Applications of parallel synthetic lead hopping and pharmacophore-based virtual screening in the discovery of efficient glycine receptor potentiators.

Glycine receptors (GlyRs) are pentameric glycine-gated chloride ion channels that are enriched in the brainstem and spinal cord where they have been demonstrated to play a role in central nervous system (CNS) inhibition. Herein we describe two novel classes of glycine receptor potentiators that have been developed using similarity- and property-guided scaffold hopping enabled by parallel synthesis and pharmacophore-based virtual screening strategies. This effort resulted in the identification of novel, efficient and modular leads having favorable in vitro ADME profiles and high CNS multi-parameter optimization (MPO) scores, exemplified by azetidine sulfonamide 19 and aminothiazole sulfone (ent2)-20.

GlyT1 Inhibitor NFPS Exerts Neuroprotection via GlyR Alpha1 Subunit in the Rat Model of Transient Focal Cerebral Ischaemia and Reperfusion.

BACKGROUND/AIMS: Glycine is a strychnine-sensitive inhibitory neurotransmitter in the central nervous system (CNS), especially in the spinal cord, brainstem, and retina. The objective of the present study was to investigate the potential neuroprotective effects of GlyT1 inhibitor N [3-(4'-fluorophenyl)-3-(4'-phenylphenoxy) propyl] sarcosine (NFPS) in the rat model of experimental stroke. METHODS: In vivo ischaemia was induced by transient middle cerebral artery occlusion (tMCAO). The methods of Western Blotting, Nissl Staining and Morris water maze methods were applied to analyze the anti-ischaemia mechanism. RESULTS: The results showed that high dose of NFPS (H-NFPS) significantly reduced infarct volume, neuronal injury and the expression of cleaved caspase-3, enhanced Bcl-2/Bax, and improved spatial learning deficits which were administered three hours after transient middle cerebral artery occlusion (tMCAO) induction in rats, while, low dose of NFPS (L-NFPS) exacerbated the injury of ischaemia. These findings suggested that low and high dose of NFPS produced opposite effects. Importantly, it was demonstrated that H-NFPS-dependent neuronal protection was inverted by salicylate (Sal), a specific GlyR x0251;1 antagonist. Such effects could probably be attributed to the enhanced glycine level in both synaptic and extrasynaptic clefts and the subsequently altered extrasynaptic GlyRs and their subtypes. CONCLUSIONS: These data imply that GlyT1 inhibitor NFPS may be a novel target for clinical treatment of transient focal cerebral ischaemia and reperfusion which are associated with altered GlyR alpha 1 subunits.

Brucine suppresses ethanol intake and preference in alcohol-preferring Fawn-Hooded rats.

AIM: Brucine (BRU) extracted from the seeds of Strychnos nux-vomica L is glycine receptor antagonist. We hypothesize that BRU may modify alcohol consumption by acting at glycine receptors, and evaluated the pharmacodynamic profiles and adverse effects of BRU in rat models of alcohol abuse. METHODS: Alcohol-preferring Fawn-Hooded (FH/Wjd) rats were administered BRU (10, 20 or 30 mg/kg, sc). The effects of BRU on alcohol consumption were examined in ethanol 2-bottle-choice drinking paradigm, ethanol/sucrose operant self-administration paradigm and 5-d ethanol deprivation test. In addition, open field test was used to assess the general locomotor activity of FH/Wjd rats, and conditioned place preference (CPP) was conducted to assess conditioned reinforcing effect. RESULTS: In ethanol 2-bottle-choice drinking paradigm, treatment with BRU for 10 consecutive days dose-dependently decreased the ethanol intake associated with a compensatory increase of water intake, but unchanged the daily total fluid intake and body weight. In ethanol/sucrose operant self-administration paradigms, BRU (30 mg/kg) administered before each testing session significantly decreased the number of lever presses for ethanol and the ethanol intake, without affecting the number of sucrose (10%) responses, total sucrose intake, and the number of lever presses for water. Acute treatment with BRU (30 mg/kg) completely suppressed the deprivation-induced elevation of ethanol consumption. Treatment with BRU (10, 20, and 30 mg/kg) did not alter locomotion of FH/Wjd rats, nor did it produce place preference or aversion. CONCLUSION: BRU selectively decreases ethanol consumption with minimal adverse effects. Therefore, BRU may represent a new pharmacotherapy for alcoholism.

