Le glutamate induit la production de radicaux libres oxygénés dans le striatum de rat (étude par microdialyse intracérébrale)

L'excitotoxicité glutamatergique et la formation de radicaux libres oxygénés sont associées à de nombreuses pathologies neurodégénératives. Malheureusement, les approches thérapeutiques pour lutter contre l'excitotoxicité et le stress oxydatif n'ont pas pour le moment apporté de réponse satisfaisante, du fait du manque de connaissances fondamentales quant aux mécanisme les reliant. Afin de mieux comprendre ces mécanismes, nous avons choisi d'étudier, in vito, la libération des radicaux libres oxygénés, hydroxyles en particulier, en réponse à une stimulation glutamatergique, au moyen d'une sonde de microdialyse implantée dans le striatum de rat vigile. En utilisant une approche pharmacologique, nous avons montré dans une première étape, que l'intervention des récepteurs de type NMDA est limitée aux circonstances où le glutamate est introduit ( ou libéré par un événement pathologique) de façon massive. Dans une deuxième étape, nous avons mies en lumière le rôle prépondérant du transport astrocytaire du glutamate dans la génération de radicaux hydroxyles lors d'une stimulation par des concentrations plus modérées de glutamate. Pour confirmer que les astrocytes sont probablement responsables de la libération des radicaux hydroxyles dans ces conditions, nous avons utilisé une troisième approche ( basée sur des perfusions répétitives des animaux ). Compte tenu des données de la littérature et de l'ensemble de nos résultats, nous proposons à ce stade de considérer que le transport du glutamate dans les astrocytes joue un rôle-si ce n'est le rôle- fondamental dans la production des radicaux libres oxygénés en réponse à une perturbation de la transmission glutamatergique. Un tel mécanisme pourrait fournir une base conceptuelle à de nouvelles stratégies de protection au moyen de pièges des radicaux libres.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Contrôle glutamatergique de la production des radicaux libres oxygénés chez le rat nouveau-né (mécanismes et contribution à la pathologie développemntales dans le système nerveux central)

Depuis une dizaine d'années on assiste en France à une amélioration régulière et très significative de la survie des grands prématurés. Par contre, la prévalence des séquelles neurologiques à long terme reste stable ou pourrait légèrement augmenter. De nombreux travaux suggèrent l'implication de trois facteurs neurochimiques principaux dans la genèse des lésions du système nerveux central en période périnatale: excitotoxicité glutamatergique, surproduction de radicaux libres oxygénés et de cytokines pro-inflammatoires. Afin de mieux comprendre les liens entre ces trois facteurs nous avons étudié, par microdialyse in vivo, les mécanismes contrôlant la libération des radicaux hydroxyles dans le striatum de rat nouveau-né. Ces expériences nous ont permis de constater un rôle central du glutamate, qui découple la production des radicaux libres oxygénés de l'activation du récepteur NMDA, par son effet conjoint sur les récepteurs métabotropiques de groupe I. L'effet inhibiteur de ces derniers récepteurs sur la libération extracellulaire des radicaux hydroxyles ne s'exprime que transitoirement, disparaissant entre la deuxième et la troisième semaine post-natale. La limitation du stress oxydatif sous l'influence tonique des récepteurs métabotropiques de groupe I semble compatible avec l'intervention fondamentale du récepteur NMDA immature dans l'ontogenèse du système nerveux central. Une telle régulation est cependant fragile puisqu'elle est affectée par l'exposition anténatale au lipopolysaccharide d'Escherichia coli. Nos résultats nous permettent de souligner l'aspect critique du couplage fonctionnel entre les différents récepteurs glutamatergiques dans le stress oxydatif au cours de la maturation. Un tel mécanisme pourrait fournir une base conceptuelle à de nouvelles stratégies neuroprotectrices en période périnataleMONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Dysfonction glutamatergique et GABAergique dans l'hippocampe après un stress immuno-inflammatoire prénatal chez le rat

