RESUMEN
Fibroblast growth factor-2 (FGF-2) plays a fundamental role in brain functions. This role may be partly achieved through the control of its expression at the translational level via an internal ribosome entry site (IRES)-dependent mechanism. Transgenic mice expressing a bicistronic mRNA allowed us to study in vivo and ex vivo where this translational mechanism operates. Along brain development, we identified a stringent spatiotemporal regulation of FGF-2 IRES activity showing a peak at post-natal day 7 in most brain regions, which is concomitant with neuronal maturation. At adult age, this activity remained relatively high in forebrain regions. By the enrichment of this activity in forebrain synaptoneurosomes and by the use of primary cultures of cortical neurons or cocultures with astrocytes, we showed that this activity is indeed localized in neurons, is dependent on their maturation, and correlates with endogenous FGF-2 protein expression. In addition, this activity was regulated by astrocyte factors, including FGF-2, and spontaneous electrical activity. Thus, neuronal IRES-driven translation of the FGF-2 mRNA may be involved in synapse formation and maturation.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Biosíntesis de Proteínas , Ribosomas/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Sinapsis Eléctricas/fisiología , Ratones , Ratones Transgénicos , Modelos Animales , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Receptores de Glutamato/metabolismoRESUMEN
Naturally occurring polyphenols are potent antioxidants. Some of these compounds are also ligands for the GABA(A) receptor benzodiazepine site. This feature endows them with sedative properties. Here, the anxiolytic activity of the green tea polyphenol (-)-epigallocatechin gallate (EGCG) was investigated after acute administration in mice, using behavioral tests (elevated plus-maze and passive avoidance tests) and by electrophysiology on cultured hippocampal neurons. Patch-clamp experiments revealed that EGCG (1-10 muM) had no effect on GABA currents. However, EGCG reversed GABA(A) receptor negative modulator methyl beta-carboline-3-carboxylate (beta-CCM) inhibition on GABA currents in a concentration dependent manner. This was also observed at the level of synaptic GABA(A) receptors by recording spontaneous inhibitory synaptic transmission. In addition, EGCG consistently inhibited spontaneous excitatory synaptic transmission. Behavioral tests indicated that EGCG exerted both anxiolytic and amnesic effects just like the benzodiazepine drug, chlordiazepoxide. Indeed, EGCG in a dose-dependent manner both increased the time spent in open arms of the plus-maze and decreased the step-down latency in the passive avoidance test. GABA(A) negative modulator beta-CCM antagonized EGCG-induced amnesia. Finally, state-dependent learning was observable after chlordiazepoxide and EGCG administration using a modified passive avoidance procedure. Optimal retention was observed only when animals were trained and tested in the same state (veh-veh or drug-drug) and significant retrieval alteration was observed in different states (veh-drug or drug-veh). Moreover, EGCG and chlordiazepoxide fully generalized in substitution studies, indicating that they induced indistinguishable chemical states for the brain. Therefore, our data support that EGCG can induce anxiolytic activity which could result from an interaction with GABA(A) receptors.
