Fig. 2.

A purinergic pathway underlies Aβ-evoked Ca²⁺ signal. Representative responses evoked by Aβ25–35 in astrocytes loaded with the chemical Ca²⁺ indicator OGB-1 AM, in control condition (a), in Ca²⁺-free extracellular solution (b), following thapsigargin (TG, 0.5 µM) depletion of ER Ca²⁺ store (c, top trace reflecting the Ca²⁺ leak signal upon TG application), in the presence of the IP3 receptor antagonist 2-APB (d, 200 µM), and of the mGluR5 antagonist MPEP (e, 50 µM). Each trace represents the response of a single astrocyte. f Aβ-evoked Ca²⁺ responses were fully abolished by blocking purinergic P2 receptors with the combination of wide-spectrum antagonists PPADS (100 µM) and suramin (50 µM). g The P2Y1 antagonist MRS2179 (5 µM) attenuated Aβ-induced Ca²⁺ responses. h Aβ25–35 enhanced Ca²⁺ influx via store-operated channels (SOCs). SOCs were activated by fully depleting the ER store with TG in Ca²⁺-free solution. SOC-mediated Ca²⁺ influx was induced by re-supplying Ca²⁺ in the extracellular solution. Ca²⁺ influx was significantly increased in the presence of Aβ25–35 (n = 11 cells per condition). i Effect of blocking connexin hemichannels with CBX (50 µM). j Aβ-evoked astrocyte Ca²⁺ responses in different conditions. Ca²⁺ signal strength was derived from the temporal integral of individual normalized traces (dF/F₀*s). Wide-spectrum P2X receptor antagonist TNP-ATP, P2X7 antagonist A740003 and pannexin blocker probenecid were applied at 10 µM, 20 µM and 500 µM, respectively. Control experiments were performed for a defined set of experiments as shown (n = 9–20 cells per condition)