Introduction: L'injection ip de lipopolysaccharide (LPS) d'E.coli à la rate gestante aboutit à un phénotype cognitivo-comportemental de pathologies neuropsychiatriques chez la progéniture mâle. L'objectif principal était de vérifier l'hypothèse d'une atteinte structurelle et d'un déséquilibre entre excitation et inhibition dans l'hippocampe. L'objectif secondaire était de dégager des stratégies thérapeutiques ciblées.Méthodes: 500 g/kg de LPS d'E.coli de sérotype O55:B5 ou 2 ml/kg de sérum physiologique étaient injectés ip à la rate au 19e jour de gestation. La progéniture mâle était étudiée à différents stades du développement. L'étude structurelle reposait sur de l'immunohistochimie, l'étude fonctionnelle sur des enregistrements électro-physiologiques de l'activité des cellules pyramidales de l'aire CA1. L'effet protecteur de la N-acétylcystéine (NAC) donnée po à la rate gestante après l'injection de LPS était testé. Résultats: Les animaux soumis à un stress prénatal par le LPS présentaient une désorganisation durable de la couche pyramidale de l'aire CA3, un déficit transitoire de neurones exprimant la reeline, une altération de la dépression à long terme des synapses glutamatergiques (LTDe) liée à un déficit des récepteurs NMDA et du système GABAergique. Un inhibiteur de la recapture du GABA parvenait à corriger les anomalies de la LTDe. La NAC prévenait les anomalies cyto-architecturales.Conclusion: Cette thèse confirme l'impact d'un stress immuno-inflammatoire maternel sur la structure et la fonction hippocampique. Elle démontre l'intérêt d'un traitement prénatal par la NAC et de la modulation du tonus GABAergique pour corriger les troubles cognitifs associés.Introduction: A late gestational exposure to lipopolysaccharide (LPS) leads to a behavioral and cognitive phenotype of neuropsychiatric disorders in male offspring. The main goal was to test the hypothesis of structural damage and imbalance between excitation and inhibition in the hippocampus. The secondary goal was to identify targeted therapeutic strategies.Methods: Pregnant rats were ip injected with either 500 g/kg LPS from E.coli O55:B5 or 2 ml/kg saline vehicle on gestational day 19. Male offspring were studied at different developmental stages. The structural study was based on immunohistochemistry, the functional study on electrophysiological recordings of the activity of pyramidal cells in the CA1 area. The protective effect of N-acetylcysteine (NAC) given to pregnant rats after LPS injection was tested.Results: In male offspring, LPS induced late gestational immune challenge led to sustainable disarray of the pyramidal layer in the CA3 area, transient deficit of reelin expressing neurons, impaired long term depression of glutamatergic synapses (LTDe), due to NMDA receptor and GABAergic system dysfunction. An inhibitor of GABA reuptake completely restored plasticity lost after prenatal stress. NAC prevented cyto-architectural abnormalities.Conclusion: This thesis confirms the impact of a late prenatal immune challenge on hippocampal structure and function. It demonstrates that prenatal treatment with NAC and GABAergic tone modulation are valuable therapeutic strategies for the cognitive impairment associated with prenatal immune challenge.MONTPELLIER-BU Médecine UPM (341722108) / SudocSudocFranceF

Metabotropic glutamate receptors as drug targets.

International audienceL-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

Prenatal infection obliterates glutamate-related protection against free hydroxyl radicals in neonatal rat brain

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Neuroprotection induced by vitamin E against oxidative stress in hippocampal neurons: involvement of TRPV1 channels.

International audiencePretreatment of cultured hippocampal neurons with a low concentration of alpha-tocopherol (alpha-TP), the major component of vitamin E, results in a long-lasting protection against oxidative damages, via genomic effects. This neuroprotection is associated with the attenuation of a calcium influx triggered by oxidative agents such as Fe(2+) ions. This Ca(2+) influx is supported by a TRP-like channel, also partly involved in capacitive calcium entry within neurons. Here, we evidence the contribution of TRPV1 channels in this mechanism. TRPV1 channels are activated by various agents including capsaicin, the pungent component of hot chili peppers and blocked by capsazepine (CPZ) or 5'-iodo-resiniferatoxin. Both TRPV1 inhibitors strongly reduced Fe(2+) ion-mediated toxicity and Ca(2+) influx, in the same way as to alpha-TP pretreatment. Moreover, CPZ also decreased capacitive calcium entry in hippocampal neurons. Finally, both CPZ and 5'-iodo-resiniferatoxin reduced spontaneous excitatory synaptic transmission; this depression of synaptic transmission being largely occluded in alpha-TP-pretreated neurons. In conclusion, in our experimental model, TRPV1 channels are involved in the Fe(2+) ion-induced neuronal death and a negative modulation of this channel activity by alpha-TP pretreatment may account, at least in part, for the long-lasting neuroprotection against oxidative stress

A subpopulation of corticotropin-releasing hormone neurosecretory cells in the paraventricular nucleus of the hypothalamus also contain NADPH-diaphorase.

