strychnine and 7-chlorokynurenic-acid

N-Methyl-D-aspartate (NMDA) receptors are important targets for drugs of abuse such as ethanol, toluene, and ketamine. Ligand-gated ion channels assembled from the NR1 and NR3 subunits have functional and pharmacological properties that are distinct from those of conventional NMDA receptors containing NR2 subunits. In the present study we used voltage-clamp electrophysiology to characterize excitatory glycine-activated receptors assembled from NR1, NR3A, and NR3B subunits expressed in human embryonic kidney (HEK) 293 cells. These glycine-activated receptors were not stimulated by glutamate or kainic acid and were resistant to magnesium block. A wide variety of NMDA receptor antagonists including d-2-amino-5-phosphonovaleric acid, ifenprodil, memantine, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) or acamprosate did not inhibit glycine-activated NR1/NR3A/NR3B receptors. Likewise, these receptors were not affected by antagonists of inhibitory glycine receptors or glycine transporters. The NMDA receptor glycine site agonist, d-serine, partially activated NR1/NR3A/NR3B receptors, whereas the antagonist, 5,7-dichloro-kynurenic acid, inhibited receptor currents. Conversely, the antagonist, 7-chlorokynurenic acid, and the partial agonist, R-(+)-3-amino-1-hydroxy-2-pyrrolidinone (HA-966), potentiated glycine-stimulated currents of these receptors. NR1/NR3A/NR3B receptor currents were inhibited by 10 to 21% by ethanol and toluene but were relatively insensitive to ketamine. Ethanol inhibition was enhanced in receptors expressing the NR1(L819A) mutant, whereas those containing NR1(F639A) or NR1(M813A) showed no change relative to the wild-type NR1. The results of this study indicate that coexpression of NR1, NR3A, and NR3B subunits in HEK 293 cells results in glycineactivated receptors with novel functional and pharmacological properties.

Topics: Alanine; Animals; Cell Line; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Glycine Plasma Membrane Transport Proteins; Humans; Kynurenic Acid; Membrane Potentials; Mice; Mutation; Patch-Clamp Techniques; Pyrrolidinones; Rats; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Sarcosine; Strychnine; Transfection

The role of the neutral amino acid glycine in excitotoxic neuronal injury is unclear. Glycine coactivates glutamate N-methyl-D-aspartate (NMDA) receptors by binding to a distinct recognition site on the NR1 subunit. Purely excitatory glycine receptors composed of NR1 and NR3/NR4 NMDA receptor subunits have recently been described, raising the possibility of excitotoxic effects mediated by glycine alone. We have previously shown that exposure to high concentrations of glycine causes extensive neurotoxicity in organotypic hippocampal slice cultures by activation of NMDA receptors. In the present study, we investigated further properties of in vitro glycine-mediated toxicity. Agonists of the glycine recognition site of NMDA receptors (D-serine and D-alanine) did not have any toxic effect in hippocampal cultures, whereas competitive blockade of the glycine site by 7-chlorokynurenic acid was neuroprotective. Stimulation (taurine, beta-alanine) or inhibition (strychnine) of the inhibitory strychnine-sensitive glycine receptors did not produce any neurotoxicity. The toxic effects of high-dose glycine were comparable in extent to those produced by the excitatory amino acid glutamate in our model. When combined with sublethal hypoxia/hypoglycemia, the threshold of glycine toxicity was decreased to less than 1 mM, which corresponds to the range of concentrations of excitatory amino acids measured during in vivo cerebral ischemia. Taken together, these results further support the assumption of an active role of glycine in excitotoxic neuronal injury.

Topics: Alanine; Animals; Animals, Newborn; Brain Ischemia; Dose-Response Relationship, Drug; Drug Resistance; Glutamic Acid; Glycine; Hippocampus; Kynurenic Acid; Nerve Degeneration; Neurons; Neuroprotective Agents; Neurotoxins; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Strychnine; Taurine

The amplitude and shape of EPSC waveforms are thought to be important determinants of information processing and storage in the brain, yet relatively little is known about the origins of EPSC variability or how it affects synaptic signaling. We investigated the stochastic determinants of AMPA receptor-mediated EPSC variability at cerebellar mossy fiber-granule cell (MF-GC) connections by combining multiple-probability fluctuation analysis (MPFA) and deconvolution methods. The properties of MF connections with a single release site and the effects of the rapidly equilibrating competitive antagonist kynurenic acid on EPSCs suggest that receptors are not saturated by glutamate during a quantal event and that quanta sum linearly over a wide range of release probabilities. MPFA revealed an average of five vesicular release sites per MF-GC connection. Our results show that the time course of vesicular release is rapid (decay, tau = 75 micros) and independent of release probability, introducing little jitter in the shape or timing of the quantal component of the EPSC at physiological temperature. Moreover, the peak vesicular release rate per release site after an action potential (AP) (approximately 3 ms(-1)) is substantially higher than previously reported for central synapses. Interaction of amplitude fluctuations arising from quantal release and quantal size with the slower, low variability spillover-mediated current produce substantial variability in EPSC shape. Our simulations of MF-GC transmission suggest that quantal variability and transmitter spillover extend the voltage from which AP threshold can be crossed, improving reliability, and that fast vesicular release allows precise signaling across MF connections with heterogeneous weights.

