Scopolamine methyl bromide

SE was induced by systemic injection of pilocarpine, as previously described [35 (link),36 (link),37 (link)]. Briefly, animals were pretreated with lithium chloride (127 mg/kg, i.p.; Sigma-Aldrich, Ciudad de México, México) 22 h before pilocarpine administration. On the day of SE induction, animals were injected with scopolamine methyl-bromide (1 mg/kg, i.p.; Sigma-Aldrich, México) to avoid peripheral cholinomimetic effects, and 30 min later, they received a single dose of pilocarpine hydrochloride (30 mg/kg, i.p.; Sigma-Aldrich, México). Behavioral seizures were scored according to the Racine scale [38 (link)]; SE was defined as sustained convulsive behavior (stage 4 or 5 on the Racine scale) for more than 30 min [20 (link)]. Ninety minutes after SE began, rats were administered an intramuscular (i.m.) injection of 5 mg/kg of diazepam (PISA, Ciudad de México, México) and were placed on an ice bed for 1 h to reduce the hyperthermia produced by SE. CONT rats received saline solution (NaCl 0.9%) instead of pilocarpine. A second dose of diazepam was administered eight hours later, and then the rats received a rehydrating injection of saline solution (5 mL, 0.9%, subcutaneous (s.c.)) on the first night. Finally, the rats were housed overnight in a room at 17 ± 0.5 °C. Beginning one day after SE, the room temperature was restored to 22 ± 2 °C.

The LiCl–pilocarpine rat model of TLE was established, as previously described [60 (link),103 (link),104 (link)]. Briefly, the rats were injected intraperitoneally (ip) with 127 mg/kg LiCl (Sigma-Aldrich, St. Louis, MO, USA) 24 h before pilocarpine injection. One hour before pilocarpine injection, (-)-scopolamine methyl bromide (1 mg/kg, ip; Sigma-Aldrich) was administered to block peripheral muscarinic receptors. pilocarpine (Sigma-Aldrich) was administered at a total dose of 20–40 mg/kg (ip, 2 to 4 injections, 10 mg/kg, at 30 min intervals) to achieve stage 4 seizures on the Racine scale [105 (link)]. Diazepam (10 mg/kg, ip; Sigma-Aldrich) was administered 75 min after the onset of stage-4 seizures to terminate the seizures. Only rats that developed stage 4 seizures were included in the study. Control animals received the same drugs, except pilocarpine.