Asunto(s)
Ansiolíticos/uso terapéutico , Ansiedad/tratamiento farmacológico , Catequina/análogos & derivados , Animales , Ansiedad/etiología , Reacción de Prevención/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Carbolinas/farmacología , Catequina/uso terapéutico , Células Cultivadas , Clordiazepóxido/farmacología , Convulsivantes/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Electrochoque/efectos adversos , Embrión de Mamíferos , Hipocampo/citología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Tiempo de Reacción/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Neuroprotection exerted by alpha-tocopherol against oxidative stress was investigated in cultured rat hippocampal neurons. In addition to its direct action as a radical scavenger revealed at concentrations above 10 microM, a transient application of 1 microM alpha-tocopherol phosphate (alpha-TP) to neurons induced a complete delayed long-lasting protection against oxidative insult elicited by exposure to Fe2+ ions, but not against excitotoxicity. A minimal 16-h application of alpha-TP was required to observe the protection against subsequent oxidative stress. This delayed protection could last up to a week after the application of alpha-TP, even when medium was changed after the alpha-TP treatment. Cycloheximide, added either 2 h before or together with alpha-TP, prevented the delayed neuroprotection, but not the acute. However, cycloheximide applied after the 16-h alpha-TP pretreatment did not alter the delayed neuroprotection. Neither Trolox, a cell-permeant analogue of alpha-tocopherol, nor other antioxidants, such as epigallocatechin-gallate and N-acetyl-L-cysteine, elicited a similar long-lasting protection. Only tert-butylhydroquinone could mimic the alpha-TP effect. Depletion of glutathione (GSH) by L-buthionine sulfoximine did not affect the delayed alpha-TP protection. Thus, in addition to its acute anti-radical action, alpha-TP induces a long-lasting protection of neurons against oxidative damage, via a genomic action on antioxidant defenses apparently unrelated to GSH biosynthesis.
Asunto(s)
Depuradores de Radicales Libres/farmacología , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , alfa-Tocoferol/farmacología , Animales , Células Cultivadas , Cicloheximida/toxicidad , Genoma/efectos de los fármacos , Glutatión/deficiencia , Hipocampo/citología , Hierro/toxicidad , Estrés Oxidativo/genética , Inhibidores de la Síntesis de la Proteína/toxicidad , RatasRESUMEN
1. Stress during pregnancy results in complex neurochemical and behavioral alterations throughout the offspring lifetime. We here examined the impact of prenatal stress (PS) on memory functions in male and female offspring and report the efficacy of a selective sigma(1) (sigma(1)) receptor agonist, igmesine, in alleviating the observed deficits. 2. Dams received an unpredictable 90-min duration restraint stress from gestational day E17 to E20. Learning was examined in offspring between day P24 and P36 using spontaneous alternation in the Y-maze, delayed alternation in the T-maze, water-maze learning and passive avoidance. 3. Both male and female PS rats showed impairments of spontaneous and delayed alternation performances. Acquisition of a fixed platform position in the water-maze was unchanged in PS rats, but the probe test revealed a diminution of time spent in the training quadrant. Acquisition of a daily changing platform position demonstrated impaired working memory for male and female PS rats. Finally, passive avoidance deficits were observed. 4. Pretreatment with the selective sigma(1) agonist igmesine (1-10 mg x kg(-1) i.p.) reversed the PS-induced learning deficits in offspring rats for each test. The sigma(1) antagonist BD1063 failed to affect performances alone but blocked the igmesine effect, confirming the involvement of the sigma(1) receptor. 5. PS thus induces delayed memory deficits, affecting spatial and nonspatial, short- and long-term memories in juvenile male and female offspring rats. Activation of the sigma(1) neuromodulatory receptor allows a significant recovery of the memory functions in PS rats.
Asunto(s)
Discapacidades para el Aprendizaje/tratamiento farmacológico , Exposición Materna/efectos adversos , Trastornos de la Memoria/tratamiento farmacológico , Receptores sigma/agonistas , Receptores sigma/uso terapéutico , Restricción Física/efectos adversos , Animales , Animales Recién Nacidos/fisiología , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Cinamatos/antagonistas & inhibidores , Cinamatos/farmacología , Cinamatos/uso terapéutico , Ciclopropanos/antagonistas & inhibidores , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Femenino , Francia , Edad Gestacional , Inyecciones Intraperitoneales , Discapacidades para el Aprendizaje/etiología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/etiología , Piperazinas/farmacología , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Sprague-Dawley , Receptores sigma/administración & dosificación , Restricción Física/métodos , Factores de TiempoRESUMEN
To evaluate the involvement of AMPA receptor activation in neuronal cell death and survival, rat hippocampal neurons in culture were treated with AMPA receptor antagonists. A 46 h treatment with 6,7-dinitroquinoxaline-2,3-dione (DNQX), added 2 h after cell plating, induces a dose-dependent neurotoxicity. Similar effects are also observed in more mature hippocampal neurons (treatment at 14 days in vitro). DNQX toxic effect is neuron-specific since cultured hippocampal glial cells are unaffected. Attempts to characterise the site of action of DNQX suggest that ionotropic glutamate receptors would not be implicated. Indeed, (i) other AMPA receptor antagonists are either ineffective or only moderately efficient in mimicking DNQX effects; (ii) AMPA alone or in the presence of cyclothiazide, as well as, other AMPA receptor agonists, do not reverse DNQX action; (iii) DNQX neurotoxicity is not likely to involve blockade of NMDA receptor glycine site, since this effect is neither mimicked by 7-chlorokynurenate nor reversed by D-serine. Thus, DNQX toxicity in cultured hippocampal neurons is apparently mediated through an ionotropic glutamate receptor-independent way.
