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. 2019 Aug 23;10:3830. doi: 10.1038/s41467-019-11739-z

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Optogenetic activation of glial cells excites nearby neurons. a Scheme for patch-clamp recording of glial cells expressing channelrhodopsin2 (left). Average membrane potential of a representative optogenetically activated glia (right), n = 3 glia. b Zebrafish brain sparsely expressing channelrhodopsin2-mCherry in GFAP positive glial cells (magenta, indicated with * and #). A single glial cell filled with neurobiotin (green) using patch-clamp electrode, at location *. Note the neurobiotin coupling across glia. c, e Dorsal view of brain-explant preparation showing sparse expression of channelrhodopsin2 in GFAP positive glial cells (magenta), and individual neurons filled with neurobiotin (green) during intracellular recordings. d, f Membrane potential of example neurons with processes nearby channelrhodopsin2 expressing glial cells (c, d), or far away from channelrhodopsin2 expressing glial cells (e, f) during 1 s optogenetic stimulation (blue shade). Overlaid sweeps (top) and 30 Hz low-pass filtered and averaged membrane potential (bottom). g Average change in membrane potential of neurons during the first 300 milliseconds of optogenetic stimulation of glial cells. n = 43 connected, n = 7 not connected neurons. h Distribution of excitation amplitude and excitation delay of connected neurons, upon optogenetic activation of glial cells. n = 43 connected neurons. i, j Examples of neurons with fast (i) and slow (j) excitation upon optogenetic activation before (black) and after (gray) addition of ionotropic glutamate receptor blockers NBQX (5 μM) and AP5 (25 μM). k Average change in membrane potential of neurons during first 1 sec of optogenetic stimulation of glial cells, before and after the addition of NBQX and AP5, n = 9 neurons. l, m Examples of neurons with fast (l) and slow (m) excitation upon optogenetic activation before (black) and after (red) addition of gap junction blocker carbenoxolone (CBX, 100 μM). n Average change in membrane potential of neurons during the first 1 s of optogenetic stimulation of glial cells, before and after addition of carbenoxolone, n = 9 neurons. **p = < 0.01, ***p = < 0.001, Wilcoxon ranksum (g) or sign-rank test (k, n). Shaded regions denote s.e.m. of 25 repetitions (a, d, f, i, j, l, m), error bars denote mean ± s.d. (g). White bars reflect 50 µm (b) or 100 µm (c, e)

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