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XI European Meeting on Glial Cell Function in Health and Disease

Overview Session Overview Sessionprint print  
 

July 3, 2013 - Hall A 11:10am - 12:55pm
Workshop I (continued): Imaging of Glial Cell Activity

Organized by
Hajime Hirase (RIKEN Brain Science Institute, Wako, Japan)
Frank Kirchhoff (University of Saarland, Homburg, Germany)

11:10am
W01-05

Optogenetic control of glial cell activity

*Ko Matsui 1
1 National Institute for Physiological Sciences, Division of Cerebral Structure, Okazaki, Japan
Abstract text :

Recent accumulating evidence shows that there are direct and rapid mechanisms for neurons to communicate with glial cells; however, without the evidence for a signaling pathway leading back from glial activity to neuronal activity, we remain uncertain of the glial participation in rapid information processing. Release of transmitters from glial cells (gliotransmitter) has been proposed to mediate such glia-to-neuron communication. Extracellular electrical stimulation used to study synaptic transmission between neurons inevitably stimulates glial cells as well, thus gliotransmitter release could have been unintentionally evoked in these studies but its effect overlooked. Here, we introduced a transgenic mouse line in which channelrhodopsin-2 (ChR2), a light-gated non-selective cation channel, was selectively expressed in astrocytes including cerebellar Bergmann glial cells. Selective photostimulation of these astrocytes lead to release of glutamate which was sufficient to activate AMPA receptors on Purkinje cells (PCs) and to induce long-term depression of parallel fiber to PC synapses through activation of mGluRs on PCs. We also show that neuronal activation by astrocyte stimulation also works in vivo and can lead to perturbation of cerebellar modulated motor behavior. In contrast to the point-to-point communication provided by neuronal release of synaptic vesicles, astrocyte activation likely causes preferential activation of perisynaptic and extrasynaptic receptors expressed on neurons as these receptors directly apposes astrocyte membrane. These results provide evidence that astrocyte activation can serve as a modulatory mechanism for setting the tone of neuronal activity and behavior.



11:45am
W01-06

Influence of blood supply on glioblastoma progression: A two photon imaging study

*Franck  Debarbieux 1,2 , Clément  Ricard 1,2 , Fabio  Stanchi 1,3 , Thieric  Rodriguez 1 , Marie-Claude  Amoureux 1,2 , Geneviève  Rougon 1,2
1 Aix Marseille University, Developmental Biology Institute of Marseille-Luminy CNRS 7288 , Marseille, France
2 European Center for Medical Imaging (CERIMED) , , Marseille, France
3 KU Leuven , VIB Vesalius Onderzoekscentrum, Leuven, France
Abstract text :

Anti-angiogenic treatments are commonly used to treat Glioblastoma multiforme (GBM) brain tumors under the assumption that tumor progression critically depends on the oxygen and metabolites supplied by blood vessels. Using quantitative intravital multiphoton imaging of orthotopic GBM tumor models in mice, we challenge this view by showing the independence of tumor cell densities and tumor cell proliferation from local levels of angiogenesis.


We optimized protocols for orthotopic GBM grafting in mice to recapitulate the biophysical constraints normally governing tumor invasion at a depth suitable for intravital multiphoton microscopy. We repeatedly imaged tumor cells and blood vessels during GBM development. Having established methods for quantitative correlative analyses of dynamic imaging data over wide fields covering the entire tumor, we searched for correlations between blood supply, tumor cell density and proliferation in control tumors. Extensive vascular remodeling and the formation of new vessels accompanied tumor growth but no strong correlation was found between local cell density and the extent of local blood supply irrespective of the tumor area or time points.


The technique moreover allowed to perform comparative analysis of mice subjected to Bevacizumab anti-angiogenic treatment that targets VEGF. Bevacizumab treatment massively reduced tumoral vessel densities but only transiently reduced tumor growth rate. Again, there was no correlation between local blood supply and local cell density and tumor growth could be sustained without additional blood supply. Bev did not directly impair tumor cells proliferation, and its antitumor effect was recapitulated if pre-treating the mouse prior tumor grafting, which suggested an action on the stroma. Moreover AMD3100 that inhibits the Stromal Derived Factor 1 alpha pathway produced larger and sustained tumor inhibition without significantly affecting blood supply.



12:20pm
W01-07

Dual-color superresolution imaging of synapses and glia cells in living brain slices using STED microscopy

*Valentin  Nägerl 1,2
1 Université Bordeaux Segalen, Interdisciplinary Institute for Neuroscience (IINS), Bordeaux, France
2 UMR 5297, Centre National de la Recherche Scientifique (CNRS), , Bordeaux, France
Abstract text :

Neuronal synapses are complex structures composed of pre- and postsynaptic membrane specializations ensheated by glia processes, forming elementary functional compartments for rapid and flexible signaling in the central nervous system. Understanding how synapses are built during development and modified by experience is a central theme and challenge for neuroscience.


            As synapses and glial processes are typically very small (<< 1 µm), dynamic and reside inside three-dimensional, light-scattering tissue, it is difficult to study them by conventional, diffraction-limited light microscopy.


            However, major advances in superresolution imaging and fluorescence labeling are greatly improving our ability to investigate the inner life and dynamics of synapses using life-cell imaging approaches. We have shown that superresolution STED microscopy is a powerful technique for live-cell imaging of synapse morphology using YFP as a genetically encoded volume-label.


            We will review our recent progress in developing STED microscopy for live-cell nanoscale imaging of neuronal and glial structures deep inside brain slices and in two colors simultaneously. Specifically, we will demonstrate the powerful potential of these methodological advances for several applications concerning superresolution imaging of synapses: 1) nanoscale imaging up to 100 µm deep below tissue surface in acute brain slices by a novel combination of two-photon and STED microscopy; 2) dual-color nanoscale imaging of synapses interacting with astrocytic and microglial processes; 3) spine structure - function analysis combining nanoscale imaging of spine morphology with two-photon fluorescence recovery after photobleaching (FRAP) measurements.