Asunto(s)
Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Quinoxalinas/toxicidad , Receptores AMPA/metabolismo , Animales , Células Cultivadas , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/citología , Hipocampo/metabolismo , Inmunohistoquímica , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidoresRESUMEN
Glutamate extracellular levels are regulated by specific transporters. Five subtypes have been identified. The two major ones, GLAST and GLT (glutamate transporters 1 and 2, respectively), are localized in astroglia in normal mature brain. However, in neuron-enriched hippocampal cultures, these proteins are expressed in neurons during the early in vitro development (Plachez et al., 2000). Here, we show that, in these cultures, GLAST and GLT neuronal expression is transient and no longer observed after 7 days in vitro, a stage at which the few astrocytes present in the culture are maturing. Moreover, we demonstrate that these few astrocytes are responsible for the repression of this neuronal expression. Indeed, addition of conditioned medium prepared from primary cultures of hippocampal astrocytes, to cultured hippocampal neurons, rapidly leads to the suppression of neuronal GLAST expression, without affecting neuronal GLT expression. However, when neurons are seeded and co-cultured on a layer of hippocampal astrocytes, they do not develop any immunoreactivity towards GLAST or GLT antibodies. Altogether, these results indicate that glia modulate the expression of GLAST and GLT glutamate transporters in neurons, via at least two distinct mechanisms. Neuronal GLAST expression is likely repressed via the release or the uptake of soluble factors by glia. The repression of neuronal GLT expression probably results from glia-neuron interactions. This further reinforces the fundamental role of direct or indirect neuron-glia interactions in the development of the central nervous system.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/biosíntesis , Astrocitos/metabolismo , Transportador 2 de Aminoácidos Excitadores/biosíntesis , Hipocampo/metabolismo , Neuronas/metabolismo , Sistema de Transporte de Aminoácidos X-AG/antagonistas & inhibidores , Animales , Astrocitos/citología , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Embrión de Mamíferos , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Regulación del Desarrollo de la Expresión Génica , Hipocampo/citología , Neuronas/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Stress during pregnancy results in neurochemical and behavioral alterations observed throughout adulthood and aging. We here examined the impact of prenatal stress (PS) on cognitive functions in juvenile-4-week-old-rats, focusing on putative sex differences. Dams received an unpredictable 90-min duration contention stress between gestational day E17 and E20. Locomotion and learning ability were examined in offsprings between day P24 and P29 using actimetry, spontaneous alternation in the Y-maze, delayed alternation in the T-maze, and passive avoidance. Both male and female PS rats showed increased activity. In the Y-maze, diminished spontaneous alternation (males: -20%; females: -29%) were seen for PS rats compared to non-PS rats. The number of arm entries was unchanged among groups. In the T-maze, PS rats failed to perform delayed alternation, as shown by equal time spent and number of entries in both the novel and previously explored arms. In the passive avoidance test, PS resulted in significant impairments for female offspring only of both step-through latency and percentage of animals to criterion. PS thus induced severe learning impairments affecting both short-term and long-term memories that could be observed early in lifetime, in 4-week-old, juvenile rats. In addition, marked sex differences were evidenced, particularly in the passive avoidance response that may reflect differential developmental neuroadaptations in precise brain structures.
