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Thursday, 28 June 2007


Cell Calcium. 2005 Jun;37(6):565-72. Epub 2005 Apr 11.

Flash photolysis using a light emitting diode: An efficient, compact, and affordable solution.

Bernardinelli Y, Haeberli C, Chatton JY.

Department of Physiology, University of Lausanne, Rue du Bugnon 7, CH-1005 Lausanne, Switzerland.

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Flash photolysis has become an essential technique for dynamic investigations of living cells and tissues. This approach offers several advantages for instantly changing the concentration of bioactive compounds outside and inside living cells with high spatial resolution. Light sources for photolysis need to deliver pulses of high intensity light in the near UV range (300-380nm), to photoactivate a sufficient amount of molecules in a short time. UV lasers are often required as the light source, making flash photolysis a costly approach. Here we describe the use of a high power 365nm light emitting diode (UV LED) coupled to an optical fiber to precisely deliver the light to the sample. The ability of the UV LED light source to photoactivate several caged compounds (CMNB-fluorescein, MNI-glutamate, NP-EGTA, DMNPE-ATP) as well as to evoke the associated cellular Ca(2+) responses is demonstrated in both neurons and astrocytes. This report shows that UV LEDs are an efficient light source for flash photolysis and represent an alternative to UV lasers for many applications. A compact, powerful, and low-cost system is described in detail.


PNAS - October 12, 2004 - vol. 101 - n°41 - 14937-14942

Astrocytes generate Na+ mediated metabolic waves

Yann Bernardinelli*, Pierre J. Magistretti*†, and Jean-Yves Chatton*‡§
*Department of Physiology, †Center for Psychiatric Neurosciences, and ‡Cellular Imaging Facility, University of Lausanne, CH-1005 Lausanne, Switzerland

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Glutamate-evoked Na+ increase in astrocytes has been identified as a signal coupling synaptic activity to glucose consumption. Astrocytes participate in multicellular signaling by transmitting intercellular Ca2+ waves. Here we show that intercellular Na+ waves are also evoked by activation of single cultured cortical mouse astrocytes in parallel with Ca2+ waves; however, there are spatial and temporal differences. Indeed, maneuvers that inhibit Ca2+ waves also inhibit Na+ waves; however, inhibition of the Na+ glutamate cotransporters or enzymatic degradation of extracellular glutamate selectively inhibit the Na+ wave. Thus, glutamate released by a Ca2+ wave-dependent mechanism is taken up by the Na+ glutamate cotransporters, resulting in a regenerative propagation of cytosolic Na+ increases. The Na+ wave gives rise to a spatially correlated increase in glucose uptake, which is prevented by glutamate transporter inhibition. Therefore, astrocytes appear to function as a network for concerted neurometabolic coupling through the generation of intercellular Na+ and metabolic waves.


Nature Neuroscience 7, 613 - 620 (2004)
Published online: 23 May 2004; | doi:10.1038/nn1246

Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate

Paola Bezzi, Vidar Gundersen, José Luis Galbete, Gerald Seifert, Christian Steinhäuser, Ethel Pilati & Andrea Volterra.

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Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca2+- and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca2+-dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.


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