Surgery

Anesthesia was induced by IP injection of ketamine (120 mg/kg) and xylazine (10 mg/kg). Considering the antiseptic technique, the stereotaxic surgery (Stoelting Co, USA) was done with a hand driller to implant four hand-made guides (needles 22-gauge) (two for monopolar electroencephalogram [EEG] recording electrodes, one for reference electrode, and one guide for injection needle) in addition to two small anchoring screws in lateral sides. The epidural EEG electrodes located on parietal bones and the reference electrode on the cerebellum. This assembly was fixed together by dental acrylic cement. The injection guide was 4 mm in length while other guides inserted in 1 mm holes. A stylet was inserted into each guide to prevent the occlusion. The relevant coordinates according to the rat brain atlas[17] were as follows: the left ventral hippocampal commissure, AP: −1.56, L: +1.4, H: 4 mm. Animals were kept warm and closely observed during surgery; then, individually put in a Plexiglas cage for 2 days to regain the normal conditions. On the 3^rd day, animals were anesthetized by urethane, 2 g/kg IP, the stylet was removed. Then, the injection needle (27-gauge dental needles) and monopolar electrodes (ADInstruments, Australia) were inserted in their place. The end of the injection needle was 1 mm beyond the tip of its guide to ensure easy access to brain tissue. After 2 min, DMSO, diazepam, or SB-334867 were administered, each 0.5 μl in 5 min, using a syringe pump. The injecting needle was left in place for at least 10 min to prevent the drug backflow. Initially, we recorded the EEG for 20–30 min until stabilization. After 20 min, we injected 4-AP, 6 mg/kg IP.[18,19] The EEG recording continued for 1.5 h. Finally, Evans blue, 0.5 μl, was injected for verification of the injection site, animals were rapidly decapitated, and the brain was removed and placed in formalin/saline 10%. The spike trains were measured as previously published data.[20] Briefly, the spike train was determined if it included the spikes and waves with an amplitude of at least 2 times the basal level, duration of at least 1 s, and frequency of 5–9 Hz.

Furthermore, an expert observer in a nonblinded manner recorded the behavioral manifestations at the beginning of the study; after injection of DMSO, diazepam, SB-334867, and 4-AP, obtaining 30–60 s videotapes every 15 min; at least six films for each rat. We used the films for data analysis to prevent the experiment's bias. Latency to initiate tonic–clonic SLEs and the total duration of such severe seizure-like behaviors in each animal were measured. We considered the onset of repeated generalized convulsions in whole body and the duration of such condition.