Asunto(s)
Discapacidades para el Aprendizaje/etiología , Efectos Tardíos de la Exposición Prenatal , Estrés Psicológico/psicología , Animales , Reacción de Prevención/fisiología , Femenino , Discapacidades para el Aprendizaje/psicología , Masculino , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Memoria a Corto Plazo/fisiología , Actividad Motora/fisiología , Embarazo , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Estrés Psicológico/complicacionesRESUMEN
L-glutamate (Glu), the main excitatory amino acid neurotransmitter in the mammalian central nervous system, is involved in many physiological functions, including learning and memory, but also in toxic phenomena occurring in numerous degenerative or neurological diseases. These functions mainly result from its interaction with Glu receptors (GluRs). The broad spectrum of roles played by glutamate derived from the large number of membrane receptors, which are currently classified in two main categories, ionotropic (iGluRs) and metabotropic (mGluRs) receptors. The iGluRs are ion channels, permeant to Na(+) (Ca(2+)) while the mGluRs belongs to the superfamily of G-protein coupled receptors (GPCRs). Despite continuous efforts over more than two decades, the use of iGluR agonists or antagonists to improve or inhibit excitatory transmission in pathological states still remains a major challenge, though the discovery and development of recent molecules may prove it worthwhile. This probably results form the vital role of fast excitatory transmission in many fundamental physiological functions. Since the discovery of mGluRs, hope has emerged. Indeed, mGluRs are mainly involved in the regulation of fast excitatory transmission. Consequently, it was logically thought that modulating mGluRs with agonists or antagonists might lead to more subtle regulation of fast excitatory transmission than by directly blocking iGluRs. As a result of intensive investigation, new drugs permitting to discriminate between these receptors have emerged. Moreover, a new class of molecules acting as negative or positive allosteric modulators or mGluRs is now available and appears to be promising. In the following, we will review the classification of mGluRs and the functions in which mGluRs are involved. We will focus on their potential as therapeutic targets for improving numerous physiological functions and for different neurodegenerative and neuropsychiatric disorders, which are related to malfunction of Glu signaling in human beings.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Animales , Sistemas de Liberación de Medicamentos/tendencias , Fármacos actuantes sobre Aminoácidos Excitadores/metabolismo , Fármacos actuantes sobre Aminoácidos Excitadores/uso terapéutico , Humanos , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
Low frequency-induced short-term synaptic plasticity was investigated in hippocampal slices with 60-electrode recording array. Remarkably, the application of low-frequency stimulation (1 Hz) for a short duration (3-5 min) resulted in the induction of a slow-onset long-term potentiation (LTP) in the immediate vicinity of the stimulated electrode. This phenomenon was observed exclusively in the CA1 subfield, neither in the CA3 area nor in the dentate gyrus. The induction of this slow-onset LTP required neither N-methyl-D-aspartate (NMDA) nor non-NMDA ionotropic receptor activation but was strongly dependent on metabotropic glutamate mGlu(5) receptor stimulation and [Ca(2+)]i increase. In addition, this form of synaptic plasticity was associated with an increase in cAMP concentration and required protein kinase A activation. Paired-pulse facilitation ratio and presynaptic fiber volley amplitude were unaffected when this LTP was triggered, suggesting the involvement of postsynaptic modifications. Although mitogen activated protein kinase pathway was stimulated after the application of low frequency, the induction and maintenance of this slow-onset LTP were not dependent on the activation of this intracellular pathway. The direct activation of adenylyl cyclase with forskolin also induced a synaptic enhancement displaying similar features. This new form of LTP could represent the mnesic engram of mild and repetitive stimulation involved in latent learning.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Colforsina/farmacología , AMP Cíclico/metabolismo , Estimulación Eléctrica/métodos , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Hipocampo/efectos de los fármacos , Aprendizaje/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Memoria/fisiología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Técnicas de Cultivo de Órganos , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