Potentiation of NMDA receptors by Withania somnifera on hippocampal CA1 pyramidal neurons.

In Ayurveda,Withania somnifera (WS) is used as a medicine to maintain mental and physical health as well as to enhance memory. In this study, the methanolic extract of WS(mWS) was tested for its electrical influence on hippocampal CA1 pyramidal neurons using a patch clamp technique. In current clamp mode under a high chloride pipette solution, mWS (400 ng/µl) induced remarkable membrane depolarization (9.75 ± 2.54 mV, n = 6) of CA1 neurons. The mWS-induced depolarization was dose-dependent, reproducible, and persistent in the presence of 0.5 µM tetrodotoxin (TTX, 10.17 ± 0.04 mV, n = 6). In voltage clamp mode (holding potential = -60 mV), mWS induced a dose-dependent non-desensitizing inward current that persisted in the presence of TTX (0.5 µM), suggesting that the response induced by mWS was purely a postsynaptic event. Interestingly, these inward currents were partially blocked by strychnine, a glycine receptor blocker. Further, mWS potentiated the NMDA response in hippocampal CA1 neurons at low concentrations. Overall, these results suggest that there are compounds in WS with possible glycine mimetic activities, which may be potential targets for inducing memory consolidation in hippocampal CA1 neurons.

New insights in endogenous modulation of ligand-gated ion channels: histamine is an inverse agonist at strychnine sensitive glycine receptors.

Histamine is involved in many physiological functions in the periphery and is an important neurotransmitter in the brain. It acts on metabotropic H1-H4 receptors mediating vasodilatation, bronchoconstriction and stimulation of gastric acid secretion. In the brain histamine is produced by neurons in the tuberomamillary nucleus (TMN), which controls arousal. Histamine is also a positive modulator of the inhibitory Cys-loop ligand-gated ion channel GABAA. We investigated now its effect on the second member of inhibitory Cys-loop ligand-gated ion channels, the strychnine sensitive glycine receptor. We expressed different human and rat glycine receptor subunits in Xenopus laevis oocytes and characterized the effect of histamine using the two electrode voltage clamp technique. Furthermore we investigated native glycine receptors in hypothalamic neurons using the patch-clamp technique. Histamine inhibited α1ß glycine receptors with an IC50 of 5.2±0.3 mM. In presence of 10 mM histamine the glycine dose-response curve was shifted, increasing the EC50 for glycine from 25.5±1.4 µM to 42.4±2.3 µM. In addition, histamine blocked the spontaneous activity of RNA-edited α3ß glycine receptors. Histamine inhibited glycine receptors expressed in hypothalamic TMN neurons with an IC50 of 4.6±0.3 mM. Our results give strong evidence that histamine is acting on the same binding site as glycine, being an inverse agonist that competitively antagonizes glycine receptors. Thus, we revealed histamine as an endogenous modulator of glycine receptors.

Subunit-specific inhibition of glycine receptors by curcumol.

Emerging evidence has suggested that inhibitory glycine receptors (GlyRs) are an important molecular target in the treatment of numerous neurological disorders. Rhizoma curcumae is a medicinal plant with positive neurological effects. In this study, we showed that curcumol, a major bioactive component of R. curcumae, reversibly and concentration-dependently inhibited the glycine-activated current (IGly) in cultured rat hippocampal neurons. The inhibitory effect was neither voltage- nor agonist concentration-dependent. Moreover, curcumol selectively inhibited homomeric α2-containing, but not α1- or α3-containing, GlyRs. The addition of ß subunit conferred the curcumol sensitivity of α3-containing, but not α1-containing, GlyRs. Site-directed mutagenesis analysis revealed that a threonine at position 59 of the α2 subunit is critical for the susceptibility of GlyRs to curcumol-mediated inhibition. Furthermore, paralleling a decline of α2 subunit expression during spinal cord development, the degree of IGly inhibition by curcumol decreased with prolonged culture of rat spinal dorsal horn neurons. Taken together, our results suggest that the GlyRs are novel molecular targets of curcumol, which may underlie its pharmaceutical effects in the central nervous system.

