Optogenetic activation of LiGluR-expressing astrocytes evokes anion channel-mediated glutamate release

INSERM U603, CNRS UMR 8154, Laboratoire de Neurophysiologie et Nouvelles Microscopies, 45 rue des Saints Pères, Paris, F-75006 France.
The Journal of Physiology (Impact Factor: 5.04). 01/2012; 590(Pt 4):855-73. DOI: 10.1113/jphysiol.2011.219345
Source: PubMed


Increases in astrocyte Ca(2+) have been suggested to evoke gliotransmitter release, however, the mechanism of release, the identity of such transmitter(s), and even whether and when such release occurs, are controversial, largely due to the lack of a method for selective and reproducible stimulation of electrically silent astrocytes. Here we show that photoactivation of the light-gated Ca(2+)-permeable ionotropic GluR6 glutamate receptor (LiGluR), and to a lesser extent the new Ca(2+)-translocating channelrhodopsin CatCh, evokes more reliable Ca(2+) elevation than the mutant channelrhodopsin 2, ChR2(H134R) in cultured cortical astrocytes. We used evanescent-field excitation for near-membrane Ca(2+) imaging, and epifluorescence to activate and inactivate LiGluR. By alternating activation and inactivation light pulses, the LiGluR-evoked Ca(2+) rises could be graded in amplitude and duration. The optical stimulation of LiGluR-expressing astrocytes evoked probabilistic glutamate-mediated signalling to adjacent LiGluR-non-expressing astrocytes. This astrocyte-to-astrocyte signalling was insensitive to the inactivation of vesicular release, hemichannels and glutamate-transporters, and sensitive to anion channel blockers. Our results show that LiGluR is a powerful tool to selectively and reproducibly activate astrocytes.

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Available from: Nicole Ropert, Sep 30, 2015
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    • "Prior to imaging, the photoswitch D-MAG0 was conjugated to LimGluR3 by incubating transfected astrocytes in D-MAG0-containing solution (50 μM) for 15 min, followed by 30 min washing with control solution. An LED-based light source (385 nm, 1.2 mW mm −2 estimated at the sample plane) was used to photoactivate LimGluR3, similar to what was described previously for LiGluR (Li et al. 2012). Ionomycin and jasplakinolide were obtained from Invitrogen, GABA, cytochalasin D, TFB-TBOA, SQ22536 and LY354740 from Tocris (Ellisville, MO, USA), dynasore from Ascent Scientific (Avonmouth, UK), forskolin and isoprenaline hydrochloride from Abcam (Cambridge, MA, USA), and all other compounds from Sigma-Aldrich. "
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    ABSTRACT: Previous works suggest that small synaptic-like vesicles in astrocytes carry vesicle-associated vSNARE proteins, VAMP3 (cellubrevin) and VAMP2 (synaptobrevin 2) both contributing to the Ca(2+) -regulated exocytosis of gliotransmitters, thereby modulating brain information processing. Here, using cortical astrocytes taken from VAMP2 and VAMP3 knock-out mice, we find that astrocytes express only VAMP3. The morphology and function of VAMP3 vesicles were studied in cultured astrocytes at single-vesicle level with stimulated emission depletion (STED) and total internal reflection fluorescence (TIRF) microscopies. We show that VAMP3 antibodies label small diameter (∼80 nm) vesicles and that VAMP3 vesicles undergo Ca(2+) -independent exo-endocytosis. We also show that this pathway modulates the surface expression of plasma membrane glutamate transporters and the glutamate uptake by astrocytes. Finally, using pharmacological and optogenetic tools, we provide evidence suggesting that the cytosolic cAMP level influences astrocytic VAMP3 vesicle trafficking and glutamate transport. Our results suggest a new role for VAMP3 vesicles in astrocytes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    The Journal of Physiology 04/2015; 593(13). DOI:10.1113/JP270362 · 5.04 Impact Factor
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    • "Interestingly, we also noticed that even if the currents elicited in Ca2+ translocating ChR2 (CatCh) positive astrocytes where on average 15 times larger than ChR2 (measured as area under the curve (AUC), Fig. S3) the neural network modulation was successfully achieved with ChR2, although previous reports suggest a better and stronger Ca2+ elevation by means of Ca2+-permeable light-gated glutamate receptor (LiGluR) [25] and CatCh [25], [26]. "
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    ABSTRACT: In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (mu LEDs) with an excitation peak at 470 nm. We combined Ca2+ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the mu LEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing mu LED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the mu LEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and mu LEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.
    PLoS ONE 09/2014; 9(9). DOI:10.1371/journal.pone.0108689 · 3.23 Impact Factor
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    • "Several floxed (Slezak et al., 2012; Zariwala et al., 2012) and tetO (Fiacco et al., 2007; Agulhon et al., 2010) mouse lines of interest have been generated. With a hGFAP-CreER T2 mouse line in which the recombination can be induced in juvenile or adult mice by tamoxifen injections, astrocyte-specific targeting has been obtained in cortex, hippocampal CA1 region, cerebellum, diencephalon and brain stem, with weaker levels of recombination in cortex (Hirrlinger et al., 2006; Lioy et al., 2011). "
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    ABSTRACT: Gray matter protoplasmic astrocytes extend very thin processes and establish close contacts with synapses. It has been suggested that the release of neuroactive gliotransmitters at the tripartite synapse contributes to information processing. However, the concept of calcium (Ca(2+))-dependent gliotransmitter release from astrocytes, and the release mechanisms are being debated. Studying astrocytes in their natural environment is challenging because: (i) astrocytes are electrically silent; (ii) astrocytes and neurons express an overlapping repertoire of transmembrane receptors; (iii) the size of astrocyte processes in contact with synapses are below the resolution of confocal and two-photon microscopes (iv) bulk-loading techniques using fluorescent Ca(2+) indicators lack cellular specificity. In this review, we will discuss some limitations of conventional methodologies and highlight the interest of novel tools and approaches for studying gliotransmission. Genetically encoded Ca(2+) indicators (GECIs), light-gated channels, and exogenous receptors are being developed to selectively read out and stimulate astrocyte activity. Our review discusses emerging perspectives on: (i) the complexity of astrocyte Ca(2+) signaling revealed by GECIs; (ii) new pharmacogenetic and optogenetic approaches to activate specific Ca(2+) signaling pathways in astrocytes; (iii) classical and new techniques to monitor vesicle fusion in cultured astrocytes; (iv) possible strategies to express specifically reporter genes in astrocytes.
    Frontiers in Cellular Neuroscience 10/2013; 7:193. DOI:10.3389/fncel.2013.00193 · 4.29 Impact Factor
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