RESUMO
Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the vertebrate CNS. Classified as AMPA, kainate, delta and NMDA receptors, iGluRs are central drivers of synaptic plasticity widely considered as a major cellular substrate of learning and memory. Surprisingly however, five out of the eighteen vertebrate iGluR subunits do not bind glutamate but glycine, a neurotransmitter known to mediate inhibitory neurotransmission through its action on pentameric glycine receptors (GlyRs). This is the case of GluN1, GluN3A, GluN3B, GluD1 and GluD2 subunits, all also binding the D amino acid d-serine endogenously present in many brain regions. Glycine and d-serine action and affinities broadly differ between glycinergic iGluR subtypes. On 'conventional' GluN1/GluN2 NMDA receptors, glycine (or d-serine) acts in concert with glutamate as a mandatory co-agonist to set the level of receptor activity. It also regulates the receptor's trafficking and expression independently of glutamate. On 'unconventional' GluN1/GluN3 NMDARs, glycine acts as the sole agonist directly triggering opening of excitatory glycinergic channels recently shown to be physiologically relevant. On GluD receptors, d-serine on its own mediates non-ionotropic signaling involved in excitatory and inhibitory synaptogenesis, further reinforcing the concept of glutamate-insensitive iGluRs. Here we present an overview of our current knowledge on glycine and d-serine agonism in iGluRs emphasizing aspects related to molecular mechanisms, cellular function and pharmacological profile. The growing appreciation of the critical influence of glycine and d-serine on iGluR biology reshapes our understanding of iGluR signaling diversity and complexity, with important implications in neuropharmacology.
Assuntos
Glicina/agonistas , Receptores Ionotrópicos de Glutamato/fisiologia , Animais , Sítios de Ligação , Ácido Glutâmico/metabolismo , Humanos , Ligantes , Camundongos , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Transmissão Sináptica/fisiologia , Xenopus laevis/metabolismoRESUMO
Frontocortical NMDA receptors are pivotal in regulating cognition and mood, are hypofunctional in schizophrenia, and may contribute to autistic spectrum disorders. Despite extensive interest in agents potentiating activity at the co-agonist glycine modulatory site, few comparative functional studies exist. This study systematically compared the actions of the glycine reuptake inhibitors, sarcosine (40-200 mg/kg) and ORG24598 (0.63-5 mg/kg), the agonists, glycine (40-800 mg/kg), and D-serine (10-160 mg/kg) and the partial agonists, S18841 (2.5 mg/kg s.c.) and D-cycloserine (2.5-40 mg/kg) that all dose-dependently prevented scopolamine disruption of social recognition in adult rats. Over similar dose ranges, they also prevented a delay-induced impairment of novel object recognition (NOR). Glycine reuptake inhibitors specifically elevated glycine but not D-serine levels in rat prefrontal cortical (PFC) microdialysates, while glycine and D-serine markedly increased levels of glycine and D-serine, respectively. D-Cycloserine slightly elevated D-serine levels. Conversely, S18841 exerted no influence on glycine, D-serine, other amino acids, monamines, or acetylcholine. Reversal of NOR deficits by systemic S18841 was prevented by the NMDA receptor antagonist, CPP (20 mg/kg), and the glycine modulatory site antagonist, L701,324 (10 mg/kg). S18841 blocked deficits in NOR following microinjection into the PFC (2.5-10 µg/side) but not the striatum. Finally, in rats socially isolated from weaning (a neurodevelopmental model of schizophrenia), S18841 (2.5 and 10 mg/kg s.c.) reversed impairment of NOR and contextual fear-motivated learning without altering isolation-induced hyperactivity. In conclusion, despite contrasting neurochemical profiles, partial glycine site agonists and glycine reuptake inhibitors exhibit comparable pro-cognitive effects in rats of potential relevance to treatment of schizophrenia and other brain disorders where cognitive performance is impaired.
