The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-beta 2

Functional activation of the neuronal K+-Cl- co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor beta 2 (TGF-beta 2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-beta 2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl- extrusion. We also identify the signaling pathway from TGF-beta 2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-beta 2-mediated KCC2 trafficking and functional activation. TGF-beta 2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-beta 2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-beta 2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission.Peer reviewe

The adipocyte hormone leptin sets the emergence of hippocampal inhibition in mice

This is the author accepted manuscript. The final version is available from eLife Sciences Publications via the DOI in this record.Brain computations rely on a proper balance between excitation and inhibition which progressively emerges during postnatal development in rodent. g-aminobutyric acid (GABA) neurotransmission supports inhibition in the adult brain but excites immature rodent neurons. Alterations in the timing of the GABA switch contribute to neurological disorders, so unveiling the involved regulators may be a promising strategy for treatment. Here we show that the adipocyte hormone leptin sets the tempo for the emergence of GABAergic inhibition in the newborn rodent hippocampus. In the absence of leptin signaling, hippocampal neurons show an advanced emergence of GABAergic inhibition. Conversely, maternal obesity associated with hyperleptinemia delays the excitatory to inhibitory switch of GABA action in offspring. This study uncovers a developmental function of leptin that may be linked to the pathogenesis of neurological disorders and helps understanding how maternal environment can adversely impact offspring brain development.This work was supported by the Ministère de la Recherche et de l’Enseignement Supérieur, Neurochlore (CD) and the National Institutes of Health (Grant MH086032, GW)

Developmental mapping of small-conductance calcium-activated potassium channel expression in the rat nervous system

Early electrical activity and calcium influx regulate crucial aspects of neuronal development. Small-conductance calcium- activated potassium (SK) channels regulate action potential firing and shape calcium influx through feedback regulation in mature neurons. These functions, observed in the adult nervous system, make them ideal candidates to regulate activity- and calcium-dependent processes in neurodevelopment. However, to date little is known about the onset of expression and regions expressing SK channel subunits in the embryonic and postnatal development of the central nervous system (CNS). To allow studies on the contribution of SK channels to different phases of development of single neurons and networks, we have performed a detailed in situ hybridization mapping study, providing comprehensive distribution profiles of all three SK subunits (SK1, SK2, and SK3) in the rat CNS during embryonic and postnatal development. SK channel transcripts are expressed at early stages of prenatal CNS development. The three SK channel subunits display different developmental expression gradients in distinct CNS regions, with time points of expression and up- or downregulation that can be associated with a range of diverse developmental events. Their early expression in embryonic development suggests an involvement of SK channels in the regulation of developmental processes. Additionally, this study shows how the postnatal ontogenetic patterns lead to the adult expression map for each SK channel subunit and how their coexpression in the same regions or neurons varies throughout development

Intracellular chloride concentration influences the GABAA receptor subunit composition

GABAA receptors (GABAARs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABAAR are largely unknown. The intracellular concentration of chloride ([Cl−]i), the main ion permeating through GABAARs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl−]i could modulate the sequential expression of specific GABAARs subtypes in primary cerebellar neurons. We show that [Cl−]i regulates the expression of α3-1 and δ-containing GABAA receptors, responsible for phasic and tonic inhibition, respectively. Our findings highlight the role of [Cl−]i in tuning the strength of GABAergic responses by acting as an intracellular messenger

