Microglial cells expressing DREADD (hM3Dq) respond to DREADD agonist C21 with a biphasic [Ca ²⁺]_i response and reduced ATP responsiveness. (a) Representative calcium signals of cultured MicroDREADD^Dq+ and MicroDREADD^Dq− microglia cells repeatedly exposed to 1 µM C21 and 10 µM ATP for 1 min. (b) Differences of average baseline values determined within 50 s before the onset of the responses. Mann-Whitney U test; ^*, P < 0.05; ****, P < 0.0001. (c–e) Peak amplitudes (c), half-width values (d), and peak areas (e) of a, b, c and d peaks denoted in the line graph (a). n = 64 for MicroDREADD^Dq+, n = 73 for MicroDREADD^Dq−; Mann–Whitney U test; ****, P < 0.0001; **, P < 0.01; *, P < 0.05 (c–e). (f) Automated analysis shows marked morphological changes in MicroDREADD^Dq+ microglia in TMX-treated mice 1 h after i.p. CNO administration, compared with littermates in which hM3Dq DREADD expression was not induced with TMX (control). n = 275 control (MicroDREADD^Dq−) and n = 122 DREADD (MicroDREADD^Dq+) microglia from n = 3 mice from each group were analysed. Mann–Whitney U test; ****, P < 0.0001 for cell volume (CellVol), soma volume (SomaVol), branch volume (branchVol), ending nodes, and branching nodes. Scale bar, 20 μm. Data are expressed as mean ± SEM.