Measurement of pentylenetetrazol-induced seizures

Adult mice (10-week-old Scn2a^RX/+, Scn2a^KO/+ and Scn2a^+/+ littermates, both sex, C57BL/6J congenic background) were used. Experiments of PTZ injection were performed between 9:00 a.m. and 6:00 p.m. A freshly prepared PTZ (Sigma-Aldrich) solution was administered intraperitoneally at a dosage of 25 or 50 mg per kg body weight in a total volume of 100–250 μL. After PTZ injection, the mice were placed in a clear plastic cage and observed for 30 min or earlier if the animal died before 30 min. Video monitoring system was used to determine the latency to the first behavioral seizures from the time of PTZ injection. The behavioral seizure events were determined based on the behavioral and motor activities. We classified the seizures as absence seizure-like immediate behavioral arrest, myoclonic, clonic, and generalized tonic-clonic seizures. Absence-like seizures consisted of > 2 sec behavioral arrest with or without vibrissal and facial myoclonus. Myoclonic seizures consisted of body twitching. Clonic seizures consisted of a whole body jerk with or without irregular, bilateral forelimb movements. Generalized tonic-clonic seizures consisted of fully generalized clonic seizures sometimes followed by a tonic phase involving entire body and result in death. In calculations, 1800 s were assigned for mice without seizures 30 min after PTZ administration. Latency of behavioral seizures by PTZ injection was determined in a genotype-blinded manner.