Assuntos
Transtornos Cognitivos/tratamento farmacológico , Cognição/efeitos dos fármacos , Glicinérgicos/farmacologia , Glicina/metabolismo , Memória de Curto Prazo/efeitos dos fármacos , Inibidores da Captação de Neurotransmissores/farmacologia , Nootrópicos/farmacologia , Aminoácidos/análise , Animais , Transtorno do Espectro Autista/tratamento farmacológico , Ciclosserina/farmacologia , Relação Dose-Resposta a Droga , Reação de Congelamento Cataléptica/efeitos dos fármacos , Glicina/agonistas , Glicina/análogos & derivados , Glicina/farmacologia , Masculino , Atividade Motora/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Glicina/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/fisiologia , Reconhecimento Psicológico/efeitos dos fármacos , Sarcosina/farmacologia , Esquizofrenia/tratamento farmacológico , Escopolamina/antagonistas & inibidores , Serina/farmacologia , Comportamento SocialRESUMO
N-methyl-d-aspartate (NMDA)-type ionotropic glutamate receptors mediate excitatory neurotransmission in the central nervous system and are critically involved in brain function. NMDA receptors are also implicated in psychiatric and neurological disorders and have received considerable attention as therapeutic targets. In this regard, administration of d-cycloserine (DCS), which is a glycine site NMDA receptor agonist, can enhance extinction of conditioned fear responses. The intriguing behavioral effects of DCS have been linked to its unique pharmacological profile among NMDA receptor subtypes (GluN1/2A-D), in which DCS is a superagonist at GluN2C-containing receptors compared with glycine and a partial agonist at GluN2B-containing receptors. Here, we identify (R)-2-amino-3-(4-(2-ethylphenyl)-1H-indole-2-carboxamido)propanoic acid (AICP) as a glycine site agonist with unique GluN2-dependent differences in agonist efficacy at recombinant NMDA receptor subtypes. AICP is a full agonist at GluN1/2A (100% response compared with glycine), a partial agonist at GluN1/2B and GluN1/2D (10% and 27%, respectively), and a highly efficacious superagonist at GluN1/2C receptors (353%). Furthermore, AICP potencies are enhanced compared with DCS with EC50 values in the low nanomolar range (1.7 nM at GluN1/2C). We show that GluN1/2C superagonism of AICP and DCS is mediated by overlapping but distinct mechanisms and that AICP selectively enhances responses from recombinant GluN1/2C receptors in the presence of physiological glycine concentrations. This functional selectivity of AICP for GluN2C-containing NMDA receptors is more pronounced compared with DCS, suggesting that AICP can be a useful tool compound for uncovering the roles of GluN2C subunits in neuronal circuit function and in the development of new therapeutic strategies.
Assuntos
Agonistas de Aminoácidos Excitatórios/metabolismo , Glicina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/farmacologia , Glicina/agonistas , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/agonistas , Ratos , Receptores de N-Metil-D-Aspartato/agonistas , Xenopus laevisRESUMO
The extensive use of copper oxide nanoparticles (CuO NPs) in many applications has raised concerns over their toxicity on environment and human health. Herein, the embryotoxicity of CuO NPs was assessed in the black sea urchin Arbacia lixula, an intertidal species commonly present in the Mediterranean. Fertilized eggs were exposed to 0.7, 10 and 20ppb of CuO NPs, until pluteus stage. Interferences with the normal neurotransmission pathways were observed in sea urchin embryos. In detail, evidence of cholinergic and serotoninergic systems affection was revealed by dose-dependent decreased levels of choline and N-acetyl serotonin, respectively, measured by nuclear magnetic resonance (NMR)-based metabolomics, applied for the first time to our knowledge on sea urchin embryos. The metabolic profile also highlighted a significant CuO NP dose-dependent increase of glycine, a component of matrix proteins involved in the biomineralization process, suggesting perturbed skeletogenesis accordingly to skeletal defects in spicule patterning observed previously in the same sea urchin embryos. However, the expression of skeletogenic genes, i.e. SM30 and msp130, did not differ among groups, and therefore altered primary mesenchyme cell (PMC) migration was hypothesized. Other unknown metabolites were detected from the NMR spectra, and their concentrations found to be reflective of the CuO NP exposure levels. Overall, these findings demonstrate the toxic potential of CuO NPs to interfere with neurotransmission and skeletogenesis of sea urchin embryos. The integrated use of embryotoxicity tests and metabolomics represents a highly sensitive and effective tool for assessing the impact of NPs on aquatic biota.
Assuntos
Arbacia/efeitos dos fármacos , Cobre/toxicidade , Nanopartículas Metálicas/toxicidade , Morfogênese/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Zigoto/efeitos dos fármacos , Exoesqueleto/efeitos dos fármacos , Exoesqueleto/crescimento & desenvolvimento , Animais , Arbacia/citologia , Arbacia/crescimento & desenvolvimento , Arbacia/fisiologia , Colina/antagonistas & inibidores , Colina/metabolismo , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Cobre/química , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glicina/agonistas , Glicina/metabolismo , Espectroscopia de Ressonância Magnética , Mar Mediterrâneo , Metabolômica/métodos , Nanopartículas Metálicas/química , Neurônios Serotoninérgicos/citologia , Neurônios Serotoninérgicos/efeitos dos fármacos , Serotonina/análogos & derivados , Serotonina/química , Serotonina/metabolismo , Sicília , Propriedades de Superfície , Teratogênicos/toxicidade , Poluentes Químicos da Água/química , Zigoto/citologia , Zigoto/crescimento & desenvolvimentoRESUMO
Taurine acts as a partial agonist at the glycine receptor (GlyR) in some brain regions such as the hippocampus, striatum, and nucleus accumbens. Ethanol, volatile anesthetics, and inhaled drugs of abuse are all known positive allosteric modulators of GlyRs, but their effects on taurine-activated GlyRs remain poorly understood, especially their effects on the high concentrations of taurine likely to be found after synaptic release. Two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes was used to compare the enhancing effects of ethanol, anesthetics, and inhalants on human homomeric α1-GlyR activated by saturating concentrations of glycine versus taurine. Allosteric modulators had negligible effects on glycine-activated GlyR while potentiating taurine-activated currents. In addition, inhaled anesthetics markedly enhanced desensitization rates of taurine- but not glycine-activated receptors. Our findings suggest that ethanol, volatile anesthetics, and inhalants differentially affect the time courses of synaptic events at GlyR, depending on whether the receptor is activated by a full or partial agonist.