International audienceThe coexistence of ND with CRH 41 was explored in the parvicellular neurons of the PVN, using dual histochemical and radioimmunocytochemical labelling with the light microscope, in rats treated with colchicine. Even though the ND staining was scarce, a clear colocalization was evidenced in the parvicellular part of the PVN. Under these conditions, the ratio of neurons expressing both markers, ND and CRH, amounted about 15% of the CRH-containing neuron population. This result provides a useful tool to study morphological plastic changes in the PVN in response to environmental variations

Involvement of Central Histamine in the Early Phase of ACTH and Corticosterone Responses to Endotoxin in Rats

International audienceThe involvement of histaminergic transmission in the rapid and sustained plasma ACTH and corticosterone (CORT) responses induced in conscious rats by intra-arterial infusions of 25 micrograms.kg-1 Escherichia coli lipopolysaccharide (LPS) was investigated. LPS challenge produced a rapid and transient increase (+ 62%) in the amount of histamine (HA) in the median eminence 15 min after LPS administration, which contrasted with constant concentrations of plasma HA throughout the entire study (up to 480 min). Blockade of histaminergic receptors by intra-arterial pretreatment with H1 or H2 antagonists (mepyramine, 1 mg/rat, and cimetidine, 2 mg/rat), administered separately, did not affect either ACTH or CORT responses to LPS. Pretreatment with the same doses of the two antagonists in combination very significantly but transiently impaired the earliest phase (30 min) of the ACTH and CORT responses, without any apparent effect on the late phase of these responses. Pretreatment of the animals with an H3-receptor agonist (R alpha-methylhistamine dihydrochloride, 1 mg/rat) similarly blunted the early corticotropic responses to LPS, and also slightly depressed the long-lasting CORT response. These findings support the view that activated central HA transmission may be a key intermediate mechanism triggering the CRH41-ACTH-CORT responses to LPS, in addition to the previously demonstrated activating role of catecholaminergic afferences to the CRH41 neurons during this early complex phase of corticotropic response to LPS

A transient treatment of hippocampal neurons with alpha-tocopherol induces a long-lasting protection against oxidative damage via a genomic action.

International audienceNeuroprotection 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

N-acetyl-cysteine prevents pyramidal cell disarray and reelin-immunoreactive neuron deficiency in CA3 after prenatal immune challenge in rats.

International audienceBACKGROUND: Prenatal infection is a major risk factor for the occurrence of neuropsychiatric disorders. These have been associated with hippocampal neuroanatomical and functional abnormalities. In the present study, we evaluated the occurrence of pyramidal cell disarray and reelin neuronal deficit in the hippocampus, and the protective role of N-acetyl-cysteine (NAC) in a rodent experimental model of prenatal immune challenge. METHODS: Sprague-Dawley rats received either 500 μg/kg of endotoxin (lipopolysaccharide, LPS) or 2 ml/kg of isotonic saline by i.p. injection on day 19 of gestation. After LPS injection, rats were or were not maintained on a preventive treatment of NAC (5 g/l in tap water), up to delivery. The pyramidal cell orientation and the number and type of reelin-expressing neurons were determined in male offspring. RESULTS: Prenatal LPS challenge led to permanent pyramidal cell disarray and to an early and transient decreased density of reelin-immunoreactive neurons. These disorders, more pronounced in the CA3 area, were prevented by NAC. CONCLUSION: Hippocampal cytoarchitectural alterations and reelin deficiency may be involved in the development of remote cognitive impairments in this model. The antioxidant NAC is an efficient neuroprotective drug that underlines the role of oxidative stress in prenatal infection and associated neurodevelopmental damage