Optogenetic activation of LiGluR-expressing astrocytes evokes anion channel-mediated glutamate release.
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.
INSERM U603, CNRS UMR 8154, Laboratoire de Neurophysiologie et Nouvelles Microscopies, 45 rue des Saints Pères, Paris, F-75006 France. dongdong.li@parisdescartes.fr
Dongdong
D
Li
Karine
K
Hérault
Ehud Y
EY
Isacoff
Martin
M
Oheim
Nicole
N
Ropert
D000818:Animals; D001253:Astrocytes; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology*; D002478:Cells, Cultured; D000075402:Channelrhodopsins; D004622:Embryo, Mammalian; D005453:Fluorescence; D018698:Glutamic Acid / Q000502:physiology*; D051379:Mice; D019579:Neocortex / Q000166:cytology; D009474:Neurons; D017470:Receptors, Glutamate / Q000502:physiology*; D014466:Ultraviolet Rays; D050994:Voltage-Dependent Anion Channels / Q000502:physiology*
D015220:Calcium Channels; D000075402:Channelrhodopsins; C508971:LiGluR receptor; D017470:Receptors, Glutamate; D050994:Voltage-Dependent Anion Channels; D018698:Glutamic Acid; D002118:Calcium