Assuntos
Receptores de Glicina/agonistas , Regulação Alostérica/efeitos dos fármacos , Anestésicos/agonistas , Anestésicos/farmacologia , Animais , Agonismo Parcial de Drogas , Etanol/agonistas , Etanol/farmacologia , Glicina/agonistas , Glicina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Receptores de Glicina/metabolismo , Taurina/agonistas , Taurina/farmacologia , Xenopus laevis/metabolismoRESUMO
Inhibitory glycine receptors (GlyRs) are pentameric ligand gated ion channels composed of α and ß subunits assembled in a 2:3 stoichiometry. The α1/ßheteromer is considered the dominant GlyR isoform at 'native' adult synapses in the spinal cord and brainstem. However, the α3 GlyR subunit is concentrated in the superficial dorsal horn (SDH: laminae I-II), a spinal cord region important for processing nociceptive signals from skin, muscle and viscera. Here we use the spasmodic mouse, which has a naturally occurring mutation (A52S) in the α1 subunit of the GlyR, to examine the effect of the mutation on inhibitory synaptic transmission and homeostatic plasticity, and to probe for the presence of various GlyR subunits in the SDH.We usedwhole cell recording (at 22-24â¦C) in lumbar spinal cord slices obtained from ketamine-anaesthetized (100 mg kg⻹, I.P.) spasmodic and wild-type mice (mean age P27 and P29, respectively, both sexes). The amplitude and decay time constants of GlyR mediated mIPSCs in spasmodic micewere reduced by 25% and 50%, respectively (42.0 ± 3.6 pA vs. 31.0 ± 1.8 pA, P <0.05 and 7.4 ± 0.5 ms vs. 5.0 ± 0.4 ms, P <0.05; means ± SEM, n =34 and 31, respectively). Examination of mIPSC amplitude versus rise time and decay time relationships showed these differences were not due to electrotonic effects. Analysis of GABAAergic mIPSCs and A-type potassium currents revealed altered GlyR mediated neurotransmission was not accompanied by the synaptic or intrinsic homeostatic plasticity previously demonstrated in another GlyR mutant, spastic. Application of glycine to excised outside-out patches from SDH neurones showed glycine sensitivity was reduced more than twofold in spasmodic GlyRs (EC50 =130 ± 20 µM vs. 64 ± 11 µM, respectively; n =8 and 15, respectively). Differential agonist sensitivity and mIPSC decay times were subsequently used to probe for the presence of α1-containing GlyRs in SDHneurones.Glycine sensitivity, based on the response to 1-3 µM glycine, was reduced in>75% of neurones tested and decay times were faster in the spasmodic sample. Together, our data suggest most GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design therapies that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.
Assuntos
Potenciais Pós-Sinápticos Inibidores/fisiologia , Células do Corno Posterior/fisiologia , Receptores de Glicina/fisiologia , Animais , Feminino , Glicina/agonistas , Glicina/farmacologia , Glicinérgicos/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Mutação Puntual , Células do Corno Posterior/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Receptores de GABA/efeitos dos fármacos , Receptores de GABA/fisiologia , Receptores de Glicina/agonistas , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
The rare amino acid isovaline has analgesic properties in pain models and is a structural analogue of the inhibitory neurotransmitter glycine. Glycinergic inhibition is prevalent in pain pathways. In this paper, we examined the possibility that isovaline inhibits neurons by activating strychnine (Str)-sensitive glycine(A) receptors in ventrobasal thalamus. Sagittal brain sections containing ventrobasal nuclei were prepared from P10-P15 rats. Whole-cell recordings were made in current-clamp and voltage-clamp modes. R-isovaline (R-Iva) increased input conductance and hyperpolarized the membrane. The conductance increase shunted action potentials and low-threshold Ca(2+) spikes evoked by current pulse injection. Unlike the Cl(-)-mediated responses to glycine, isovaline responses were insensitive to Str antagonism and usually not reversible. The concentration-response curve was non-sigmoidal, rising to a maximum at approximately 100 microM, and thereafter declining in amplitude. Current-voltage relationships showed that isovaline increased inward and outward rectification. The isovaline current reversed polarity close to the K(+) equilibrium potential. The relationships were negligibly affected by tetrodotoxin (TTX), chelation of intracellular Ca(2+) or blockade of the hyperpolarization-activated current, I(h). Internal Cs(+) and external Ba(2+) or Cs(+) prevented isovaline responses. In conclusion, isovaline inhibited firing mainly by activating rectifying and possibly leak K(+) currents. Isovaline-induced changes shunted action potentials and suppressed rebound excitation in ventrobasal neurons, as expected for analgesic actions.
