P6503

Epilepsy was triggered following Pilo-SE in 180–200 g male rats at the age of 7 weeks, as previously described^{33 (link),49 (link)}. Scopolamine methylnitrate (1 mg × kg⁻¹, s.c.; S-2250, Sigma-Aldrich) was administered 30 min prior to pilocarpine hydrochloride (350 mg × kg⁻¹, i.p.; P-6503, Sigma-Aldrich). After an initial period of immobility, the onset of SE was characterized by repetitive tonic–clonic activity of the trunk and the limbs, occurring after repeated rearing with forelimb clonus and falling. Because SE event is a life-threating condition in rats, there is a need to stop it after 2 h by a single injection of diazepam (Valium®, 10 mg × kg⁻¹, i.p.; Roche). Rats were then hydrated with 2 mL of saline solution (0.9% NaCl; s.c.) and transferred to individual cages. After a one-week recovery period, all rats (control and treated rats) were housed in groups of 5 to avoid any stress that may result from social isolation.

Spontaneous activities were recorded for 10 min before treatment and after the cumulative addition to the culture medium of one of the following convulsant agents or receptor antagonists: 4-AP (0.1, 1, 3, 10, or 30 µm; 016–02,781, Wako), pilocarpine (0.1, 1, 3 10, or 30 µm; P6503, Sigma-Aldrich), picrotoxin (0.1, 0.3, 1, 3, or 10 µm; 2,800,471, Nacalai tesque), and AP-5 (1, 3, 10, 30, and 100 µm; 165,304, Sigma-Aldrich). All chemicals were dissolved in DMSO (0.2%–0.6%), which was used as control.
For frequency analysis experiments, 4-AP (0.3, 3, or 30 µm) was administrated into the culture medium, and spontaneous firing was recorded for 10 min.
For propagation analysis experiments using neural spheroids, spontaneous activities were recorded for 10 min before and 20 min after the addition of one of the following typical convulsant agents or receptor antagonists to the culture medium: 4-AP (0.3, 1, 3, 10, or 30 µm), picrotoxin (0.3, 1, 3, 10, or 30 µm), and CNQX (0.3, 1, 3, 10, or 30 µm; C-140, ALOMONE). All chemicals were dissolved in DMSO (0.2%–0.6%), which was used as control.
During all recordings and drug administration, the cultures were kept at 37 °C under a 5% CO₂ atmosphere.

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.