2-carboxycyclopropylglycine-I nor the putative presynaptic metabotropic glutamate receptor agonist, L-2-amino-4-phosphonobutyrate, mimicked the effects of 1S,3R-ACPD or quisqualate. Bath application of the putative metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, competitively antagonized the excitatory actions of 1S,3R-ACPD. Another putative antagonist, L-2-amino-3-phosphonopropionate, failed to antagonize the reduction in spike frequency adaptation. Intracellular injection of guanosine-5'-O-(2-thiodiphosphate), a non-hydrolysable analog of GTP, inhibited the postsynaptic metabotropic glutamate receptor-mediated effects. However, the depression of synaptic transmission by 1S,3R-ACPD was not antagonized by this compound. The decrease in spike frequency adaptation by 1S,3R-ACPD was not prevented by prior exposure to the non-specific protein kinase inhibitors H-7 or H-8 (10 microM), the protein kinase A inhibitor H-89 (0.25 microM) or the protein kinase C inhibitor staurosporine (0.10 microM). These data suggest that the metabotropic glutamate receptor-mediated reduction in spike adaptation requires the activation of specific G-protein-coupled metabotropic glutamate receptor subtypes located on postsynaptic sites. The increase in neuronal excitability observed in the adult neocortex may be mediated either by an unidentified G-protein-coupled second messenger or via a membrane-delimited G-protein action.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adult; Alanine; Amino Acids, Dicarboxylic; Animals; Benzoates; Cycloleucine; Enzyme Inhibitors; Frontal Lobe; Glycine; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Isoquinolines; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Protein Kinase Inhibitors; Protein Kinases; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Signal Transduction; Staurosporine; Sulfonamides; Thionucleotides

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acids, Dicarboxylic; Animals; Electric Stimulation; Ibotenic Acid; In Vitro Techniques; Inositol Phosphates; Kainic Acid; N-Methylaspartate; Piperazines; Quinoxalines; Quisqualic Acid; Rats; Rats, Inbred F344; Receptors, Glutamate; Receptors, Neurotransmitter; Synaptosomes