Assuntos
Analgésicos/farmacologia , Neurônios/efeitos dos fármacos , Dor/tratamento farmacológico , Canais de Potássio/efeitos dos fármacos , Valina/farmacologia , Núcleos Ventrais do Tálamo/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Analgésicos/uso terapêutico , Animais , Animais Recém-Nascidos , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Relação Dose-Resposta a Droga , Glicina/agonistas , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Dor/metabolismo , Dor/fisiopatologia , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/metabolismo , Valina/uso terapêutico , Núcleos Ventrais do Tálamo/metabolismoRESUMO
RATIONALE: Many N-methyl-D-aspartate (NMDA) antagonists produce phencyclidine (PCP)-like side effects that limit their clinical utility. NMDA glycine-site antagonists may be less likely to produce these effects than other site-selective NMDA antagonists. OBJECTIVES: The objective of the study is to compare the discriminative stimulus effects of novel NMDA glycine-site drugs to those of channel blocking and competitive NMDA antagonists. MATERIALS AND METHODS: Drug discrimination studies were performed in separate groups of rats trained with saline vs. PCP (2 mg/kg i.p.) or the competitive antagonist NPC 17742 (4 mg/kg i.p.) using a standard two-lever operant conditioning procedure under an FR32. RESULTS: Neither the partial glycine-site agonists aminocyclopropane carboxylic acid methyl ester and (+)-HA-966 nor the antagonists L701,324; MDL 100,458; MDL 100,748; MDL 103,371; MDL 104,472; MDL 105,519; MRZ 2/571; MRZ 2/576; and ACEA 0762 produced >50% PCP-lever selection, though all were tested over a sufficient dose range to produce response rate decreasing effects. All of the antagonists, except MDL 100,458 and MDL 100,748, were also tested for NPC 17742-like effects, producing somewhat more variable results than in PCP-trained rats. ACEA-0762 produced full substitution for NPC 17742, whereas MDL 105,519 produced no substitution. The remaining compounds engendered between 20% and 80% drug-lever selection. CONCLUSION: These results provide evidence that NMDA glycine-site partial agonists and antagonists generally do not produce discriminative stimulus effects similar to those of representative NMDA channel blockers or competitive antagonists. This suggests that these NMDA glycine-site antagonists should be less likely to produce the undesirable behavioral side effects seen in clinical trials with many other NMDA antagonists.
Assuntos
Aminoácidos/farmacologia , Discriminação Psicológica/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/antagonistas & inibidores , Fenciclidina/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Comportamento Animal/efeitos dos fármacos , Sítios de Ligação , Relação Dose-Resposta a Droga , Glicina/agonistas , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/agonistasRESUMO
The anticonvulsant effect of felbamate (FBM) is ascribable to inhibition of N-methyl-d-aspartate (NMDA) currents. Using electrophysiological studies in rat hippocampal neurons to examine the kinetics of FBM binding to and unbinding from the NMDA channel, we show that FBM modifies NMDA channel gating via a one-to-one binding stoichiometry and has quantitatively the same enhancement effect on NMDA and glycine binding to the NMDA channel. Moreover, the binding rates of FBM to the closed and the open/desensitized NMDA channels are 187.5 and 4.6 x 10(4) M(-1) s(-1), respectively. The unbinding rates of FBM from the closed and the open/desensitized NMDA channels are approximately 6.2 x 10(-2) and approximately 3.1 s(-1), respectively. From the binding and unbinding rate constants, apparent dissociation constants of approximately 300 and approximately 70 microM could be calculated for FBM binding to the closed and the open/desensitized NMDA channels, respectively. The slight (approximately fourfold) difference in FBM binding affinity to the closed and to the open/desensitized NMDA channels thus is composed of much larger differences in the binding and unbinding kinetics (approximately 250- and approximately 60-fold difference, respectively). These findings suggest that the effects of NMDA and glycine binding coalesce or are interrelated before or at the actual activation gate, and FBM binding seems to modulate NMDA channel gating at or after this coalescing point. Moreover, the entrance zone of the FBM binding site very likely undergoes a much larger conformational change along the gating process than that in the binding region(s) of the binding site. In other words, the FBM binding site becomes much more accessible to FBM with NMDA channel activation, although the spatial configurations of the binding ligand(s) for FBM themselves are not altered so much along the gating process.
