Local brain injury-induced migration of microglia in larval zebrafish in vivo.

(A) Whole-brain projection images showing the distribution of microglia (GCaMP5-positive, green) and neurons (jRGECO1a-positive, red) in a double transgenic larva Tg(coro1a:GCaMP5);Tg(HuC:NES-jRGECO1a) at 5 dpf. The green signals on the eyeballs were auto-fluorescence. C, caudal; L, lateral. Scale bar, 50 μm

(B) In vivo time-lapse (with a 5-s interval) two-photon images showing microglia's directional migration evoked by two-photon laser-induced local brain injury (red dot). Only time series at a 15-min interval were shown. The time point of 0 min indicates the onset of injury induction. The numbers and arrowheads indicate the microglia traced across the whole imaging. The image field is outlined in (A), and the images were obtained from a 7-dpf larva. Scale bar, 20 μm

(C) In vivo time-lapse (with a 4-s interval) two-photon images showing microglia's directional migration evoked by micropipette (dashed orange lines)-induced local brain injury. Only time series at a 5-min interval were shown. The time point of 0 min indicates the onset of micropipette insertion. The numbers and arrowheads indicate the microglia traced across the whole imaging. The image field is outlined in (A), and the images were obtained from a 5-dpf larva. Scale bar, 10 μm. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Association between the Ca²⁺ activity and migration of activated microglia.

**p < 0.01, ***p < 0.001 (unpaired Student's t-test for (C) and for comparison between migrating and non-migrating microglia in (D), paired Student's t-test for comparison within migrating or non-migrating microglia in (D)). Error bars, SEM.

(A) Up, in vivo time-lapse two-photon images showing Ca²⁺ activities (in heatmaps) accompanying with microglia migration induced by laser-based local brain injury (red dot) at time 0. The data were obtained from the same larvae with that in Fig. 1B. Red numbers and arrowheads, microglia responsive to the injury; green numbers and arrowheads, microglia located in the contralateral hemisphere and non-responsive to the injury. Dashed white lines represent the midline between two hemispheres. Down, raw images showing the morphology of non-responsive microglia in the contralateral hemisphere. Scale bar, 50 μm

(B) Traces of Ca²⁺ activities of microglia numbered in (A). The vertical line indicates the onset of local brain injury. Arrow, immediate Ca²⁺ transient induced by the injury.

(C) Summary of the fraction of cells showing injury-induced immediate Ca²⁺ transient for the migrating (red) and non-migrating (green) microglia. Data were collected from 31 microglial cells in 4 zebrafish larvae, and each dot on the bar represents the data from an individual larva.

(D) Summary of injury-induced later Ca²⁺ transient number per minute before and after the local injury in migrating (red) and non-migrating (green) microglia. The number in the brackets represents the number of microglia examined, and each dot on the bar represents the data from an individual microglial cell. Data were collected from 31 microglial cells in 4 zebrafish larvae.

(E) Association between Ca²⁺ transient number per minute and migration speed before microglia stop moving on the injury site. The black linear line represents the simple linear regression slope (Y = 0.9932*X + 0.9130). Each dot on the graph represents the data from an individual microglial cell. Data were collected from 31 microglial cells in 4 zebrafish larvae. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Requirement of Ca²⁺ activity for injury-induced migration of activated microglia.

*p < 0.05 (paired Student's t-test for comparison within each group before and after the puff, unpaired Student's t-test for comparison between groups of 2-APB and ES). Error bars, SEM.

(A) In vivo time-lapse two-photon images showing Ca²⁺ activity (in heatmaps) and migration of microglia evoked by micropipette-induced local brain injury (dashed orange lines) applied at time −10 min before the local puff of 2-APB (just after the time of 0 min). Scale bar, 10 μm

(B) Traces of both the Ca²⁺ activity (black) of the microglial cell and the distance (red) between the microglial cell body and micropipette tip for the case in (A). Gray area, application of 2-APB.

(C and D) Control case with puff of extracellular solution (ES) instead of 2-APB. Scale bar in (C), 10 μm

(E and F) Summary of changes in the Ca²⁺ activity (E) and migration speed (F) induced by puffing of ES or 2-APB. Each dot on the bar represents the data from an individual microglial cell examined. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)