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. 2020 Feb 12;40(7):1373–1388. doi: 10.1523/JNEUROSCI.0218-19.2019

Figure 3.

Figure 3.

High doses of ADP induce microglial process retraction and membrane ruffling. A, ADP induces membrane ruffling of amoeboid microglia. Control (left), ADP (2 mm, 20 min, middle). Right, Merge image represents color-coded differences. Red represents new fluorescent signal; green represents lost signal; yellow represents maintained signal. B, Transition from 2 mm ADP back to control for the same cell (wash, left) induces process extension. Right, Merge image represents differences from ADP. Blue represents new signal; red represents lost signal. C, ADP induces process retraction and membrane ruffling of ramified microglia. Control (left), ADP (2 mm, 20 min, middle). Right, Merge represents differences. Blue represents new signal; green represents lost signal; yellow represents maintained signal. D, Transition from ADP back to control for the cell in C induces process extension (wash, left). Right, Merge image represents color-coded differences. Blue represents new signal; red represents lost signal. A–D, Maximal projections from z stacks (50 μm, 2 μm step). E, Time course of changes in mean cross-sectional area in 7 initially amoeboid cells. Little change occurs during application of 2 mm ADP application. Processes extend when ADP is removed. F, Time course of the reduction in mean cross-sectional area induced by 2 mm ADP in 9 ramified microglia. Processes extend when ADP is removed. G, Different latencies to process extension (increase of 20% for cross-sectional area) during the transition from 2 mm ADP back to control ACSF. The latency is significantly longer for ramified microglia (t test, p = 0.004**). Movie 2 shows membrane ruffling of amoeboid microglia induced by 2 mm ADP. Movie 3 shows process retraction of a ramified microglia induced by 2 mm ADP. H, Fluorescence at 0, 5, and 20 min after focal ejection of the fluorescent purine EDA-ADP-ATTO-488 at 1 mm from a patch pipette into middle regions of a 300-μm-thick PTC slice. I, The decay followed a double exponential time course with fastest decay in the first 3–5 min. The fit was y = 435.0 × exp(−x/0.9) + 158.9 × exp(−x/3.4) + 14.6, where y is fluorescence and x is time. Fluorescence was maintained longer than 20 min after ejection into the slice but decayed in 2–3 s after ejection into the bath.