Topics: Animals; Cerebellum; Evoked Potentials; Excitatory Amino Acid Antagonists; GABA Antagonists; In Vitro Techniques; Kynurenic Acid; Models, Neurological; Pyridazines; Quantum Theory; Rats; Rats, Sprague-Dawley; Strychnine; Synapses; Synaptic Transmission

Dopaminergic and non-dopaminergic neurons of the ventral tegmental area (VTA) were recorded intracellularly in slices of rat midbrain. Glycine (0.1-3 mM) caused a strychnine-sensitive and chloride-dependent reduction in membrane input resistance in both types of neuron. However, glycine also reduced the frequency of spontaneous bicuculline-sensitive inhibitory postsynaptic potentials (IPSPs) when recorded in dopaminergic cells. We conclude that glycine inhibits both types of VTA neuron by activating a strychnine-sensitive chloride conductance. Our data also raise the possibility that glycine could increase dopamine output from the VTA by a mechanism of disinhibition.

Topics: Action Potentials; Animals; Dopamine; Excitatory Amino Acid Antagonists; GABA Antagonists; gamma-Aminobutyric Acid; Glycine; Glycine Agents; Kynurenic Acid; Male; Neural Inhibition; Neurons; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Strychnine; Synaptic Transmission; Tetrodotoxin; Ventral Tegmental Area

Glutamate is the neurotransmitter of the synapse between vestibular type I hair cells and the afferent nerve calyx. This calyx may also be involved in local feedback, which may modify sensory cell activity via N-methyl-D-aspartate (NMDA) receptors. Glycine is the co-agonist of glutamate in NMDA receptor activation. Both agents have been detected by immunocytochemistry in the nerve calyx. Glutamate and NMDA stimulations cause changes in the intracellular calcium concentration ([Ca(2+)](i)) of isolated type I sensory cells. We investigated the effect of glycine stimulation on [Ca(2+)](i) in guinea pig type I sensory cells by spectrofluorimetry with fura-2. Glycine application to isolated type I sensory cells induced a rapid and transient increase in [Ca(2+)](i). The fluorescence ratio increased by 55% above the resting level. The peak was reached in 9 s and the return to basal level took about 20 s. A specific antagonist of the glycine site on NMDA receptors, 7-chlorokynurenate (10 microM), decreased the calcium response to glycine by 60%. Glycine may activate NMDA receptors. Glycine may also activate the strychnine-sensitive glycine receptor-gated channel. Strychnine (50 microM) decreased the calcium response to glycine by 60%. Thus, glycine probably induces calcium concentration changes in type I vestibular sensory cells via NMDA receptors and/or glycine receptors.

Topics: Animals; Calcium; Chelating Agents; Chlorides; Egtazic Acid; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamates; Glycine; Guinea Pigs; Hair Cells, Vestibular; Kynurenic Acid; Models, Neurological; N-Methylaspartate; Strychnine

This study examined the role of the strychnine-insensitive glycine binding site of the NMDA receptor in prepulse inhibition (PPI) of the acoustic startle response (ASR) in rats. PPI is an operational measure of gating processes which normally lead to a diminished ASR when a startling stimulus is preceded by a weak prepulse. PPI is impaired in schizophrenics and, therefore, experimentally induced PPI deficits in rats can be regarded as a model for gating deficits in schizophrenia. Local administration of 7-chlorokynurenate (7-CLKYN), an antagonist of the strychnine-insensitive glycine site of the NMDA receptor, into the nucleus accumbens reduced PPI. This sensorimotor gating deficit was antagonized by systemic pretreatment of the rats with the glycine site agonist D-cycloserine, indicating that the effect of 7-CLKYN was due to a blockade of the NMDA receptor associated glycine binding site. A similar deficit in PPI was observed after intra-accumbal administration of the competitive NMDA receptor antagonist AP-5. PPI was normal after injecting these drugs into the anterodorsal striatum. The hypothesis that the PPI deficit is accompanied by a change in dopamine release was tested by a neurochemical analysis of the effects of local injection of 7-CLKYN. Microdialysis data showed no increase of accumbal and striatal dopamine release after blockade of the glycine site with 7-CLKYN. Our data demonstrate that the glycine/NMDA receptor in the nucleus accumbens plays a important role in sensorimotor information processing that depends not on a hyperactive dopamine system.