Assuntos
Fenilcarbamatos/metabolismo , Propilenoglicóis/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Animais Recém-Nascidos , Anticonvulsivantes/metabolismo , Anticonvulsivantes/farmacologia , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , Felbamato , Glicina/agonistas , Glicina/antagonistas & inibidores , Glicina/metabolismo , Técnicas In Vitro , Ativação do Canal Iônico , Cinética , N-Metilaspartato/metabolismo , Neurônios/metabolismo , Fenilcarbamatos/farmacologia , Propilenoglicóis/farmacologia , Conformação Proteica , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
The acute administration of the noncompetitive glutamate N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK 801) is known to increase central dopaminergic activity in rats and to elicit schizophreniform behavior in human. The current study was undertaken to compare the effects of different acute or chronic neuroleptic treatments, on the response of ventral tegmental area dopamine (DA) neurons to MK 801, using the in vivo electrophysiological paradigm in anesthetized preparations. Sprague Dawley male rats were treated, acutely or chronically during 3 weeks, with saline, olanzapine (10 mg/kg), haloperidol (1 mg/kg) or the combination of haloperidol with D-serine (1 mg/kg/300 mg/kg), a gliotransmitter coagonist of the NMDA receptor that has been shown to improve the efficacy of typical neuroleptics. In control animals, the acute administration of MK 801 (0.5 mg/kg, i.v.) increased significantly both the firing and burst activity of DA neurons by 20 and 26%, respectively, the latter effect being partially reversed by the selective 5-HT2A antagonist M 100,907 (0.4 mg/kg, i.v.). The acute preadministration of haloperidol (1 mg/kg, i.p.) and olanzapine (10 mg/kg, i.p.) failed to prevent or reverse the activatory effect of MK 801 on firing activity. On the other hand, MK 801-induced burst activity, was partially prevented by olanzapine, but not by haloperidol pretreatment. All antipsychotic treatments, when administered chronically, prevent the activatory effect of MK 801 on both firing and burst activity, and occasionally convert the response to MK 801 on burst activity to an inhibitory response, the latter occurring more predominantly in rats treated with the combination haloperidol/D-serine. These results suggest that a chronic antipsychotic regime alters the function of the NMDA receptors that tonically control the firing activity of midbrain dopaminergic neurons.
Assuntos
Antipsicóticos/farmacologia , Dopamina/metabolismo , Mesencéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Serina/farmacologia , Animais , Benzodiazepinas/farmacologia , Maleato de Dizocilpina/farmacologia , Eletrofisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/agonistas , Haloperidol/farmacologia , Masculino , Mesencéfalo/metabolismo , Microeletrodos , Neurônios/metabolismo , Olanzapina , Ratos , Ratos Sprague-DawleyRESUMO
Conditioned flavor-taste preference (CFTP) is a robust form of learning in which animals acquire a preference for a flavor (e.g. Kool-Aid) previously mixed with a highly preferred tastant (e.g. fructose) over a flavor previously mixed with a less-preferred tastant (e.g. saccharin). Here, the role of the N-methyl-D-aspartate (NMDA) glutamate-glycine receptor (NR) was probed using systemic MK-801, a non-competitive antagonist, and D-cycloserine (DCS), a glycine agonist. Rats were injected with MK-801 (100 microg/kg) or vehicle 30 min prior to a daily 2-h conditioning session with 1-bottle access to a Kool-Aid flavor (grape or cherry) mixed with either 8% fructose (CS+/F) or 0.2% saccharin (CS-/S). CFTP expression was measured in 2-bottle preference tests between the Kool-Aid flavors mixed with 0.2% saccharin (CS+/S vs. CS-/S). While vehicle-treated rats acquired a preference for CS+/S over CS-/S, MK-801 prior to conditioning completely blocked CFTP learning. The effect of MK-801 was specific to CFTP acquisition, because follow-up experiments demonstrated that MK-801 did not induce a conditioned taste aversion, cause state-dependent learning, or affect CFTP expression. In a second approach, rats were injected with DCS (15 mg/kg) 60 min prior to daily conditioning. In contrast to MK-801, administration of DCS prior to conditioning enhanced CFTP learning (but not reversal conditioning). These results demonstrate that NR neurotransmission is critical for CFTP learning. Furthermore, enhancement of CFTP learning by DCS suggests that endogenous levels of glycine or D-serine may be a limiting factor in CFTP learning.
