Seizure phenotyping

Scn1a^+/− littermate mice carrying B6/129 or Edited/129 Gabra2 alleles were monitored for spontaneous generalized tonic–clonic seizures (GTCS), as previously described (Hawkins et al. 2017a). Briefly, at P18 or P19, mice were subjected to a single, brief (< 1 min) hyperthermia-induced GTCS and then immediately cooled to baseline temperature. If a GTCS did not occur, the mouse was excluded from the study (< 1%) in order to ensure all Scn1a^+/− mice under study had a similar baseline with an initial ‘priming seizure’. Two to three mice of mixed genotype and sex were placed in a monitoring cage with ad libitium access to standard rodent chow and water. Spontaneous GTCS frequency was captured by continuous video monitoring as previously described (Hawkins et al. 2017a, 2016). Mice were monitored beginning at midnight (12–16 h post priming) for 12–14 consecutive days (278–336 h) or until sudden death occurred. This window captures the period of highest seizure frequency in Dravet mouse models (Cheah et al. 2013; Favero et al. 2018; Miller et al. 2014; Mistry et al. 2014; Oakley et al. 2009). Videos were scored offline by reviewers blinded to genotype to determine the frequency and severity of spontaneous GTCS. The total number of seizures for each mouse was divided by the total hours monitored and then converted to a seizure frequency per 24 h. GTCS events were scored using a modified Racine scale adapted for the Scn1a^+/− model as follows: (1) Rearing and paddling, straub tail with no other movement; (2) Rearing and paddling, straub tail, loss of posture, short bursts of movement, often backwards; (3) Rearing and paddling with wild running and/or jumping without loss of posture; (4) Rearing and paddling with wild running and/or jumping with loss of posture; (5) Rearing and paddling with wild running and/or jumping with loss of posture progressing to tonic hindlimb extension (HLE); (6) Rearing and paddling with wild running and/or jumping with loss of posture progressing to tonic HLE ending in death. The number of mice in each score category was compared between groups using a chi-square test. The proportion of seizures with HLE (stage 5–6) was determined for each mouse based on presence or absence of tonic HLE phase for each GTCS event and proportions were averaged by genotype. Seizure frequency and average HLE proportions were first compared between sexes within genotypes. No sex difference was detected; therefore, groups were collapsed across sex for analysis of genotype effect. Seizure frequency and average HLE proportions were compared between genotypes using Mann Whitney U-Tests (GraphPad Prism). Data are presented as means ± SEM.