Scopolamine methyl nitrate

As described previously41 (link), adult male C57Bl/6-N mice received a low dose of scopolamine methyl nitrate (1 mg kg⁻¹, subcutaneous (s.c.); Sigma) 20 min before the administration of pilocarpine hydrochloride (335 mg kg⁻¹, s.c.; Sigma). Forty minutes after SE onset, the mice received 4 mg kg⁻¹ s.c. diazepam (Ratiopharm). Sham-control animals were treated identically, but received saline instead of pilocarpine.
Adult male Sabra rats (150–200 g) were kept in the animal facility of Hebrew University and Hadassah School of Medicine. All the experiments were approved by the local institution's ethical committee. Rats were injected with scopolamine methyl nitrate (1 mg kg⁻¹, s.c; Sigma). Thirty minutes afterwards, SE was induced with a single dose of pilocarpine (350 mg kg⁻¹ (i.p.); Sigma). SE was terminated after 2 h by diazepam (Ratiopharm; 0.1 mg kg⁻¹ (i.p.)).

Picrotoxin, pilocarpine, scopolamine methyl nitrate and pentobarbital were purchased from Sigma-Aldrich (St. Louis, MO, USA), Lactec D from Otsuka Pharmaceutical Factory (Tokushima, Japan), TTX from Sankyo (Osaka, Japan), D-APV and CNQX from Tocris Bioscience (Avonmouth, U.K.) and halothane from Takeda Pharmaceutical Company Limited (Osaka, Japan).

Male C57BL/6 mice 7–8 weeks of age were administered scopolamine methyl nitrate (intraperitoneally (i.p.); 1.5 mg/kg; Sigma-Aldrich) and terbutaline hemisulfate salt (i.p.; 1.5 mg/kg; Sigma-Aldrich) to block peripheral effects of PI and dilate the respiratory tract, respectively. PI hydrochloride (i.p.; Sigma-Aldrich) at 320 mg/kg was injected 30 min later (Fig. 1E). After continuous tonic clonic convulsive seizures were initiated, mice were placed at room temperature for 8 h and monitored with video recoding. Acute seizures were behaviorally scored using a modified Racine scale (Additional file 1: Videos S1–S6, stages: 0, no abnormality; 1, exploring, sniffing, and grooming ceased, becoming motionless; 2, forelimb and/or tail extension, appearance of rigid posture; 3, myoclonic jerks of the head and neck, with brief twitching movement, or repetitive movements with head bobbing; 4, forelimb clonus and partial rearing, or occasional rearing and falling; 5, forelimb clonus, continuous rearing and falling; and 6, tonic–clonic movements with loss of posture tone, often resulting in death) [27 (link)–29 (link)]. Only mice showing multiple stage 5 or above behavioral seizures were selected for further processing as status epilepticus.