Topics: Animals; Biogenic Monoamines; Chromatography, High Pressure Liquid; Dopamine; Electrochemistry; Glycine Agents; Kynurenic Acid; Male; Microdialysis; Microinjections; Neostriatum; Nucleus Accumbens; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Reflex, Startle; Strychnine

1. The effects of N-methyl-D-aspartate (NMDA) receptor glycine-binding site antagonists 7-chlorokynurenate (7-Clkyn) and (+/-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) on spinal reflexes in an isolated spinal cord that was maintained in Mg(2+)-free medium in vitro were examined. The actions of 7-Clkyn and HA-966 were compared with those of the channel-site antagonist (i.e., dizocilpine) and NMDA-binding site antagonists--that is, 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV). 2. 7-Clkyn and HA-966 produced a selective depression of the polysynaptic reflex (PSR) while negligibly affecting the activity of the monosynaptic reflex (MSR). The PSR was also differentially suppressed by dizocilpine, CPP and APV. The PSR inhibitory activity of the NMDA antagonists was in the following order: dizocilpine > CPP > APV = 7-Clkyn > HA-966. 3. The inhibitory effects of 7-Clkyn on PSR were markedly antagonized by the simultaneous application of D-serine, an agonist for the NMDA receptor glycine-binding sites. However, PSR inhibition by dizocilpine and CPP was unaffected. 4. Inhibition of the PSR by 7-Clkyn persisted in the presence of strychnine, which markedly increased the PSR activity by itself. 5. These findings suggest that the NMDA receptor glycine-binding sites play a role in generating the NMDA receptor-mediated PSR in the spinal cord in vitro.

Topics: Animals; Depression, Chemical; Dizocilpine Maleate; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; In Vitro Techniques; Kynurenic Acid; Male; Piperazines; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Reflex; Reflex, Monosynaptic; Serine; Spinal Cord; Spinal Nerve Roots; Strychnine; Valine

2,3-Dihydro-6,7-dichloro-pyrido[2,3-b]pyrazine-8-oxide was synthesized and evaluated for in vitro/in vivo antagonistic activity at the strychnine insensitive glycine binding site on the NMDA receptor revealing it to be a useful tool to evaluate the effectiveness of glycine antagonists in vivo.

Topics: Animals; Avoidance Learning; Binding Sites; Cyclic N-Oxides; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glycine; Kynurenic Acid; Mice; Motor Activity; N-Methylaspartate; Neuroprotective Agents; Pyrazines; Rats; Receptors, N-Methyl-D-Aspartate; Seizures; Stereotyped Behavior; Strychnine

The present study investigated if blockade of the modulatory glycine receptor of the NMDA receptor complex influences the expression of behavior (sniffing stereotypy and locomotion) and dopamine metabolism in rats as it has been shown for NMDA receptor antagonists. The glycine receptor antagonist, 7-chlorokynurenate (7-chloro-4-hydroxyquinoline-2-carboxylic acid), induced a dose-dependent sniffing stereotypy but had no effect on locomotion when it was given i.c.v. The glycine receptor agonist, D-cycloserine (D-4-amino-3-isoxazolidinone), antagonized the sniffing stereotypy. 7-Chlorokynurenate had no influence on dopamine metabolism in the striatum and the nucleus accumbens, but moderately decreased the metabolism in the prefrontal cortex. Comparison of behavioral and neurochemical outcomes suggests that the failure to induce locomotion correlates with the unchanged dopamine metabolism in the basal ganglia, while sniffing stereotypy does not. These results show that blockade of the glycine receptor of the NMDA receptor complex induces a behavioral and neurochemical profile similar to that of competitive NMDA receptor antagonists.

Topics: Animals; Behavior, Animal; Corpus Striatum; Cycloserine; Dopamine; Drug Interactions; Excitatory Amino Acid Antagonists; Kynurenic Acid; Locomotion; Male; Nucleus Accumbens; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Sensitivity and Specificity; Stereotyped Behavior; Strychnine

The potential for compounds acting at the strychnine-insensitive glycine receptor to injure neurons was examined using induction of a 70 kDa heat shock protein (HSP-70) as a marker. HSP-70 was consistently detected in retrosplenial and cingulate cortices after MK-801 but not glycine drug treatment. Elsewhere in the cortex, mild diffuse HSP-70 immunoreactivity was detected following 7-chlorokynurenic acid. Following HA-966, intense hippocampal HSP-70 immunoreactivity was observed. These findings indicate that even after very high doses, drugs acting at the strychnine-insensitive glycine receptor are less likely to injure cingulate cortical neurons than other classes of NMDA antagonists.

