Depolarization induces hyperalgesia in vivo. (A and C) Evaluation of mechanical nociceptive thresholds (Randall–Selitto test) 5, 15, 45, 60, 120, 180, and 1,440 min after intradermal injection of KCl (5 µl of 80 mM KCl solution in dH₂O) in the dorsum of the hindpaw of male rats. A biphasic mechanical hyperalgesia was observed in the ipsilateral paw (A, percentage reduction from baseline: F_(1,10)=186.0, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001; C, nociceptive threshold: F_(1,10)=47.75, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, when the ipsilateral and contralateral paws were compared in all time points; two-way repeated-measures ANOVA followed by Sidak's test). (B and D) Vehicle (5 µl, dH₂O + 1% ethanol) or verapamil (10 µg diluted in 5 µl dH₂O + 1% ethanol) was injected intradermally in the dorsum of the hindpaw. 30 min later, KCl (5 µl of 80 mM KCl solution in dH₂O) was injected at the same site and mechanical nociceptive threshold evaluated 5, 15, 45, 60, 120, 180, 360, and 1,440 min later. In the group that received verapamil, hyperalgesia was inhibited (B, percentage reduction in nociceptive baseline: F_(1,10) = 222.0, ****, P < 0.0001; D, nociceptive threshold: F_(1,10) = 22.38, *, P = 0.02, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, when the vehicle- and verapamil-treated groups are compared at all time points after the injection of KCl; two-way repeated-measures ANOVA followed by Sidak's test). Data represent means ± SEM, n = 6 rats per group.