Animal treatments.

The first SE was induced in 8-week-old male mice by the administration of pilocarpine and the second SE was induced 4 weeks after the first SE. A low dose of 100 mg/kg pilocarpine (161-07201, Wako) per injection was administered i.p. every 20 minutes until the onset of Racine scale stage 5 seizures. Scopolamine methyl bromide (1 mg/kg, i.p.; 198-07971, Wako) was administered 30 minutes prior to pilocarpine injection to reduce its peripheral effects (32, 33). Seizures were terminated with pentobarbital (20 mg/kg, i.p.; Kyoritu Seiyaku) when mice experienced stage 5 seizures for 30 minutes. Behavior of pilocarpine-treated mice was observed for 1 hour after SE. To examine whether the first SE increased seizure susceptibility, the second SE was induced 4 weeks after the first SE using the same protocol.

To establish whether minocycline inhibits acute seizure-induced microglial activation, mice were administered i.p. with saline or minocycline (25 mg/kg) 1 hour after pilocarpine-SE induction and for the following 2 consecutive days (37–39). Microglia were also depleted from mice by treatment with the CSF1R antagonist, PLX5622 (Plexxikon), formulated in AIN-76A rodent chow (Research Diets). Mice were treated with PLX5622 (1200 mg/kg Chow) or a matched control diet (AIN-76A) for 7 days before SE and the following 7 consecutive days (40–42).