Assuntos
Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Ciclosserina/farmacologia , Maleato de Dizocilpina/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Glicina/agonistas , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologia , PaladarRESUMO
In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277-793nM)). Eight amino acids, which correspond to amino acids that are critical for ligand binding to other NMDA receptor subunits, situated within the S1S2 predicted ligand binding domain of hNR3A were mutated, which resulted in complete or near complete loss of [(3)H]-glycine binding to hNR3A. The NMDA NR1 glycine site agonist d-serine and partial agonist HA-966 (3-amino-1-hydroxypyrrolid-2-one), similarly to glycine displaced [(3)H]-glycine monophasically, suggesting a single common binding site. However, neither the partial agonist d-cycloserine nor the antagonist 7-chlorokynurenic acid displaced [(3)H]-glycine. Using homology modelling, a model of the NR3A binding pocket was generated which we suggest can be used to identify candidate agonists and antagonists. Our data show that glycine is a ligand, and most probably the endogenous ligand, for native NR3A at a binding site with unique pharmacological characteristics.
Assuntos
Ligação Competitiva/efeitos dos fármacos , Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Linhagem Celular Transformada , Ciclosserina/farmacologia , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/agonistas , Glicina/antagonistas & inibidores , Humanos , Ácido Cinurênico/análogos & derivados , Ácido Cinurênico/farmacologia , Modelos Moleculares , Mutação/fisiologia , Ligação Proteica/efeitos dos fármacos , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , TransfecçãoRESUMO
We report the development of an intracellular recording technique for adult mouse motoneurons in sacral spinal cord. Based on a similar preparation for adult rat, we modified the cord preparation solution and filled the sharp electrode with a solution that has physiological osmolarity and pH. The viability of the preparation was examined by recording root reflexes. Short-latency reflexes mediated through monosynaptic transmission between S1 and S3 ventral root were reliably produced by dorsal root electrical stimuli and were stably recorded for more than eight hours. Long-lasting potentiation of the root reflex was observed by bath application of methoxamine, a noradrenergic alpha1 receptor agonist. Bath application of strychnine and picrotoxin, antagonists for glycine and GABA(A) receptors respectively, unmasked long-lasting reflexes that may contain polysynaptic components. In addition, on the background of strychnine and picrotoxin, adding methoxamine induced spontaneous ventral root activity. For intracellular recording, the motoneurons could be reliably penetrated and held for up to 30 min. In all 16 motoneurons recorded, resting membrane potential, input resistance, action potentials and repetitive firing were comparable to those of rat motoneurons. Thus, this preparation is viable and provides a new method for combined electrophysiological and genetic studies of the adult mouse spinal cord.
Assuntos
Neurônios Motores/fisiologia , Medula Espinal/fisiologia , Potenciais de Ação/efeitos dos fármacos , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Estimulação Elétrica , Eletrodos , Eletrofisiologia , Agonistas GABAérgicos/farmacologia , Glicina/agonistas , Técnicas In Vitro , Metoxamina/farmacologia , Camundongos , Reflexo/efeitos dos fármacos , Medula Espinal/citologia , Raízes Nervosas Espinhais/fisiologia , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
For decades neuroreceptor research has focused on the development of NMDA glycine-site antagonists, after Johnson and Ascher found out in 1987 about the co-agonistic character of this achiral amino acid at the NMDA receptor. Contrary to the inhibitory glycine receptor (glycine(A)) the glycine binding site on the NMDA receptor (glycine(B)) is strychnine-insensitive. A great diversity of diseases showing a disturbed glutamate neurotransmission have been linked to the NMDA receptor. Glycine site antagonists have been investigated for acute diseases like stroke and head trauma as well as chronic ones like dementia and chronic pain.
Assuntos
Fármacos Neuroprotetores/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Anticonvulsivantes/uso terapêutico , Sítios de Ligação , Demência/tratamento farmacológico , Epilepsia/tratamento farmacológico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Glicina/agonistas , Glicina/antagonistas & inibidores , Glicina/química , Humanos , Memantina/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Dor/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/química , Esquizofrenia/tratamento farmacológico , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.
