Differential involvement of Ca(2+) channels in survival and neurite outgrowth of cultured embryonic cockroach brain neurons.

Full text: http://jn.physiology.org/content/88/3/1475International audienceThe contribution of voltage-gated calcium channels (VGCC) to the development of cultured embryonic cockroach brain neurons was assessed using pharmacological agents. VGCC currents were recorded using the patch-clamp technique and were found to be blocked dose-dependently by micromolar concentrations of mibefradil. The activation and inactivation properties of the calcium channels enable a sizeable calcium current to flow at rest (about -30 and -20 mV in high-potassium culture media). As expected, the cytoplasmic-free calcium concentration was found to rise when the extracellular potassium concentration was raised from 3 to 15 and 30 mM. The effects of VGCC blockers and calcium chelators were different in fresh and in mature cultures in which the neurons were connected to each other to form a defined network. In fresh cultures, the two non-selective VGCC blockers (verapamil and mibefradil) induced a dose-dependent cell death that was proportional to their blocking effect on I(Ba). This effect could not be prevented by addition of fetal calf serum to the culture medium. A similar effect was obtained using intra- or extracellular calcium chelating agents (10 microM BAPTA-AM or 10 mM EGTA). Quite unexpectedly, blockade of the P/Q-like (omega-Aga WA-sensitive) component of the calcium current by 500 nM of omega-AgaTx IVA had no lethal effect, suggesting that the corresponding channels are not involved in the survival mechanism. As expected from their lack of effect on I(Ba), isradipine, nifedipine, and omega-CgTx GVIA did not induce cell death. When the neurons started growing neurites, their sensitivity to calcium channel blockade by mibefradil decreased, indicating a correlation between neurite outgrowth and resistance to calcium depletion. In mature cultures, the neurons became resistant to mibefradil, verapamil, and BAPTA-AM. However, these agents, as well as omega-AgaTx IVA, had a significant inhibitory effect on the increase in diameter of the connectives that linked adjacent clusters of neurons. This effect has been shown to result, in the case of mibefradil, from an inhibition of neurite outgrowth characterized by a significant reduction of the number of primary neurites and secondary branchings but not to a significant modification of the diameter of individual neurites. These results support the view that, as in vertebrates, calcium influx through VGCC plays an important role in survival and neurite outgrowth of cultured embryonic insect neurons. The differential contribution of the P/Q-like and R-like (omega-Aga WA-sensitive) calcium channels in these processes is discussed

Dynamic reorganization of functional brain networks during picture naming.

International audienceFor efficient information processing during cognitive activity, functional brain networks have to rapidly and dynamically reorganize on a sub-second time scale. Tracking the spatiotemporal dynamics of large scale networks over this short time duration is a very challenging issue. Here, we tackle this problem by using dense electroencephalography (EEG) recorded during a picture naming task. We found that (i) the picture naming task can be divided into six brain network states (BNSs) characterized by significantly high synchronization of gamma (30–45 Hz) oscillations, (ii) fast transitions occur between these BNSs that last from 30 msec to 160 msec, (iii) based on the state of the art of the picture naming task, we consider that the spatial location of their nodes and edges, as well as the timing of transitions, indicate that each network can be associated with one or several specific function (from visual processing to articulation) and (iv) the comparison with previously-used approach aimed at localizing the sources showed that the network-based approach reveals networks that are more specific to the performed task. We speculate that the persistence of several brain regions in successive BNSs participates to fast and efficient information processing in the brain

Connectivité de sources en EEG-hr et dynamique des réseaux cérébraux fonctionnels

National audienceLe traitement l'information par le cerveau est un processus dynamique qui met en jeu une réorganisation rapide des réseaux cérébraux fonctionnels, sur une échelle de temps très courte (> nombre d'électrodes), (ii) estimer les dépendances statistiques (connectivité fonctionnelle) entre les sources reconstruites , (iii) caractériser les réseaux identifiés (sous forme des noeuds connectés par des liens formant un graphe) par des analyses basées sur la théorie des graphes et (vi) segmenter, dans le temps, le processus cognitif sous la forme d'une séquence d'états de connectivité fonctionnelle (fcSs : 'functional connectivity states'). Les résultats montrent qu'un traitement approprié du signal EEG permet d'identifier une dynamique spatio-temporelle dans les réseaux fonctionnels mis en jeu durant la tâche avec une excellente résolution temporelle (de l'ordre de la ms) et spatiale (~ 1000 régions d'intérêt). Cette dynamique correspond à une séquence de six fcSs (durée : 30 ms à 160 ms) caractérisés par une corrélation de phase significative des oscillations gamma (30-45 Hz). Des transitions rapides entre ces fcS sont observées et les réseaux associés à chaque fcS se recouvrent partiellement. Ces réseaux s'instancient sur des régions cérébrales pertinentes par rapport à la tâche de dénomination d'objets, depuis la perception de l'image jusqu'à l'articulation du nom. La méthode proposée ouvre de nombreuses perspectives quant à l'identification, à partir des données d'EEG de scalp, de réseaux cérébraux mis en jeu transitoirement lors d'activités cognitives. Abstract-The information processing in the human brain is a dynamic process that involves a rapid reorganization of functional brain networks, in a very short time scale (> number of electrodes), (ii) estimating the statistical dependencies (functional connectivity) between reconstructed sources (iii) characterizing the identified networks (in the form of nodes connected by edges forming a graph) by graph theory based analysis and (vi) segmenting, in time, the cognitive process as a sequence of functional connectivity states (fcSs). The results show that appropriate processing of the EEG signals can reveal the spatiotemporal dynamics of functional brain networks involved in the task with excellent temporal (on the order of ms) and spatial (~ 1000 regions of interest) resolution. This corresponds to a dynamic sequence of six fcSs (duration: 30 ms to 160 ms) with significant gamma phase synchronization (30-45 Hz). Rapid transitions between these fcS are observed and the networks associated with each fcS partially overlap. These networks disclose relevant brain regions related to picture naming task, from the perception of the image until the naming. The proposed method offers many opportunities in the identification, from the EEG data, of brain networks involved in cognitive activities

