2.3. SE Induction and EEG Analysis

Three days after surgery, SE was induced by a single dose (30 mg/kg) of pilocarpine in rats pretreated (24 h before pilocarpine injection) with 127 mg/kg LiCl, as previously described [14,15]. Before pilocarpine injection, animals were given atropine methylbromide (5 mg/kg i.p.) to block the peripheral effect of pilocarpine. As controls, rats were treated with saline instead of pilocarpine. After injection, animals were monitored continuously for 2 h to register the extent of behavioral seizure activity. Behavioral seizure severity was also evaluated according to Racine’s scale [19]: (1) immobility, eye closure, twitching of vibrissae, sniffing, or facial clonus; (2) head nodding associated with more severe facial clonus; (3) clonus of one forelimb; (4) rearing, often accompanied by bilateral forelimb clonus; and (5) rearing with loss of balance and falling accompanied by generalized clonic seizures. Within 20–45 min of treatment with pilocarpine, animals became catatonic and began staring, followed by myoclonic twitching and often frequent rearing and falling. The behavioral seizure score reached 4–5 in all groups. There was no difference in the behavioral seizure score induced by pilocarpine among all the groups. In some animals, EEG signals were also recorded with a DAM 80 differential amplifier (0.1–3000 Hz bandpass, World Precision Instruments, Sarasota, TL, USA), digitized (sampling rates, 1000 Hz) and analyzed using LabChart Pro v7 (AD Instruments, Bella Vista, NSW, Australia). Total EEG power and spectrograms were automatically calculated in 2-hour recording session using a Hanning sliding window with 50% overlap [14,15]. Two hours after SE, animals received diazepam (Valium; Roche, France; 10 mg/kg, i.p.) to terminate SE.