Assuntos
Resistência a Medicamentos/genética , Glicina/metabolismo , Hipercinese/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Antipsicóticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/genética , Aminas Biogênicas/metabolismo , Corpo Estriado/química , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Agonistas GABAérgicos/farmacologia , Marcação de Genes , Ácido Glutâmico/farmacocinética , Glicina/agonistas , Glicina/farmacocinética , Heterozigoto , Hipocampo/fisiopatologia , Hipercinese/fisiopatologia , Técnicas In Vitro , Potenciação de Longa Duração/genética , Camundongos , Camundongos Mutantes Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Fenótipo , Mutação Puntual , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Serina/análogos & derivados , Serina/farmacologia , Estereoisomerismo , Comportamento Estereotipado/efeitos dos fármacosRESUMO
NMDA receptor currents desensitize in an agonist-dependent manner when either the glutamate or glycine agonist is subsaturating. This may result from a conformational change in the NMDA receptor protein that lowers glutamate and glycine binding site affinity induced by co-agonist binding, channel opening, or ion permeation. We have used whole-cell voltage clamp of cultured hippocampal neurons with agonist paired-pulse protocols to demonstrate that glutamate and glycine dissociate 7.9- and 6.8-fold slower in the absence of their respective co-agonists than when their co-agonists are present. Paired-pulse and desensitization protocols were used to show that co-agonist binding and channel opening are sufficient to cause a reduction in glycine affinity, but extracellular sodium or magnesium binding was required in addition to conformational changes leading to channel opening to reduce glutamate binding-site affinity. Use of cesium or potassium as the major extracellular cation prevented the reduction of glutamate affinity. In addition, the use of choline-, sodium-, or cesium-based intracellular solutions did not alter desensitization characteristics, indicating that the site responsible for reduction of glutamate affinity is not in the intracellular domain. The fact that the reduction of glutamate affinity is dependent on certain small extracellular cations whereas the reduction of glycine affinity is insensitive to such cations indicates that conformational changes induced by the binding of glutamate are not completely paralleled by the conformational changes induced by glycine. Although glutamate and glycine are essential co-agonists, these data suggest that they have differential roles in the process of NMDA receptor activation.
Assuntos
Cátions/metabolismo , Agonistas de Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Glicina/agonistas , Glicina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Magnésio/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Conformação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sódio/metabolismoRESUMO
D-Cycloserine, a partial agonist at the glycine recognition site of the N-methyl-D-aspartate (NMDA) receptor complex, has been shown to facilitate certain forms of memory formation and to improve visual recognition memory in normal monkeys. In the present study, the effects of D-cycloserine on spatial short-term memory deficits in monkeys induced by chronic low-dose 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) administration were examined. Chronic low-dose MPTP administration resulted in deficits in the performance of a variable delayed-response task (VDR). Single administration of D-cycloserine (320 or 1000 microgram/kg) significantly improved the performance on this task. High-dose D-cycloserine (8000 microgram/kg) or MK-801 (10-32 microgram/kg) administration had no effects on delayed-response performance but impaired performance on a visual discrimination (VD) task that was not adversely affected by MPTP administration. These results show that at low doses, D-cycloserine has cognition-enhancing properties in this model of early Parkinsonism.
Assuntos
Cognição/efeitos dos fármacos , Ciclosserina/uso terapêutico , Glicina/agonistas , Intoxicação por MPTP/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson Secundária/tratamento farmacológico , Desempenho Psicomotor/efeitos dos fármacos , Animais , Ciclosserina/farmacologia , Ciclosserina/toxicidade , Discriminação Psicológica/efeitos dos fármacos , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/toxicidade , Glicina/antagonistas & inibidores , Intoxicação por MPTP/psicologia , Macaca fascicularis , Masculino , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/toxicidade , Doença de Parkinson Secundária/induzido quimicamente , Tempo de Reação/efeitos dos fármacos , Percepção Visual/efeitos dos fármacosRESUMO
Previously, it was shown that glycine prevented increases in intracellular calcium ([Ca2+]i) in Kupffer cells. Since Kupffer cells and T lymphocytes are derived from the same pluripotent stem cell, it was hypothesized that glycine would prevent increases in [Ca2+]i in lymphocytes and inhibit cell proliferation. Lymphocyte proliferation was measured in one-way MLC with spleen cells from DA and Lewis rats and in enriched T lymphocyte preparations stimulated by immobilized anti-CD3 Ab. Glycine caused a dose-dependent decrease in cell proliferation to about 40% of control. Con A caused a dose-dependent increase in [Ca2+]i in Jurkat cells which was blunted maximally with 0.6 mM glycine. The effect of glycine was dependent on extracellular chloride and reversed by strychnine, an antagonist of the glycine-gated chloride channel. Similar results were obtained with rat T lymphocytes stimulated by anti-CD3 Ab. Surprisingly, glycine had no effect on IL-2 production in the mixed lymphocyte culture; therefore, the effect of glycine on IL-2-dependent proliferation was tested. Glycine and rapamycin caused dose-dependent decreases in IL-2-stimulated growth of Ctll-2 cells to about 60% and 40%, respectively, of control. Moreover, glycine also inhibited the IL-2-stimulated growth of rat splenic lymphocytes. It is concluded that glycine blunts proliferation in an IL-2-independent manner. This is consistent with the hypothesis that glycine activates a glycine-gated chloride channel and hyperpolarizes the cell membrane-blunting increases in [Ca2+]i that are required for transcription of factors necessary for cell proliferation.
