Fig. 9

P2X7R-mediated activation of a chloride channel is responsible for human microglial permeabilization. a Microglia were pre-incubated for 30 min with the non-selective chloride channel inhibitors tannic acid (TA, 20 μM) or DIDS (100 μM), and YO-PRO-1 uptake was measured after 15 min in the presence of BzATP (300 μM) in normal extracellular solution. Both antagonists significantly inhibited dye uptake; p < 0.0001; ANOVA. The independence of YO-PRO-1 uptake on calcium flux was assessed by loading the cells with 10 μM BAPTA-AM and measuring dye uptake in Ca²⁺-free extracellular solution with 1 mM EDTA (-Ca²⁺; no significant difference from control). YO-PRO uptake in Cl⁻-free solution (-Cl⁻; where extracellular Cl⁻ was replaced with gluconate) was not significantly different from control. n = 4–8 separate experiments for all conditions. b Representative tracing of whole-cell currents (holding voltage = − 60 mV) activated by 10 mM ATP in primary adult human microglia before and after 2-min incubation with TA. c Current amplitude (as fraction of control #1: the current amplitude before antagonist application) induced by ATP (10 mM) in the presence of tannic acid (20 μM, n = 12 cells) or DIDS (100 μM, n = 7 cells) was not significantly different from control #2. Control #2 represents the I_ATP measured after 2-min incubation in control ECS solution. d Representative tracing of microglial 10 mM ATP-gated current that continues to facilitate even in the constant presence of TA (20 μM). e, f Fluorescence images of YO-PRO uptake in microglia incubated for 30 min in the presence of 20 μM nigericin (e) + 20 μM TA (f). Scale bar: 20 μm. g Quantification of nigericin-stimulated YO-PRO-1 uptake by microglia reveals significant inhibition of dye uptake by the chloride channel inhibitor TA, n = 3 separate experiments, significant inhibition by TA (p < 0.0001; unpaired t test)