T2528

We created a C57/BL6 male mouse SE model, as previously described^{30 (link)}. Seven-week-old mice received 2 mg/kg of scopolamine methyl bromide (intraperitoneally [i.p.]; S8502; Sigma-Aldrich, St. Louis, MO, USA) and terbutaline hemisulfate salt (2 mg/kg, i.p.; T2528; Sigma-Aldrich) to block the peripheral effects of pilocarpine and dilate the respiratory tract to reduce mortality, respectively. After 30 min, the muscarinic agonist pilocarpine hydrochloride (320 mg/kg, freshly prepared, i.p.; P6503; Sigma-Aldrich) was injected to induce SE. Seizure activity was scored using Racine’s classification^{31 (link)}. Only mice with a Racine’s scale stage of 5 or above for behavioral seizures were selected for the experiments.

Following 2 consecutive days of retro-hM4Di-GFP injections, we performed the pilo induced SE model for TLE based on the previously published methods^{14 (link)}. Briefly, scopolamine methyl nitrate at 2 mg/kg (Sigma-Aldrich S2250) and terbutaline hemisulfate salt at 2 mg/kg (Sigma-Aldrich T2528) were injected intraperitoneally (i.p.) to aid in respiration and peripheral effects due to pilo. 30 min later, pilo hydrochloride (Sigma-Aldrich P6503) was injected i.p. at 300 mg/kg. Mice were then placed in an incubated chamber (ThermoCare) at 31 °C until SE was reached (about 20 min). Once in SE, mice were transferred to a room temperature cage for 3 h where SE was terminated with diazepam (10 mg/kg; Sigma-Aldrich D0899; i.p.). Mice were given 1 ml of 5% dextrose solution (i.p.) and 1 mL of 0.9% NaCl saline (i.p.) to aid in recovery. Mice were monitored in the incubated chamber for 2 days then returned to their home cage and group housed. Mice were excluded from further experiments if SE was not reached within 1 hr of pilo administration and a full duration of 3 h. For mice injected with retro-hM3Dq or hM4Di, CNO (Sigma-Aldrich C0832) was injected (i.p.) daily or twice daily as noted per experiment at 1 mg/kg. To account for any off-target effects of CNO^{60 (link)}, appropriate controls were included in all analysis.