Assuntos
Glicina/fisiologia , Inibidores do Crescimento/fisiologia , Interleucina-2/fisiologia , Linfócitos T/citologia , Animais , Complexo CD3/imunologia , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Agonistas dos Canais de Cloreto , Concanavalina A/farmacologia , Ciclosporina/farmacologia , Feminino , Glicina/agonistas , Inibidores do Crescimento/agonistas , Inibidores do Crescimento/farmacologia , Humanos , Soros Imunes/farmacologia , Líquido Intracelular/metabolismo , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Ratos , Ratos Endogâmicos Lew , Estricnina/agonistas , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
N-Methyl-D-aspartate receptor channels are composed of an NR1 subunit and at least one of the NR2 subunits (NR2A-D). Activation of the N-methyl-d-aspartate receptor requires the co-agonists glycine and glutamate. It has been proposed that the NR1 subunit possesses a glycine-binding site. We have expressed a soluble form of the NR1 subunit, which was produced by connecting the N-terminal extracellular region with the extracellular loop between the third and fourth membrane segments, by a baculovirus system along with full-length and truncated membrane-bound forms. The soluble NR1 receptor was efficiently secreted into the culture medium and showed a high affinity for ligands. The Kd of a glycine-site antagonist, [3H]MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4, 6-dichloro-1H-indole-2-carboxylic acid], for the soluble receptor was 3.89+/-0.97 nM, which was comparable to the Kd of 4.47+/-1.39 nM for the membrane-bound full-length form. These values were close to the values reported previously with the use of rat brain membranes and Chinese hamster ovary cells expressing the full-length form of the NR1 subunit. The Ki values of other glycine-site antagonists, L-689,560 (trans-2-carboxy-5,7-dichloro - 4 - phenylaminocarbonylamino - 1,2,3,4 - tetrahydroquinoline), 5, 7-dichlorokynurenate and 5,7-dinitroquinoxaline-2,3-dione, for the soluble receptor were also similar to those for the full-length form of NR1. [3H]MDL 105,519 binding was also inhibited by the agonists glycine and d-serine. Thus the affinity and selectivity of ligand-binding characteristics of the NR1 subunit is conferred on the soluble form of the NR1 subunit. This soluble receptor provides a good experimental tool for initiating a biophysical analysis of the N-methyl-d-aspartate receptor channel protein.
Assuntos
Glicina/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Amidoidrolases/metabolismo , Aminoquinolinas/metabolismo , Animais , Baculoviridae/genética , Sítios de Ligação , Western Blotting , Linhagem Celular , Vetores Genéticos/genética , Glicina/agonistas , Glicina/antagonistas & inibidores , Glicosilação , Indóis/antagonistas & inibidores , Indóis/metabolismo , Concentração Inibidora 50 , Insetos/citologia , Insetos/genética , Ligantes , Peso Molecular , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/biossíntese , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
1-Aminocyclopropanecarboxylic acid (ACPC) is a high affinity ligand at strychnine-insensitive glycine sites of the N-methyl-D-aspartate (NMDA) channels and exhibits partial agonist properties in both biochemical and electrophysiological measures. While ACPC was reported active in animal models used to evaluate potential antidepressants and anxiolytics, its effects on learning and memory are unknown. In the present study we investigated the effects of ACPC on spatial learning in the Morris water maze. On a schedule of 12 learning trials, one trial per day, mature male Wistar rats (3 months of age) rapidly acquired the task. Electroconvulsive shocks applied after each of the learning trials markedly inhibited the consolidation of spatial memory. Administration of either a muscarinic agonist, arecoline (1 mg/kg) or ACPC (250 or 400 mg/kg) 20 min before each of the learning trials did not affect the acquisition of spatial learning. Aged (16 months old) male Wistar rats demonstrated difficulties in the acquisition of spatial learning task. In these subjects, ACPC administered 20 min before each of the learning trials at a dose of 400, but not 250 mg/kg, facilitated the acquisition of spatial memory as indicated on trials 3-5. ACPC did not affect the strength of spatial memory as assessed at the end of conditioning, by measuring swimming behavior of rats in the pool with platform removed. It is suggested that ACPC may alleviate learning deficits observed in the elderly.