Topics: Amino Acids; Amino Acids, Cyclic; Animals; Dizocilpine Maleate; Gyrus Cinguli; Heat-Shock Proteins; Hippocampus; Immunohistochemistry; Kynurenic Acid; Male; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Strychnine

This study evaluated a possible mechanism by which glycine potentiates the activity of diazepam (DZP) and valproic acid (VAL) against the clonic seizures induced by pentylenetetrazol (PTZ) in rats. Neither 7-chlorokynurenic acid (7-CLKYNA) nor strychnine in doses of 10, 50, or 100 nmol ICV significantly altered the clonic seizure response to PTZ. However, 7-CLKYNA (100 nmol, ICV), but not strychnine (100 nmol, ICV), antagonized the anticonvulsant activity induced by coadministration of DZP (1.0 mg/kg, IP) and glycine (40 mmol/kg, PO). Neither 7-CLKYNA (100 nmol, ICV) nor strychnine (100 nmol, ICV) significantly altered the anticonvulsant activity induced by coadministration of VAL and glycine. 7-CLKYNA (100 nmol, ICV) had no effect on the anticonvulsant activity of DZP or VAL in the absence of glycine. These results provide evidence that the glycine potentiation of the anticonvulsant activity of DZP in clonic seizures induced by PTZ may be mediated by the strychnine-insensitive glycine receptor.

Topics: Animals; Diazepam; Drug Synergism; Glycine; Injections, Intraventricular; Kynurenic Acid; Male; Pentylenetetrazole; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Seizures; Strychnine

Glutamic acid and glycine were quantified in cells and medium of cultured rostral rhombencephalic neurons derived from fetal rats. In the presence of 1 mM Mg2+, NMDA (50 microM) significantly stimulated (by 69%) release of newly synthesized 5-[3H]hydroxytryptamine ([3H]5-HT). D-2-Amino-5-phosphonopentanoate (AP-5; 50 microM) blocked the stimulatory effect of NMDA. AP-5 by itself inhibited [3H]5-HT release (by 25%), suggesting a tonic control of 5-HT by glutamate. In the absence of Mg2+, basal [3H]5-HT release was 60% higher as compared with release with Mg2+. AP-5 blocked the increased [3H]5-HT release observed without Mg2+, suggesting that this effect was due to the stimulation of NMDA receptors by endogenous glutamate. Glycine (100 microM) inhibited [3H]5-HT release in the absence of Mg2+. Strychnine (50 microM) blocked the inhibitory effect of glycine, indicating an action through strychnine-sensitive inhibitory glycine receptors. The [3H]5-HT release stimulated by NMDA was unaffected by glycine. In contrast, when tested in the presence of strychnine, glycine increased NMDA-evoked [3H]5-HT release (by 22%), and this effect was prevented by a selective antagonist of the NMDA-associated glycine receptor, 7-chlorokynurenate (100 microM). 7-Chlorokynurenate by itself induced a drastic decrease in [3H]5-HT release, indicating that under basal conditions these sites were stimulated by endogenous glycine. These results indicate that NMDA stimulated [3H]5-HT release in both the presence or absence of Mg2+. Use of selective antagonists allowed differentiation of a strychnine-sensitive glycine response (inhibition of [3H]5-HT release) from a 7-chlorokynurenate-sensitive response (potentiation of NMDA-evoked [3H]5-HT release).

Topics: Animals; Cells, Cultured; Embryo, Mammalian; Female; Gestational Age; Glutamates; Glutamic Acid; Glycine; Kinetics; Kynurenic Acid; N-Methylaspartate; Pregnancy; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Rhombencephalon; Serotonin; Strychnine; Valine

