The P2Y₁₂ receptor regulates microglial activation by extracellular nucleotides

Sharon E Haynes, Gunther Hollopeter, Guang Yang, Dana Kurpius, Michael E Dailey, Wen-Biao Gan & David Julius

Supplementary Fig. 1 (pdf 16 k)
Microglia from P2Y₁₂-deficient mice have normal morphology and prevalence within the CNS.

Supplementary Fig. 2 (pdf 45 k)
Pretreatment with LPS diminishes ATP-induced microglial ruffling.

Supplementary Fig. 3 (pdf 94 k)
Microglia express P2Y₁₃ transcripts, but not detectable receptor protein.

Supplementary Video 1 (mov 541 k)
Wild-type (left) microglia in the Dunn chemotaxis chamber undergo membrane ruffling, polarization, and directed motility in the presence of a 50 to 0 M ATP gradient (top to bottom) whereas P2Y₁₂-deficient (right) microglia show dramatically reduced stimulation. The chemotaxis chamber was placed in a heated, C0₂ buffered humidified microscope incubator and phase contrast images were acquired every 150 s for 30 min. Scale bar represents 20 m.

Supplementary Video 2 (mov 9,981 k)
Acutely prepared hippocampal slices from P7 neonatal wild-type (left) and P2Y₁₂-deficient (right) mice were bathed in imaging media with 1 mM ADP. Wild-type microglia respond within minutes by sending out long, cellular projections towards the periphery of the slice (top, right, and bottom edges of field of view). Some cells actually undergo whole-cell body displacement as they travel towards the nucleotide source while other cells extend elaborately branched ramifications. Some cells first respond by sending out branches before exhibiting whole cell locomotion. In striking contrast, microglia from P2Y₁₂-deficient animals show a dramatically diminished behavioral response towards the nucleotide source. Images show the CA3 layer of the hippocampal slice. Cells were visualized by GFP expression, images were taken at 5 min intervals for 6.75 h, and a maximum projection of 15 z-steps spaced 2 m apart (30 m total thickness) was constructed for each frame. Scale bar represents 100 m.

Supplementary Video 3 (mov 2,433 k)
In vivo imaging of GFP labelled microglia in wild-type (left) and P2Y12-deficient (right) mice after injection of 20 mM ATP (red needle). Wild-type microglia displayed robust branch extension towards the site of injection whereas P2Y12-deficient microglia did not. Experiment is 40 min, 4 min intervals. Scale bar represents 20 m.

Supplementary Video 4 (mov 5,590 k)
In vivo imaging of GFP labelled microglia in wild-type (left) and P2Y₁₂-deficient (right) mice after focal laser ablation induced by the two-photon laser. Wild-type microglia displayed robust process extension towards the site of injury whereas P2Y₁₂-deficient microglia showed a dramatically reduced and time-delayed response. Experiment is 40 min, 4 min intervals. Scale bar represents 20 m.

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