Direct and indirect control of orexin/hypocretin neurons by glycine receptors.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward seeking, while reduction in their activity has been linked to narcolepsy, obesity and depression. However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem and spinal cord, elicits dose dependent postsynaptic Cl⁻ currents in hcrt/orx cells in acute mouse brain slices. The effect was blocked by the glycine receptor (GLyR) antagonist strychnine and mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functional during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings revealed that they progressively switch from excitatory to inhibitory during the first two postnatal weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/ß-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits, whereas α2-subunits are present in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine and increased by glycine in ~50% of hcrt/orx neurons. Together, these results provide the first evidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs located on all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some hcrt/orx cells.

Potentiation and inhibition of glycine receptors by tutin.

In the present study we characterized the effects of the South American neurotoxin tutin on recombinant glycine receptors (GlyR) expressed in HEK 293 cells using whole-cell patch-clamp techniques. Tutin induced a concentration-dependent inhibition of α(1) and α(2) homomeric GlyRs, with IC(50)s of 35 ± 1 and 15 ± 3 µM, respectively. The co-expression of αß subunits reduced the potency of tutin, thus increasing the IC(50) to 51 ± 4 and 41 ± 8 µM for α(1)ß and α(2)ß GlyRs, respectively. The inhibitory effect of tutin was competitive, independent of membrane potential and reversible suggesting a pore independent site. On the other hand, low tutin concentrations enhanced the current, which was not synergic with Zn(2+) or ethanol. A mutation in Lys385 altered ethanol but not tutin sensitivity, suggesting different sites for modulation of α1-containing GlyRs. Our results suggest that tutin affects the GlyR by a mechanism distinct to that of picrotoxin and ethanol, and that the pharmacological profile of tutin exhibits a "Zn-like" behaviour. In conclusion, these results provide information on molecular mechanisms important for understanding the toxic effects of a recently discovered South American neurotoxin.

Competitive inhibition at the glycine site of the N-methyl-D-aspartate receptor mediates xenon neuroprotection against hypoxia-ischemia.

BACKGROUND: The general anesthetic gas xenon is neuroprotective and is undergoing clinical trials as a treatment for ischemic brain injury. A small number of molecular targets for xenon have been identified, the N-methyl-D-aspartate (NMDA) receptor, the two-pore-domain potassium channel TREK-1, and the adenosine triphosphate-sensitive potassium channel (KATP). However, which of these targets are relevant to acute xenon neuroprotection is not known. Xenon inhibits NMDA receptors by competing with glycine at the glycine-binding site. We test the hypothesis that inhibition of the NMDA receptor at the glycine site underlies xenon neuroprotection against hypoxia-ischemia. METHODS: We use an in vitro model of hypoxia-ischemia to investigate the mechanism of xenon neuroprotection. Organotypic hippocampal brain slices from mice are subjected to oxygen-glucose deprivation, and injury is quantified by propidium iodide fluorescence. RESULTS: We show that 50% atm xenon is neuroprotective against hypoxia-ischemia when applied immediately after injury or after a delay of 3 h after injury. To validate our method, we show that neuroprotection by gavestinel is abolished when glycine is added, confirming that NMDA receptor glycine site antagonism underlies gavestinel neuroprotection. We then show that adding glycine abolishes the neuroprotective effect of xenon, consistent with competitive inhibition at the NMDA receptor glycine site mediating xenon neuroprotection. CONCLUSIONS: We show that xenon neuroprotection against hypoxia- ischemia can be reversed by increasing the glycine concentration. This is consistent with competitive inhibition by xenon at the NMDA receptor glycine site, playing a significant role in xenon neuroprotection. This finding may have important implications for xenon's clinical use as an anesthetic and neuroprotectant.