Glutamate neurotoxicity was examined in cultured cerebellar granule neurons following both prolonged (20-24 h) and brief (45 min) exposure to compounds acting at strychnine-insensitive glycine receptors. Glutamate neurotoxicity was reduced in a concentration-dependent fashion by brief exposure to the glycine partial agonists 1-aminocyclopropanecarboxylic acid (ACPC) and (+-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) and the competitive antagonist, 7-chlorokynurenic acid (7-CK) with a rank order efficacy: 7-CK > HA-966 > ACPC. Neither D-cycloserine (D-CS) nor glycine affected neurotoxicity produced by maximum glutamate concentrations, while glycine but not D-CS augmented the effects of submaximum glutamate concentrations. Prolonged exposure of cultures to either full (glycine) or partial agonists (ACPC, D-CS, HA-966) abolished the neuroprotective effects of ACPC and significantly diminished the neuroprotective effects of HA-966. In contrast, the neuroprotective effects of 7-CK were only marginally reduced by prolonged exposure to glycinergic ligands, while the neuroprotection afforded by compounds acting at other loci on the NMDA receptor complex (e.g. 2-amino-5-phosphonopentanoate (APV) and dizocilpine (MK-801)) were unaltered. These effects may represent homologous desensitization of the NMDA receptor complex at its strychnine-insensitive glycine receptor induced by prolonged exposure to glycinergic agonists and partial agonists. Nonetheless, levels of the NMDA receptor subunit zeta 1 mRNA were unaffected by prolonged exposure to ACPC, indicating the apparent desensitization could involve a post-translational modification of the NMDA receptor complex.

Topics: 2-Amino-5-phosphonovalerate; Amino Acids; Amino Acids, Cyclic; Analysis of Variance; Animals; Base Sequence; Blotting, Northern; Cells, Cultured; Cerebellum; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glutamates; Glutamic Acid; Glycine; Kinetics; Kynurenic Acid; Molecular Sequence Data; Neurons; Neurotoxins; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Strychnine; Time Factors

The effects of systemic lidocaine (3-4 mg/kg) on the responses of 60 wide dynamic range neurons (WDR) to iontophoretically applied N-methyl-D-aspartic acid (NMDA) and quisqualic acid (QUIS) were studied in anesthetized, paralysed rats. The results show that lidocaine induced (i) potentiation of the NMDA excitation, reversible by 7-chloro-kynurenate (7-Cl-KYNA), a selective antagonist of the glycine binding site on the NMDA receptor; (ii) reduction of the QUIS excitation, reversible by strychnine (STRYCH), a glycine antagonist at its receptor. These findings, supporting a glycine-like action of lidocaine, are discussed together with data on the role of excitatory amino acids (EAAs) and the analgesic effect of lidocaine on neuropathic pain.

Topics: Animals; Drug Synergism; Glycine; Injections, Intravenous; Iontophoresis; Kynurenic Acid; Lidocaine; Male; N-Methylaspartate; Quisqualic Acid; Rats; Rats, Wistar; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Strychnine

Repeated administration of methamphetamine (METH) results in damage to nigrostriatal dopaminergic neurons. Both competitive N-methyl-D-aspartate (NMDA) receptor antagonists and use-dependent cation channel blockers attenuate METH-induced damage. The objectives of the present study were to examine whether comparable reductions in METH-induced damage could be obtained by compounds acting at strychnine-insensitive glycine receptors on the NMDA receptor complex. Four injections of METH (5 mg/kg i.p.) resulted in a approximately 70.9% depletion of striatal dopamine (DA) and approximately 62.7% depletion of dihydroxyphenylacetic acid (DOPAC) content, respectively. A significant protection against METH-induced DA and DOPAC depletion was afforded by the use-dependent channel blocker, MK-801. The competitive glycine antagonist 7-chlorokynurenic acid (7-Cl-KA), the low efficacy glycine partial agonist (+)-3-amino-1-hydroxy-2-pyrrolidone ((+)-HA-966), and the high efficacy partial glycine agonist 1-aminocyclopropane-carboxylic acid (ACPC) were ineffective against METH-induced toxicity despite their abilities to attenuate glutamate-induced neurotoxicity under both in vivo and in vitro conditions. These results indicate that glycinergic ligands do not possess the same broad neuroprotective spectrum as other classes of NMDA antagonists.

Topics: 3,4-Dihydroxyphenylacetic Acid; Amino Acids; Amino Acids, Cyclic; Animals; Corpus Striatum; Dizocilpine Maleate; Dopamine; Kynurenic Acid; Male; Methamphetamine; Mice; Mice, Inbred Strains; Receptors, Glycine; Strychnine; Substantia Nigra

Two experiments examined the involvement of the strychnine-insensitive glycine binding site (SIGBS) of the N-methyl-D-aspartate (NMDA) receptor/channel complex in feeding behavior. The first experiment demonstrated that the SIGBS antagonist, 7-chlorokynurenic acid (7CK), dose-dependently increased food intake in rats without producing significant locomotor deficits or stereotypies. The second experiment showed that D-serine, a SIGBS agonist, dose-dependently antagonized 7CK induced feeding, such that at the highest dose of D-serine used 7CK induced feeding was completely abolished, demonstrating that the effects of 7CK are specific to an action at the SIGBS.

