Pilocarpine-Induced Status Epilepticus

A total of 160FVB/NJ mice (all males JAX stock #001800) were purchased from The Jackson Laboratory (Bar Harbor, ME) and received at 5–7 weeks of age. Prior to experiments, littermates (on average, 5) were group housed in temperature- and humidity-controlled rooms on a 12 h light/dark cycle with access to food and water ad libitum. All mice were allowed to acclimate to the environment and altitude for 1 week prior to handling. Prior to the start of experiments, mice (18–24 g) were briefly handled once daily for at least 1 week to reduce the stress induced by experimental protocols. On the day of the injections, mice were transferred to an experimental room, marked, weighed, and allowed to habituate undisturbed for at least 1 h. Entire litters (an average of 5 mice) were randomly assigned to a single treatment/time point group. Only in cases where fewer than 5 mice comprised a single litter were mice from two litters combined for a single treatment/time point. For pilocarpine treated mice, 15 min before the first pilocarpine injection, mice were given an intraperitoneal injection of 1 mg/kg scopolamine methyl bromide (Sigma-Aldrich) to block the peripheral muscarinic effects of pilocarpine. The initial dose of pilocarpine HCl (200 mg/kg; Sigma-Aldrich) was followed, after 1 h, by subsequent doses (100 mg/kg) given at 30 min intervals until the onset of SE, defined as the appearance of repeated behavioral seizures (stage four or higher with at least one seizure being five or higher) according to a modified Racine scale (22, 23). In the majority animals, SE initiated within 1 h of the first pilocarpine injection; thus these animals received a single injection. The maximum number of injections given was three. SE persisted for at least 90 min. Control mice were given injections of saline at identical time intervals. After SE induction, mice were returned to their home room, singly housed, and given free access to water and moistened chow. Cohorts (5 per group) of pilocarpine treated mice were sacrificed after SE, at 15 min (onset), 1 and 6 h, 1 and 5 days, and 2 weeks. Groups of saline-injected mice were sacrificed at the same time points (see Supplementary Figure 1).