Glycine receptors contribute to hypnosis induced by ethanol.

BACKGROUND: Glycine is a major inhibitory neurotransmitter in the adult central nervous system (CNS), and its receptors (GlyRs) are well known for their effects in the spinal cord and the lower brainstem. Accumulating evidence indicates that GlyRs are more widely distributed in the CNS, including many supraspinal regions. Previous in vitro studies have demonstrated that ethanol potentiates the function of these brain GlyRs, yet the behavioral role of the brain GlyRs has not been well explored. METHODS: Experiments were conducted in rats. The loss of righting reflex (LORR) was used as a marker of the hypnotic state. We compared the LORR induced by systematic administration of ethanol and of ketamine in the absence and presence of the selective glycine receptor antagonist strychnine. Ketamine is a general anesthetic that does not affect GlyRs. RESULTS: Systemically administered (by intraperitoneal injection) ethanol and ketamine dose-dependently induced LORR in rats. Furthermore, systemically administered (by subcutaneous injection) strychnine dose-dependently reduced the percentage of rats exhibiting LORR induced by ethanol, increased the onset time, and decreased the duration of LORR. Strychnine had no effect, however, on the LORR induced by ketamine. CONCLUSIONS: Given that hypnosis is caused by neuronal depression in upper brain areas, we therefore conclude that brain GlyRs contribute at least in part to the hypnosis induced by ethanol.

Theanine, gamma-glutamylethylamide, a unique amino acid in tea leaves, modulates neurotransmitter concentrations in the brain striatum interstitium in conscious rats.

Theanine (gamma-glutamylethylamide) is one of the major amino acid components in green tea and can pass through the blood-brain barrier. Recent studies suggest that theanine affects the mammalian central nervous system; however, the detailed mechanism remains unclear. In this study, we demonstrated the effect of theanine on neurotransmission in the brain striatum by in vivo brain microdialysis. Theanine injection into the rat brain striatum did not increase the concentration of excitatory neurotransmitters in the perfusate. On the other hand, theanine injection increased the concentration of glycine in the perfusate. Because it has been reported that theanine promotes dopamine release in the rat striatum, we investigated the glycine and dopamine concentrations in the perfusate. Co-injection of glycine receptor antagonist, strychnine, reduced theanine-induced changes in dopamine. Moreover, AMPA receptor antagonist, which regulates glycine and GABA release from glia cells, inhibited these effects of theanine and this result was in agreement with the known inhibitory effect of theanine at AMPA receptors.

Molecular architecture of glycinergic synapses.

Synapses can be considered chemical machines, which are optimized for fast and repeated exocytosis of neurotransmitters from presynaptic nerve terminals and the reliable electrical or chemical transduction of neurotransmitter binding to the appropriate receptors in the postsynaptic membrane. Therefore, synapses share a common repertoire of proteins like, e.g., the release machinery and certain cell adhesion molecules. This basic repertoire must be extended in order to generate specificity of neurotransmission and allow plastic changes, which are considered the basis of developmental and/or learning processes. Here, we focus on these complementary molecules located in the presynaptic terminal and postsynaptic membrane specializations of glycinergic synapses. Moreover, as specificity of neurotransmission in this system is established by the specific binding of the neurotransmitter to its receptor, we review the molecular properties of glycine receptor subunits and their assembly into functional glycine receptors with different functional characteristics. The past years have revealed that the molecular machinery underlying inhibitory and especially glycinergic postsynaptic membrane specializations is more complex and dynamic than previously anticipated from morphological studies. The emerging features include structural components as well as signaling modules, which could confer the plasticity required for the proper function of distinct motor and sensory functions.

Glycinergic inhibition in thalamus revealed by synaptic receptor blockade.

