S8502

To evoke and detect handling-induced behavioral seizures, mice (male and female) were gently handled by grasping the tail or scruff 2–3 times a week throughout the lifetime of mice. Behavioral seizure was scored by using Racine scale^{17 (link)}. In pilocarpine-induced seizure experiment, young adult mice (8–12 weeks old) were first injected with N-methyl scopolamine bromide (1 mg/kg, Sigma S8502) before the injection of pilocarpine to inhibit the peripheral cholinergic effects. 15 minutes later, mice were treated with pilocarpine (300 mg/kg, Sigma P6503). After administration of pilocarpine, behavioral seizure score was measured using modified Racine scale for 2 hours: grade (1) stiffness and rigid posture; (2) head nodding; (3) partial forelimb clonus; (4) severe whole body continuous seizures; (5) falling, forelimb clonus and jumping (generalized motor convulsions). To cease the epileptic activity, diazepam (5 mg/kg, Dongwha pharm) was injected to mice. Mortality was comparable during pilocarpine-induced seizure between the genotypes in young adult mice.

We created a C57/BL6 male mouse SE model, as previously described^{30 (link)}. Seven-week-old mice received 2 mg/kg of scopolamine methyl bromide (intraperitoneally [i.p.]; S8502; Sigma-Aldrich, St. Louis, MO, USA) and terbutaline hemisulfate salt (2 mg/kg, i.p.; T2528; Sigma-Aldrich) to block the peripheral effects of pilocarpine and dilate the respiratory tract to reduce mortality, respectively. After 30 min, the muscarinic agonist pilocarpine hydrochloride (320 mg/kg, freshly prepared, i.p.; P6503; Sigma-Aldrich) was injected to induce SE. Seizure activity was scored using Racine’s classification^{31 (link)}. Only mice with a Racine’s scale stage of 5 or above for behavioral seizures were selected for the experiments.