Kainate-Induced Hippocampal Alterations

To elicit status epilepticus, male rodents were subjected to intraperitoneal (i.p.) injection of vehicle control (autoclaved H₂O or saline) or kainate (abcam) as described^{40 (link)} at 9 mg/kg for Sprague-Dawley rats weighing 200–250 g (Fig. 8b,c), 20 and 30 mg/kg for CD001 rats weighing 110–160 g (Fig. 8d,e), 15 and 30 mg/kg for C57BL/6 J mice weighing 20 g (Supplementary Fig. S8). Rodents were returned to their home cage and video monitored. At 3 h and 8 h post injection, 2 hippocampi per rat was homogenized in modified RIPA buffer (1 mL) containing 1% SDS and protease inhibitors, sonicated briefly, and centrifuged at 14,000 rpm. Two hippocampi per mouse was homogenized as described^{65 (link)} to isolate crude homogenate (S1), soluble (S2) and membrane (P2) protein fractions. Hippocampal lysates were immunoblotted with antibodies for GIRK1 and GIRK2 N termini (1:200, Supplementary Fig. S2), c-Fos, c-Jun, anti-β-tubulin, or GAPDH (1:500–1:1,000, all Cell Signaling). Several hippocampal lysates from vehicle-treated rats did not show any immunoblot bands for cleaved GIRK (Fig. 8b–e). Therefore, background-subtracted immunoblot band intensity ratios of cleaved GIRK/loading controls (β-tubulin or GAPDH) from kainate-injected rats were taken as 100%. The ratio from vehicle-injected rats were then normalized to the ratio from kainate-injected rats to obtain % KA.