Using juvenile rat brain slices, we examined the possibility that strychnine-sensitive receptors for glycine-like amino acids contributed to synaptic inhibition in ventrobasal thalamus, where gamma-aminobutyrate (GABA) is the prevalent inhibitory transmitter. Ventrobasal nuclei showed staining for antibodies against alpha1 and alpha2 subunits of the glycine receptor. Exogenously applied glycine, taurine and beta-alanine increased membrane conductance, effects antagonized by strychnine, indicative of functional glycine receptors. Using glutamate receptor antagonists, we isolated inhibitory postsynaptic potentials and currents (IPSPs and IPSCs) evoked by high-threshold stimulation of medial lemniscus. Like the responses to glycine agonists, these synaptic responses reversed near E(Cl). In comparative tests with GABA receptor antagonists, strychnine attenuated inhibition in a majority of neurons, but did not alter slow, GABA(B) inhibition. For complete blockade, the majority of fast IPSPs required co-application of strychnine with bicuculline or gabazine, GABA(A) receptor antagonists. Strychnine acting with an IC50 approximately = 33 nM, eliminated residual fast inhibition during selective GABA(A) receptor blockade with gabazine. The latency of onset for IPSPs was compatible with polysynaptic pathways or prolonged axonal propagation time. Strychnine lacked effects on monosynaptic, GABAergic IPSPs from zona incerta. The specific actions of strychnine implicated a glycine receptor contribution to fast inhibition in somatosensory thalamus.

Levetiracetam influences human motor cortex excitability mainly by modulation of ion channel function--a TMS study.

PURPOSE: Levetiracetam (LEV) is a new compound with anticonvulsive efficacy in focal and generalized epilepsies. Recent in vitro studies suggest LEV to act as a selective N-type-calcium-channel blocker. METHODS: We used transcranial magnetic stimulation (TMS) in order to investigate if ion-channel blockade is relevant to the inhibitory CNS effects of LEV in vivo and if motor thresholds (MTs) are a valid TMS parameter to detect this mode of action. In a double blind, placebo-controlled, crossover study, the effects of single oral doses of 500 and 2000 mg LEV on motor thresholds, recruitment curves (REC), cortical induced silent period (CSP) and on intracortical inhibition (ICI) and facilitation (ICF) were studied in 10 healthy subjects. RESULTS: A significant increase of motor thresholds was noticed after 2000 mg LEV as compared to placebo. The recruitment curve showed a trend towards motor evoked potential (MEP) amplitude reduction after LEV. LEV had no significant effect on CSP or on intracortical excitability as measured by inhibition and facilitation. CONCLUSIONS: We conclude that the modulation of ion-channel function, reflected by motor threshold elevation and a trend towards recruitment curve suppression, is relevant to the inhibitory CNS effects of LEV in vivo, and therefore, may contribute to the anticonvulsive efficacy of LEV. GABAergic or glutamatergic mechanisms seem to be less important in vivo as measured by TMS.

Pentobarbital induces thalamic oscillations in brain slices, modulated by GABA and glycine receptors.

We studied the effects of pentobarbital and antagonists of glutamate, gamma-aminobutyrate (GABA), and glycine receptors on extracellular activity in ventrobasal thalamic slices. Pentobarbital at sedative-hypnotic concentration (20 microM) reversibly induced 1-15 Hz oscillations. Sustained oscillations required electrical stimulation of internal capsule, but not elevated temperature or low [Mg2+]. Anesthetic concentration (200 microM) of pentobarbital evoked only transient oscillations. Kynurenate-sensitive glutamate receptors were essential for oscillations. GABA(A) antagonism (bicuculline, 50 microM or gabazine, 20 microM) suppressed oscillations at 5-15 Hz. GABA(B) antagonism (CGP 35348, 100 nM), or antagonism of glycine receptors (strychnine, 1 microM) suppressed oscillations at 1-4 and 11-15 Hz. GABA and glycine receptors modulated oscillation frequency. For elimination, oscillations required GABA antagonists and strychnine. Receptors for glutamate and glycine mediated oscillations during GABA receptor blockade in ventrobasal nuclei, or on disconnection from nRT. Glycine receptors were critical for oscillations in dorsal thalamic network, divested of GABAergic inhibition. Glutamate and GABA receptors mediated pentobarbital-induced oscillations in nRT, disconnected from ventrobasal nuclei. Hence, pentobarbital oscillogenesis occurred in isolated networks of the ventrobasal and reticularis nuclei mediated by glutamate receptors, with frequency modulation by GABA(A), GABA(B), and glycine receptors. These stationary oscillations represent a model of sedation-hypnosis, amenable to pharmacological analysis.