Neuron to Astrocyte Communication via Cannabinoid Receptors Is Necessary for Sustained Epileptiform Activity in Rat Hippocampus

Astrocytes are integral functional components of synapses, regulating transmission and plasticity. They have also been implicated in the pathogenesis of epilepsy, although their precise roles have not been comprehensively characterized. Astrocytes integrate activity from neighboring synapses by responding to neuronally released neurotransmitters such as glutamate and ATP. Strong activation of astrocytes mediated by these neurotransmitters can promote seizure-like activity by initiating a positive feedback loop that induces excessive neuronal discharge. Recent work has demonstrated that astrocytes express cannabinoid 1 (CB1) receptors, which are sensitive to endocannabinoids released by nearby pyramidal cells. In this study, we tested whether this mechanism also contributes to epileptiform activity. In a model of 4-aminopyridine induced epileptic-like activity in hippocampal slice cultures, we show that pharmacological blockade of astrocyte CB1 receptors did not modify the initiation, but significantly reduced the maintenance of epileptiform discharge. When communication in astrocytic networks was disrupted by chelating astrocytic calcium, this CB1 receptor-mediated modulation of epileptiform activity was no longer observed. Thus, endocannabinoid signaling from neurons to astrocytes represents an additional significant factor in the maintenance of epileptiform activity in the hippocampus

The move: When neurosciences teach us to better teach neurosciences

International audienc

CARACTERISATION, EVOLUTION ET ROLES DE CANAUX CALCIQUES VOLTAGE-DEPENDANTS AU COURS DU DEVELOPPEMENT IDE NEURONES CHEZ UN INSECTE

L'OBJET DE CETTE THESE A ETE DE CARACTERISER, DE SUIVRE L'EVOLUTION, DE DETERMINER LE ROLE ET LES REGULATIONS DE CANAUX CALCIQUES VOLTAGE-DEPENDANTS (CCVD) AU COURS DU DEVELOPPEMENT NEURONAL D'UN INSECTE. LES COURANTS CALCIQUES DE NEURONES EMBRYONNAIRES (E21) DE CERVEAU DE BLATTE EN CULTURE PRIMAIRE SONT UNIQUEMENT DE TYPE HAUT-SEUIL. SELON LA CLASSIFICATION DES CCVD DE VERTEBRES, 75% DES NEURONES EXPRIMENT DU COURANT DE TYPE P/Q ET 100% DES NEURONES EXPRIMENT DU COURANT DE TYPE R. AU COURS DU DEVELOPPEMENT DES NEURONES EN CULTURE, LA DENSITE DE CES COURANTS AUGMENTE ET LES PROPRIETES D'ACTIVATION VOLTAGE-DEPENDANTE CHANGENT (UN SHIFT DE 10 MV EST OBSERVE). LA PROPORTION DE COURANT DE TYPE R AUGMENTE PROGRESSIVEMENT PAR RAPPORT A CELUI DE TYPE P/Q, JUSQU'A DEVENIR MAJORITAIRE. PARALLELEMENT, LE COURANT QUI EST TRES AGAIVA SENSIBLE (IC 5 0=0.2 NM) DANS LES CULTURES JEUNES (1 A 5 JOURS IN VITRO (JIV)) DIMINUE ET EST PROGRESSIVEMENT REMPLACE PAR UN COURANT DE TYPE P/Q MOINS SENSIBLE A CETTE TOXINE (IC 5 0=12 NM), SUGGERANT UNE EVOLUTION DU COURANT DE TYPE P EN COURANT DE TYPE Q AU COURS DU DEVELOPPEMENT DES NEURONES EN CULTURES. NOUS AVONS CONFIRME LA PRESENCE DE CES COURANTS SUR DES NEURONES FRAICHEMENT DISSOCIES A PARTIR D'EMBRYONS DE DIFFERENTS AGES. LES COURANTS APPARAISSENT A E14, LEUR DENSITE AUGMENTE AVEC L'AGE DE L'EMBRYON ET UN SHIFT DE 10 MV EST OBSERVE ENTRE E14 ET E18. EN PLUS DES COURANTS DE TYPE P/Q ET R, UN COURANT DE TYPE N A ETE IDENTIFIE. SUR DES NEURONES DE CULTURES JEUNES, L'ADDITION DANS LE MILIEU DE CULTURE DE CHELATEURS EXTRA OU INTRACELLULAIRES DE CALCIUM OU DE BLOQUEURS AFFECTANT LE COURANT DE TYPE R, OU L'UTILISATION D'INHIBITEURS DE LA CA/CALMODULINE KINASE ENTRAINENT LA MORT DE TOUS LES NEURONES EN 48 HEURES. CES EFFETS SONT FONCTION DE L'ETAT DU RESEAU NEURONAL. LORS DE LA FORMATION DU RESEAU (10-30 JIV), LA SURVIE NEURONALE DEVIENT CALCIUM INDEPENDANTE. CEPENDANT LES BLOQUEURS DE CCVD DIMINUENT DE FACON SIGNIFICATIVE LA POUSSE NEURITIQUE. LE SERUM FTAL BOVIN N'EST PAS NECESSAIRE A LA SURVIE NEURONALE A COURT TERME MAIS EST INDISPENSABLE A LA POUSSE NEURITIQUE. LE MECANISME D'ACTION DE CE SERUM SEMBLE IMPLIQUER LA CASCADE DE SIGNALISATION TK-, RAS- ET PI3K CONDUISANT A UNE AUGMENTATION DE LA DENSITE DES CCVD NOTAMMENT DE TYPE R.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Apports de la microscopie biphotonique à l étude du tissu musculaire squelettique et du système nerveux

