JUNCTIONAL COMMUNICATION AND INTERACTIONS BETWEEN GLIAL AND NEURONAL NETWORKS

Director: Christian GIAUME

The main focus of our studies is the definition of the properties and the understanding of the multiple roles of connexins in glial cells, particularly in astrocytes. Connexins are membrane proteins that form gap junction channels allowing direct exchanges (ions, energetic metabolites, cyclic nucleotides, gliotransmitters….) between the cytoplasm of adjacent cells and the ionic/biochemical coordination of these communicating cells. These gap junctions constitute the basis of astroglial networks, however, in defined conditions connexins can operate as hemichannels providing exchanges between cytoplasmic and extracellular compartments. This hemichannel function is also contributed by pannexins, another membrane protein family that is expressed in neurons and in reactive astrocytes. It is now recognized that astrocytes interact dynamically with neurons and modify their activity and survival, but most of the time these interactions are considered at an individual level. Our strategy is to study neuroglial interactions at a more integrated level as the result of an overlap of neuronal circuits and astroglial networks supported by gap junctions and/or hemichannels. A similar remark and proposed approach are also true for the gliovascular interface and the control of blood flow by astrocytes.

Visualization of Cx43 gap junctions in cultured cortical astrocytes. Dual immunofluorescence/DIC confocal microscopy showing that the finest punctate labelling of Cx43 (in green), which represent gap junctions, is aligned at contact between cell membranes while larger intracellular spots are attributed to internalized gap junctions.

Based on this, our main research topics are :

1) the characterization of connexin-mediated networks in astrocytes (molecular composition, regulation, spatial organization, relation of connexins with the cytoskeleton and other scaffolding proteins);
2) the definition of the properties and role of astroglial networks in brain regions characterized by a strong anatomo-functional organization of neuronal circuits (somatosensory cortex, olfactory bulb);
3) the identification of the role of astroglial connexins/pannexins in synaptic transmission and plasticity in the hippocampus;
4) the understanding of their contribution to brain metabolism by considering the uptake and the trafficking of energetic metabolites and their delivery to active neurons;
5) as there is a high level of connexins expression at contacts between astrocytic endfeet enwrapping blood vessels we are interested in determining their participation to the development, the integrity and the maintenance of the blood brain barrier;
6) the development of new molecular approaches with astrocyte-directed tools (conditional and cell type targeted transgenic mice, lentiviral constructs) to study the channels and non- channels functions of connexins/pannexins in neuroglial and gliovascular interactions. Finally, our researches cover also pathological situations, such as epilepsy and Alzheimer’s disease, using pharmacological and genetic animal models in which reactive gliosis is associated with changes in the expression of connexins/pannexins in astrocytes.

These projects are developed :

1) using a multidisciplinary approach combining electrophysiology, calcium imaging, dye injections, cellular and molecular biology, biochemistry, confocal and bi-photon microscopy, immunoblotting and immunohistochemistry, using in vitro (cultures, acute brain slices) and in vivo models with the requirement to associate expression of the studied proteins with their function;
2) through national (Mircen/CEA, ENS) and international (Germany, Spain, Swizzerland, Chile) collaborations that provide specific animal models and pharmacological tools as well as high-tech approaches (in vivo imaging, metabolic assays) ; 
3) mathematical modeling is also associated to several of our projects to complete experimental approaches of neuroglial networking.

In summary, our research aims at defining cellular and molecular mechanisms involved in neuroglial and gliovascular interactions as well as identifying glial targets that could represents the basis for alternative strategies to prevent, or at least reduce, neuronal dysfunction or death.

Astroglial networks in the olfactory glomeruli revealed by dialysis of sulforhodamin B (red) through a patch clamp pipette. The recorded astrocyte is identified by its electrophysiological properties (inset: a passive I/V relationship). Cell bodies stained by the Nissl reagent (blue) define the glomerular structures. Scales : 50μm, inset 30ms/1nA.

Calcium imaging in astrocytes (left) and field potential recording of a neuronal bursting activity (lower right) in intact mouse hippocampus formation during the established epilepsy model 0Mg2+ showing synchronous calcium oscillations (upper right) in astrocytes during neuronal seizures.

Shaping of astroglial metabolic networks during afferent stimulation in the CA1 region of the mouse hippocampus. A fluorescent glucose derivative (2-NBDG, left) was injected within the astroglial network through a patch-clamp pipette (upper image). Prolonged stimulation of the Schaffer collaterals induces a change in the shape of the network by recruting astrocytes located over the pyramidal layer (lower image). Calibration: 50μm.

Connexin 43 immunoreactivity (green) is enhanced in the reactive astrocytes (GFAP-stained in red) at ß-amyloid plaques (blue) in a human temporal cortex section from a patient affected by the Alzheimer's disease. Calibration: 20 μm.

SELECTED PUBLICATIONS 2007-2010

- Giaume C., Koulakoff A., Roux L., Holcman D. & Rouach N. (2010), Astroglial networks: a step further in neuroglial and gliovascular interactions. Nat Rev Neurosci, 11:87-99.
- Rouach N., Koulakoff A., Abudara V., Willecke K. & Giaume C. (2008), Astroglial metabolic networks sustain hippocampal synaptic transmission. Science, 322:1551-5.
- Koulakoff A., Ezan P. & Giaume C. (2008), Neurons control the expression of connexin 30 and connexin 43 in mouse cortical astrocytes. Glia, 56:1299-311.
- Houades V., Koulakoff A., Ezan P., Seif I. & Giaume C. (2008), Gap junction-mediated astrocytic networks in the mouse barrel cortex. Journal of Neuroscience, 28:5207-17.
- Retamal M.A., Froger N., Palacios-Prado N., Ezan P., Sáez P.J., Sáez J.C. & Giaume C. (2007), Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely by proinflammatory cytokines released from activated microglia. Journal of Neuroscience, 27:13781-92.

PEOPLE

Director :
Giaume Christian, DR1 CNRS

Researchers :
Cohen-Salmon Martine, CR1 CNRS
Koulakoff Annette, MdC CDF
Burbassi Silvia, MC associé CDF

Post-docs and Students :
Kowalewski Jacob, Post Doc ARN
Roux Lisa, PhD student Paris 6
Liu Xinhe, PhD student
Boulay Anne-Cécile, M2 ENS
Madar Antoine, M2

Technical staff :
Amigou Edwige, ITA CDF
Cazéres Joséphine, ITA CDF
Ezan Pascal, ITA CDF