Kinetic and mutational analysis of Zn2+ modulation of recombinant human inhibitory glycine receptors.

1. The effects of Zn2+ on glycine receptor (GlyR) currents were analysed in Xenopus oocytes and human embryonic kidney cells expressing homomeric human wild-type and mutant alpha1 subunit GlyRs. 2. Low concentrations (10 microM) of extracellular Zn2+ converted the partial agonist taurine into a high-efficacy agonist. Concentration-response analysis showed that the EC50 for taurine decreased whereas the Hill coefficient increased under these conditions. In contrast, 50-500 microM Zn2+ showed an increased EC50 value and reduced maximal inducible taurine currents. The potency of competitive antagonists was not affected in the presence of Zn2+. 3. Single-channel recording from outside-out patches revealed different kinetics of glycine- and taurine-gated currents. With both agonists, Zn2+ altered the open probability of the alpha1 GlyR without changing its unitary conductance. Low Zn2+ concentrations (5 microM) increased both the opening frequency and mean burst duration, whereas higher Zn2+ concentrations (> 50 microM) reduced GlyR open probability mainly by decreasing the open frequency and the relative contribution of the longest burst of the single-channel events. 4. Site-directed mutagenesis of the GlyR alpha1 subunit identified aspartate 80 and threonine 112 as important determinants of Zn2+ potentiation and inhibition, respectively, without affecting potentiation by ethanol. 5. Our data support the view that Zn2+ modulates different steps of the receptor binding and gating cycle via specific allosteric high- and low-affinity binding sites in the extracellular N-terminal region of the GlyR alpha1 subunit.

GABAergic and glycinergic inhibition sharpens tuning for frequency modulations in the inferior colliculus of the big brown bat.

Discrimination of amplitude and frequency modulated sounds is an important task of auditory processing. Experiments have shown that tuning of neurons to sinusoidally frequency- and amplitude-modulated (SFM and SAM, respectively) sounds becomes successively narrower going from lower to higher auditory brain stem nuclei. In the inferior colliculus (IC), many neurons are sharply tuned to the modulation frequency of SFM sounds. The purpose of this study was to determine whether GABAergic or glycinergic inhibition is involved in shaping the tuning for the modulation frequency of SFM sounds in IC neurons of the big brown bat (Eptesicus fuscus). We recorded the response of 56 single units in the central nucleus of the IC to SFM stimuli before and during the application of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline or the glycine receptor antagonist strychnine. To evaluate tuning to the modulation frequency, the normalized spike count (normalized according to the maximal response for each condition tested) was plotted versus the modulation frequency and the upper and lower 50% cutoff points were determined. Bicuculline increased the upper cutoff in 46% of the neurons by >/=25%. The lower cutoff decreased in 48% of the neurons tested. In some neurons (approximately 30%), a sharpening of the tuning by bicuculline was observed. Strychnine induced an increase of the upper cutoff in almost half of the neurons. Compared with bicuculline these changes were smaller. The lower cutoff decreased in 50% of the neurons with strychnine. The synchronization coefficient (SC) was calculated and compared for three modulation frequencies (50, 100, and 200 Hz) between predrug and drug condition. For all neurons, synchronization decreased (n = 36) or did not change (n = 26) during drug application. This was mainly an effect of the prolonged discharge in response to each cycle. Under predrug conditions, many neurons exhibited selectivity to the direction of the FM, hence they only responded once to each cycle. In a minority of neurons, direction selectivity was abolished by drug application. The main finding was that neuronal inhibition sharpens tuning to the modulation frequency in the majority of neurons. In general, changes induced by bicuculline or strychnine were comparable.

