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. 2021 Jan 28;18:33. doi: 10.1186/s12974-021-02093-3

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Optogenetic microglia depolarization decelerates chemotactic response kinetics. a Graphic illustration and representative images of microglia chemotaxis towards an induced laser-damage. b Two-photon maximum projections of the chemotactic response 0, 9, and 24 min after the laser-damage. c Two-photon z-projection of a patched microglia during chemotaxis. d Voltage-clamp recordings of patched microglia during chemotaxis. Gray: Individual microglial responses from four experiments, red: Average of all experiments. Left: no light stimulation during laser-damage, right: with light stimulation during laser damage. e Automated MATLAB analysis of chemotaxis quantified as the reduction in microglia-free area around the laser damage (black polygon) at different time points of the experiment. f Relative laser damage response measured as microglia-free area. Black: Control slices (no construct) with light stimulation (n = 8 areas, 5 slices). Gray: Experiments with ChETA expression in microglia, but without light stimulation (n = 7 areas, 4 slices). Blue: Slices with ChETA expression in microglia combined light stimulation (n = 11 areas, 7 slices). Insert: Graphic representation of light stimulation protocol between stack acquisitions. 2-way ANOVA (‡ control480 – ChETA480, p < 0.001, († ChETA no light – ChETA480, p < 0.01). g Time to 50% engulfment was prolonged by optogenetic depolarization. h 9 min after injury, the microglia-free area was larger when microglia were depolarized. g, h One-way ANOVA with Tukey’s post hoc comparison (*p < 0.05, **p < 0.01, ***p < 0.001)