Topics: Analysis of Variance; Animals; Ataxia; Dose-Response Relationship, Drug; Feeding Behavior; Glycine; Injections, Intraventricular; Kynurenic Acid; Male; Muscle Contraction; Rats; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Serine; Strychnine

Topics: Amygdala; Analysis of Variance; Animals; Epilepsy; Kindling, Neurologic; Kynurenic Acid; Male; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Receptors, Neurotransmitter; Strychnine

Modulation of the NMDA receptor by the strychnine-insensitive glycine site was studied both in vitro and in vivo. In vitro the glycinergic stimulation of [3H]MK801 binding was measured in three different rat forebrain membrane preparations. An increased association rate of [3H]MK801 in the presence of glycine was observed. The binding of the radioligand was also enhanced by D-serine, whereas L-serine was less potent. The concentration-effect curves were shifted to the right by the glycine antagonist 7-chlorokynurenic acid (7CKA). In vivo modulation of the N-methyl-D-aspartate (NMDA) receptor was studied using NMDA induced convulsions in 7 day old rats. The NMDA effect was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten,5,10-imine maleate (MK801) and D-(-)-2-amino-5-phosphono-pentanoic acid (AP5). The effect of a submaximal dose of NMDA was dose-dependently potentiated by 1-10 mg/kg D-serine, whereas higher doses of L-serine were needed to obtain a similar effect. 7CKA did not affect NMDA-induced convulsions but reduced the D-serine potentiation of NMDA responses. This study illustrates the ability of the strychnine-insensitive glycine site to modulate the NMDA receptor function both in vitro and in vivo.

Topics: 2-Amino-5-phosphonovalerate; Animals; Animals, Newborn; Dizocilpine Maleate; Glycine; Kynurenic Acid; Male; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Seizures; Serine; Stereoisomerism; Strychnine

We have examined the influence of strychnine-insensitive glycine (gly2) receptor agonists and antagonists on the expression of generalized seizure activity (generalized seizure threshold (GST), afterdischarge duration and motor seizure response) in fully amygdala kindled rats. Intra-amygdaloid administration of the selective gly2 receptor antagonist, 7-chlorokynurenic acid (7-C1KYN) (10-50 nmol) dose-dependently raised GSTs in a glycine-reversible manner. The same doses had no significant effect on other parameters of seizure expression. (+/-)-3-Amino-1-hydroxy-2-pyrrolidone (HA-966), a proposed low-efficacy partial agonist at the gly2 receptor17, showed similar but weaker anticonvulsant activity in this model. The active, R-(+)-enantiomer of HA-966 showed greater anticonvulsant efficacy than the racemic mixture, but was still clearly less active than 7-C1KYN. The gly2 receptor agonists glycine (10-50 nmol), D-serine (50 nmol) and D-alanine (50 nmol) failed to influence any of the parameters of the seizure response, suggesting that gly2 receptors in the basolateral amygdala are fully saturated in vivo. The behavioural impairment induced by high doses of 7-C1KYN did not appear to be associated with gly2 receptor blockade. These results support the concept that potent and selective gly2 receptor antagonists may provide a useful, novel class of anticonvulsant agents.

Topics: Alanine; Amygdala; Animals; Glycine; Kindling, Neurologic; Kynurenic Acid; Male; Pyrrolidinones; Rats; Rats, Inbred Strains; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Seizures; Serine; Strychnine

Strychnine-insensitive, glycinergic modulation of N-methyl-D-aspartate (NMDA)-evoked adenosine and [3H]noradrenaline release was investigated in superfused rat cortical slices. 7-Chlorokynurenic acid (100 microM) significantly decreased 300 microM NMDA-evoked adenosine and [3H]noradrenaline release. The addition of exogenous glycine (100 microM) reversed 7-chlorokynurenic acid antagonism. Higher concentrations of NMDA (500 microM, 3 mM) overcame the 7-chlorokynurenic acid (100 microM) block of NMDA-evoked adenosine release but not the block of NMDA-evoked [3H]noradrenaline release. Addition of exogenous glycine (100 microM) alone did not augment either adenosine or [3H]noradrenaline release. Addition of exogenous glycine show that endogenous glycine, acting at a strychnine-insensitive glycine site on the NMDA receptor, is required for NMDA receptor-mediated release of adenosine and noradrenaline. The finding that non-competitive block of NMDA-evoked adenosine release by 7-chlorokynurenic acid could be overcome by high NMDA concentrations supports the suggestion that spare NMDA receptors exist for adenosine release. Furthermore, heterogeneous endogenous glycine concentrations within the cortical slices cannot account for the observation that NMDA is 33 times more potent at releasing adenosine than at releasing [3H]noradrenaline.