Extracellular glutamate is kept below a toxic level by glial and neuronal glutamate transporters. Here we show that the transportable glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (t-PDC) induced cell death in mature, but not in immature, hippocampal neuron-enriched cultures. The cell death produced by a 24-hr treatment with t-PDC was dose-dependent and reached 85% of the cell population at a 250 microM concentration at 23 days in vitro (DIV). Immunocytochemistry experiments showed that, under these experimental conditions, t-PDC killed not only neurons as expected but also glial cells. The N-methyl-D-aspartate (NMDA) antagonist D-2-aminophosphonovalerate (D-APV; 250 microM) only partially reversed this toxicity, completely protecting the neuronal cell population but not the glial population. The antioxidant compounds alpha-tocopherol or Trolox, used at concentrations that reverse the oxidative stress-induced toxicity, did not block the gliotoxicity specifically produced by t-PDC in the presence of D-APV. The nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) elicited cell death only in mature, but not in immature, hippocampal cultures. The TBOA toxic effect was dose dependent and reached a plateau at 100 microM in 23-DIV cultures. About 50% of the cell population died. TBOA affected essentially the neuronal population. D-APV (250 microM) completely reversed this toxicity. It is concluded that nontransportable glutamate uptake inhibitors are neurotoxic via overactivation of NMDA receptors, whereas transportable glutamate uptake inhibitors induce both an NMDA-dependent neurotoxicity and an NMDA- and oxidative stress-independent gliotoxicity, but only in mature hippocampal cultures.
Asunto(s)
Ácidos Dicarboxílicos/toxicidad , Ácido Glutámico/toxicidad , Hipocampo/patología , Neuroglía/efectos de los fármacos , Neurotoxinas/farmacología , Inhibidores de la Captación de Neurotransmisores/toxicidad , Pirrolidinas/toxicidad , Sistema de Transporte de Aminoácidos X-AG/efectos de los fármacos , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Ácido Aspártico/toxicidad , Muerte Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Inhibidores de la Captación de Neurotransmisores/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
The effects of adrenergic receptor stimulation on spontaneous synaptic transmission were investigated in cultured rat hippocampal neurons by recording spontaneous excitatory and inhibitory postsynaptic currents (sEPSC and sIPSC). Noradrenaline (NA) inhibited sEPSC in a concentration-dependent manner, with maximal effect at 10 microM. The alpha(1)- and alpha(2)-adrenoceptor-selective agonists cirazoline and clonidine induced an inhibition of sEPSC appearance, whereas the beta-adrenoceptor agonist isoproterenol elicited an increase. The inhibitory effect of NA was reversed by alpha(1)-adrenoceptor blockade. The participation of gamma-aminobutyric acid (GABA)(B)-receptor stimulation in the inhibitory effect of NA was further examined. GABA(B)-receptor stimulation with baclofen induced a strong inhibition of bursting activity, which was fully reversed by the GABA(B) antagonist CGP 55845. By itself, CGP 55845 exerted a stimulatory effect on sEPSC frequency. In the presence of CGP 55845, the inhibitory effects of cirazoline and clonidine were maintained. NA (1, 10, and 100 microM) and alpha-adrenoceptor agonists decreased miniature EPSC and IPSC occurrence, whereas beta-adrenergic stimulation increased it. In 50% of the cells examined, NA (1, 10 microM) had a stimulatory effect on sIPSC, whereas, in the remaining 50% of cells, NA (1, 10 microM) had an inhibitory effect. In all the cells, 100 microM NA induced an inhibition of sIPSC. The inhibitory effect of NA was due to alpha(1)-receptor stimulation, whereas the excitatory effect was due to beta-receptor stimulation. In cultured hippocampal neurons, spontaneous excitatory and inhibitory synaptic transmissions are both similarly altered by adrenoceptor stimulation. However, in a subset of cells, low concentrations of NA mediate an increase of sIPSC via beta-adrenoceptor activation.