La microscopie biphotonique s est imposée progressivement depuis vingt ans dans le champ de la biologie, aux côtés d autres méthodologies d imagerie. Elle permet d imager à la fois des fluorophores et des molécules endogènes hyperpolarisables par des ondes électromagnétiques (harmonophores) comme la myosine, et ce, in vivo et/ou en profondeur. La première partie de cette thèse a porté sur l imagerie de seconde harmonique (SHG) du tissu musculaire, et a montré comment le signal sarcomérique, qui se présente à l état physiologique sous la forme d une simple bande brillante, se dédouble lorsque le tissu subit différentes formes de protéolyse (post-mortem, stress mécanique et chimique, photodégradation). Ce changement de profil d intensité serait causé par l extension d une zone centrale de centrosymétrie, qui empêche le signal SHG de se construire au fur et à mesure qu il se propage dans le tissu. L imagerie harmonique musculaire apparait comme un excellent outil pour l étude structurale multi-échelle de l organisation des myofilaments au sein et entre myofibrilles dans différentes conditions physiologiques et extra-physiologiques. Dans la seconde partie de ce travail, la microscopie biphotonique multicouleur a été utilisée pour caractériser in vivo la tissu-spécificité de l expression d un transgène dans la lignée de xénopes transgéniques P-nbt-EGFP. Finalement ces travaux montrent l étendue des possibilités offertes par l imagerie biphotonique dans le domaine de la biologie intégrative pour l étude de sources harmoniques ou fluorescentes dans des tissus épais et même chez le petit animal in vivo.Over the last twenty years, two-photon microscopy has gradually emerged in the field of biology, along with other imaging methods. It allows imaging of both fluorophores and endogenous molecules with non linear susceptibility (harmonophores), as myosin, either in thick tissues or in vivo. The first part of this study focused on second harmonic imaging (SHG) of muscle tissues, and showed how sarcomeric SHG intensity profile, which is single peak under physiological conditions, is converted into double peak when the tissue undergoes proteolysis (post-mortem, mechanical and chemical stresses, photodamage). This change in intensity profile is caused by expansion of a central centro-symmetry area which prevents construction of the signal SHG as it propagates through the tissue. Muscle SHG microscopy appears to be an excellent tool for studying multi-scale structural organization of myofilaments within and between myofibrils in different physiological and non-physiological conditions. In the second part of this work, two-photon multiplex microscopy was used to characterize in vivo, the tissue-specific expression of a transgene from the P-nbt-EGFP Xenopus transgenic line. Finally this work shows the wide possibilities offered by two-photon imaging in the field of integrative biology for the study of harmonic or fluorescent sources in thick tissues and even in small animals in vivo.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Muscarinic receptor stimulation reduces NMDA responses in CA3 hippocampal pyramidal cells via Ca2+-dependent activation of tyrosine phosphatase

Poste

In vitro development of P- and R-like calcium currents in insect (Periplaneta americana) embryonic brain neurons.

International audienceVoltage-gated calcium currents are important for the survival and growth of embryonic cockroach brain neurons in primary culture. In the present experiments, we have studied, using the patch-clamp technique, the evolution with time in culture of the voltage-dependency and of the pharmacological properties of the calcium conductance of these neurons during the formation of a network. We have observed a progressive increase of the high-voltage-activated calcium conductance and a 10mV shift of the voltage-dependency of activation towards more negative potentials. The proportion of the R-like calcium current component increased during network formation. At the same time, the highly omega-AgaTxIVA-sensitive P-like component of the current is progressively replaced by a component which is less sensitive to the toxin. The origin and functional implications of these modifications are discussed