Topics: Adenosine; Animals; Cerebral Cortex; Glycine; In Vitro Techniques; Kynurenic Acid; Male; N-Methylaspartate; Norepinephrine; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Strychnine

The mechanisms of action of three different glycine-site antagonists of the N-methyl-D-aspartate (NMDA)-receptor channel were analyzed employing [3H]glycine direct binding assays, as well as functional glycine- and glutamate-induced uncompetitive blocker binding assays. The latter assays measure apparent channel opening. All three antagonists tested, viz., 7-chlorokynurenic acid (7-Cl-KYNA), kynurenic acid (KYNA), and 1-hydroxy-3-aminopyrrolidone-2 (HA-966), inhibited the binding of [3H]glycine to the NMDA receptor in a dose-dependent manner. These antagonists also inhibited the glycine-induced increase in accessibility of the uncompetitive blocker [3H]N-[1-(2-thienyl)cyclohexyl]-piperidine ([3H]TCP) to the channel. 7-Cl-KYNA and KYNA, but not HA-966, completely blocked the glutamate-induced binding of [3H]TCP, in a manner similar to the non-competitive manner in which the selective NMDA antagonist D-(-)-2-amino-5-phosphonovaleric acid (AP-5) inhibited glycine-induced [3H]TCP binding. The inhibitory effects of HA-966 and of AP-5 on glutamate-induced [3H]TCP binding were overcome when glutamate concentrations were increased. Of the three antagonists, 7-Cl-KYNA appears to be the most potent (Ki = 0.4-1.0 microM) and the most selective glycine antagonist. KYNA was found to act at both the glycine (Ki = 40-50 microM) and the glutamate sites. In contrast, HA-966 (Ki = 6-17 microM) appears to act either on a domain distinct from the glutamate and the glycine sites, but tightly associated with the latter, or at the glycine site, but according to a mechanism distinct from that of 7-Cl-KYNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding Sites; Glycine; Ion Channels; Kynurenic Acid; Phencyclidine; Pyrrolidinones; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Strychnine

1. In order to determine whether the strychnine-insensitive glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex is fully activated in vivo, the ability of the selective glycine receptor agonist, D-serine, to modulate seizure susceptibility in the mouse has been examined. 2. D-Serine (10-200 micrograms per mouse, i.c.v.) dose-dependently increased the potency of NMDLA in inducing seizures in Swiss Webster mice by approximately 3 fold. L-Serine was without significant effect. 3. The potency of pentylenetetrazol in inducing seizures was also enhanced by D-, but not L-serine, although the magnitude of the shift (1.6 fold) was considerably less than for NMDLA. 4. Similar doses of D-serine were also able to block the anticonvulsant effect of the non-selective glycine receptor antagonist, kynurenic acid, against seizures induced by NMDLA, but were without effect on the anticonvulsant effect of the competitive NMDA receptor antagonist, 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). 5. D-Serine completely antagonized the protective effect of the selective glycine receptor antagonist, 7-chlorokynurenic acid, against sound-induced seizures in DBA/2 mice, but was less effective in this model against the less selective antagonist, kynurenic acid. 6 The results indicate that in vivo, NMDA receptors are not maximally potentiated by endogenous glycine and suggest an important involvement of the glycine modulatory site on the NMDA receptor/ion channel complex in the pathophysiology of epilepsy.

Topics: Acoustic Stimulation; Animals; Aspartic Acid; Diazepam; Injections, Intraventricular; Ion Channels; Kynurenic Acid; Male; Mice; Mice, Inbred DBA; N-Methylaspartate; Pentylenetetrazole; Piperazines; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Seizures; Serine; Strychnine

Intracerebroventricular (ICV) injection of N-methyl-D-aspartate (NMDA) was shown to induce generalized seizures in mice. The competitive NMDA antagonists DL-2-amino-5-phosphonovaleroate (DL-AP7) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), the NMDA "channel blocker" antagonist (+)-5-methyl-10,11-dihydro 5H-dibenzo-[a,d] cycloheptan-5,10-imine maleate (MK-801) and the strychnine-insensitive glycine antagonists kynurenic acid (KYNA) and 7-chloro-kynurenic acid (7-Cl-KYNA), when co-administered (ICV) with NMDA, antagonized NMDA-induced generalized seizures. Administration (ICV) of DL-AP7, CPP and MK-801 resulted in impared learning performance in a passive avoidance task in mice, with ED50 close to the anticonvulsant dose. The glycine antagonists KYNA and 7-Cl-KYNA at high doses significantly failed to affect performance in the same model of learning. The results indicate that compounds acting at the strychnine-insensitive glycine site may have a larger "therapeutic window" as anticonvulsants than antagonists of the NMDA receptor and channel.