Role of KCC2 in neuronal plasticity

Lors du développement, la différentiation neuronale et la création de réseaux neuronaux sont accompagnés de changements majeurs de l expression et de la fonction de canaux et transporteurs ioniques contrôlant l activité neuronale (Ben Ari, 2002). Un des plus grands changements concerne la signalisation médiée par les récepteurs GABA (GABAAR). Le GABA est le principal neurotransmetteur inhibiteur du cerveau adulte. Cependant, à un état précoce du développement (lors des deux premières semaines post-natales chez le rongeur), son effet est différent. Paradoxalement, son action est dépolarisante et excitatrice sur des neurones immatures (Leinekugel et al., 1999;Ben Ari et al., 1989). Il a été démontré plusieurs rôles physiologiques pour ce GABA dépolarisant et excitateur dans le cerveau en développement. La dépolarisation produite par le GABA provoque des potentiels d action sodiques, active les canaux calcium voltage-dépendant et facilite l activité des récepteurs NMDA par l atténuation du blocage magnésium de ces récepteurs (Leinekugel et al., 1997). Le GABA dépolarisant est un acteur critique de la génération des potentiels dépolarisants géants (GDPs), schéma d activité caractéristique de l activité de réseau dans le cortex immature (Garaschuk et al., 2000;Ben Ari et al., 1989;Ben Ari, 2002). Selon plusieurs articles, le GABA dépolarisant joue un rôle de facteur trophique sur le développement cérébral, contrôlant la différentiation neuronale (Loturco et al., 1995), la croissance neuronale (Barbin et al., 1993;Groc et al., 2002) et le phénotype neuronal (Marty et al., 1996). Le passage de l action du GABA d excitateur à inhibiteur implique non seulement le mode de fonctionnement du réseau neuronal mais aussi les effets trophiques du GABA (Represa & Ben Ari, 2005). Le GABA dépolarisant participe dans l induction de la plasticité à long terme des synapses GABAergiques (Caillard et al., 1999b) et glutamatergiques (Pavlov et al., 2004) dans l hippocampe de rat nouveau-né. Le blocage des GABAARs de l hippocampe de rats nouveau-nés inhibe la formation des synapses GABAergiques (Colin-Le Brun et al., 2004). L action dépolarisante du GABA est due à une concentration intracellulaire élevée de chlore dans les neurones immatures (Ben Ari et al., 1989), qui est elle-même due au développement postnatal du système d homéostasie du chlore. Ce système est principalement régi par le co-transporteur potassium-chlore KCC2 (Rivera et al., 1999). Le niveau d expression de KCC2 est relativement bas dans les neurones de l hippocampe de rat immature, mais augmente durant la seconde semaine postnatale. Cette augmentation est associée avec le décalage progressif du potentiel de réversion de la réponse médiée par les GABAARs. Cela change l action du GABA d excitateur à inhibiteur (Ludwig et al., 2003;Rivera et al., 2005;Stein et al., 2004). De récentes données montrent que KCC2 serait aussi impliqué dans la neurotoxicité (Rivera et al., 2004). Une des conséquences d une libération excessive de glutamate lors d une pathologie cérébrale est la modification de l homéostasie du chlore favorisant la dilatation cellulaire, la dépolarisation de la membrane et ainsi contribue à la neurotoxicité. Une telle augmentation de la concentration intracellulaire de chlore est due à l inactivation de KCC2 (Woodin et al., 2003;Palma et al., 2006). En effet, dans des neurones pyramidaux de CA1 de tranches d hippocampe adulte, une activité de type inter-ictale soutenue diminue l expression de l ARNm de KCC2 et de sa protéine. Cela conduit à une moindre sortie de chlore et change le GABA d inhibiteur à excitateur (Rivera et al., 2004). Cela facilite l activation des RNMDAs, selon le scénario décrit précédemment pour des neurones immatures (Leinekugel et al., 1997), ce qui conduit alors à la mort neuronale.L ensemble de ces données suggère donc que l activité neuronale synchrone dirigée par la neurotransmission GABA excitatrice participe à la formation du réseau neuronal lors du développement cérébral précoce. Comme, lors du développement, le passage de l effet du GABA d excitateur à inhibiteur est sous le contrôle de KCC2, nous postulons que ce transporteur joue un rôle important dans la formation du réseau neuronal. Le but principal de ce travail de thèse est de vérifier cette hypothèse en utilisant différents modèles permettant la modification de l expression de KCC2. Le travail de thèse inclut quatre parties complémentaires: I) la mise en place des modèles expérimentaux et des outils de biologie moléculaire permettant la surexpression de KCC2 dans des neurones vivants; II) l étude du rôle de régulation de KCC2 dans la synaptogenèse en utilisant la surexpression de cette protéine; III) l étude de la synaptogenèse dans un modèle comportant un déficit en KCC2; IV) l étude des implications possible de KCC2 comme agent neuroprotecteur.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Neuronal integration in the adult mouse olfactory bulb is a non-selective addition process

International audienceAdult neurogenesis in the olfactory bulb (OB) is considered as a competition in which neurons scramble during a critical selection period for integration and survival. Moreover, newborn neurons are thought to replace pre-existing ones that die. Despite indirect evidence supporting this model, systematic in vivo observations are still scarce. We used two-photon in vivo imaging to study neuronal integration and survival. We show that loss of new neurons in the OB after arrival at terminal positions occurs only at low levels. Moreover, long-term observations showed that no substantial cell death occurred at later stages. Neuronal death was induced by standard doses of thymidine analogs, but disappeared when low doses were used. Finally, we demonstrate that the OB grows throughout life. This shows that neuronal selection during OB-neurogenesis does not occur after neurons reached stable positions. Moreover, this suggests that OB neurogenesis does not represent neuronal turnover but lifelong neuronal addition

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

Efficient and long-lasting transfection of primary neurons is an essential tool to address many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (shRNA) vectors, using magnetofection, into rat hippocampal neurons (E18/19) cultured for several hours to 21 days in vitro. This protocol even allows for double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. The protocol, which uses inexpensive equipment and reagents, takes 1 h, utilizes mixed hippocampal cultures, a transfection reagent, CombiMag and a magnetic plate, shows low toxicity and is suited for single cell analysis. Modifications as done by our three laboratories are detailed