Competitive NMDA and strychnine-insensitive glycine-site antagonists disrupt prepulse inhibition.

Prepulse inhibition (PPI) is thought to reflect the operation of a sensorimotor gating system in the brain. Sensorimotor gating abnormalities have been identified in schizophrenic patients, and various neural systems are involved in this function. To study the modulation of the sensorimotor gating system by the N-methyl-D-aspartate (NMDA) receptor channel complex, the effects of noncompetitive and competitive NMDA antagonists on PPI were examined in rats. PPI was not disrupted by CGS 19755, a competitive NMDA antagonist, at 30 min after subcutaneous (s.c.) administration. However, CGS 19755 (40 mg/kg s.c.) decreased PPI at 120 min after administration with a marked decrease of startle amplitude. Late onset of the effect of CGS 19755 was also observed in the increase of spontaneous locomotor activity (SLA). On the other hand, phencyclidine, a noncompetitive NMDA antagonist, disrupted PPI at 30 min after administration and increased SLA from 20 min after administration. PPI was also disrupted by bilateral intracerebroventricular administration of 5,7-dichlorokyn urenate (10 and 20 micrograms/side X 2), an antagonist at the strychnine-insensitive glycine receptor, which is an allosteric binding site in the NMDA receptor-channel complex. It is concluded that the NMDA receptor-channel complex plays an important role in regulation of PPI.

EEG seizure activity and behavioral neurotoxicity produced by (+)-MK801, but not the glycine site antagonist L-687,414, in the rat.

The objective of the present study was to compare the in vivo effects of the anticonvulsant/neuroprotective glycine-site partial agonists L-687,414 (3R-amino-1-hydroxy-4R-methylpyrrolidin-2-one) and (+)-HA966 (3-Amino-1-hydroxypyrrolidin-2-one) and the non-competitive N-methyl-D-aspartate (NMDA) antagonist (+)-MK801 on spontaneous cortical EEG activity and behavior in the unanesthetized rat. Comprehensive dose-response assessments demonstrated that acute i.v. injections of (+)-MK801 induced a behavioral neurotoxic syndrome comprised of head-weaving, ataxia, hyperlocomotion and myoclonic/clonic behaviors and associated with disruptions in normal EEG rhythms including paroxysmal EEG spike/wave complexes. Injections of (+)-HA966 produced behavioral sedation associated with high-amplitude, slow-wave synchronized EEG patterns; signs of ictal EEG activity were minimal (33% incidence) and only seen at the highest dose tested (100 mg/kg). Both (+)-MK801 and (+)-HA966 severely delayed the latency to slow-wave sleep (SWS). In contrast, the EEG dynamics and overt behavior associated with L687,414 were essentially indistinguishable from controls. There was no disruption in the latency to SWS and mild ataxia was evident only upon awakening. The calculated protective indices (EEG seizure ED50/anticonvulsant ED50) for (+)-MK801 and L-687,414 were 1.2 and > 4.5, respectively. The results of this study confirm that valuable pharmacological actions mediated via glycine site modulation of the NMDA receptor are possible without the clinical manifestation of unwanted neurotoxic side-effects.

The glycine/NMDA receptor antagonist, L-701,324 reverses isolation-induced deficits in prepulse inhibition in the rat.

Previous studies have demonstrated that the glycine/NMDA receptor antagonist, L-701,324 (7-chloro-4-hydroxy-3(3-phenoxy)phenyl-2(H)quinolone) blocks the activation of mesolimbic dopamine systems induced following psychostimulant administration in the rat (Bristow et al. 1994). In the present study, pretreatment with L-701,324 also reversed the deficit in prepulse inhibition (PPI) observed in rats reared in social isolation after weaning. Given that PPI is also attenuated in schizophrenic patients and that isolation rearing induces both neurochemical and behavioural abnormalities suggestive of a physiologically induced state of dopaminergic hyperactivity, these results suggest that blockade of the glycine/NMDA receptor may offer a new strategy for the development of novel antipsychotic agents.