Topics: 2-Amino-5-phosphonovalerate; Animals; Dizocilpine Maleate; Injections, Intraventricular; Kynurenic Acid; Learning; Male; Mice; N-Methylaspartate; Piperazines; Receptors, Glycine; Receptors, Neurotransmitter; Seizures; Strychnine

The enantiomers of the strychnine-insensitive glycine antagonist, HA-966 (1-hydroxy-3-amino-pyrrolidone-2), stereoselectively enhance binding of the N-methyl-D-aspartate (NMDA) competitive antagonist, [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) to rat brain synaptosomal membranes. The enhancement by the more potent (R)-HA-966 is competitively inhibited by the glycine antagonist 7-chlorokynurenic acid and noncompetitively by the polyamine spermine. Thus, (R)-HA-966, apparently at the glycine site, enhances the binding of antagonist to the NMDA receptor, possibly through a mechanism partially in common with that of spermine.

Topics: Animals; In Vitro Techniques; Kynurenic Acid; Piperazines; Pyrrolidinones; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Spermine; Stereoisomerism; Strychnine; Synaptic Membranes

We present a comparative study of the modulation of the N-methyl-D-aspartate (NMDA) receptor at the strychnine-insensitive glycine site in the spinal cord and in the cortex. The excitatory effect of NMDA was potentiated by D-serine (a glycine mimetic) in the hemisected rat spinal cord. The non-competitive NMDA antagonists 7-chlorokynurenic acid (7-Cl KYNA; 10 microM) and 3-amino-1-hydroxypyrrolid-2-one (HA-966; 100 or 200 microM) antagonized the effect of NMDA in the spinal cord and cortical wedge preparation. The antagonism was reversed by the addition of D-serine. This effect was strychnine-insensitive and hence not related to the inhibitory glycine receptor known to be present in the spinal cord. Our results suggest strongly that glycine positively modulates the NMDA system not only at a supraspinal level but also at the spinal level. As the positive modulation of NMDA responses by D-serine was also seen in the presence of tetrodotoxin, we conclude that the NMDA/glycine complex is (also) located on motoneurones in addition to the known glycine-mediated inhibitory system.

Topics: Animals; Cerebral Cortex; Evoked Potentials; In Vitro Techniques; Kynurenic Acid; Male; Pyrrolidinones; Rats; Receptors, N-Methyl-D-Aspartate; Serine; Spinal Cord; Strychnine; Tetrodotoxin

Electrical kindling of the rat amygdala was performed following daily intra-amygdaloid injections of the strychnine-insensitive glycine receptor antagonist 7-chlorokynurenic acid (7-CIKYN) (10 nmol), 7-CIKYN (10 nmol) plus glycine (40 nmol), or vehicle alone. Control (vehicle-treated) animals showed their first fully kindled (Stage 5) seizure after 9.5 +/- 0.8 daily stimulations. Animals pretreated with 7-CIKYN (10 nmol) showed a significant retardation of development of both the electroencephalographic and motor (17.7 +/- 2.9 daily stimulations) components of the seizure response. This inhibitory influence of 7-CIKYN was blocked when glycine (40 nmol) was co-injected daily with the antagonist. The mean initial afterdischarge threshold (ADT) was unaffected by pretreatment with 7-CIKYN (10 nmol). These results provide the first demonstration of an antiepileptogenic action of a strychnine-insensitive glycine receptor antagonist. They further support a key involvement of NMDA receptors in generative mechanisms of epilepsy.

Topics: Amygdala; Animals; Anticonvulsants; Electric Stimulation; Kindling, Neurologic; Kynurenic Acid; Limbic System; Male; Rats; Rats, Inbred Strains; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Seizures; Strychnine

The novel glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited glutamate stimulated [3H]GABA release from cortical neurons in vitro. Kainate-induced release was blocked in a competitive fashion but N-methyl-D-aspartate (NMDA)-induced release was blocked non-competitively by CNQX. 7-Chlorokynurenate (7-CK) also inhibited NMDA evoked [3H]GABA release non-competitively, but had no effect on kainate induced release. The effects of both CNQX and 7-CK on NMDA-induced release were reversed by addition of exogenous glycine but the effects of CNQX on kainate-induced release were not altered by glycine. This suggests that both CNQX and 7-CK may interact with the glycine regulatory site of the NMDA receptor.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Aspartic Acid; Cells, Cultured; Cerebral Cortex; gamma-Aminobutyric Acid; Glycine; Kainic Acid; Kynurenic Acid; N-Methylaspartate; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Strychnine