Primary cultures of hippocampal neurons were prepared as described46, with minor modifications. Briefly, hippocampi were extracted from neonatal (P1–4) pups and placed into an ice-cold modified Hank’s Balanced Salt Solution (HBSS, Sigma-Aldrich) containing 1 mM kynurenic acid, 12 mM MgCl2, 3 mM HEPES, and 5.5 mM D-glucose. The tissue was digested for 20 min with papain and DNAse I in the modified HBSS solution at 37 °C with occasional inversion. Tissue was then washed twice with modified HBSS, twice with modified HBSS plus 20% FBS, and twice with growth medium: Neural-basal A medium containing 2% B27 supplement, 0.5 mM Glutamax, and antibiotic/antimycotic (Thermo Fisher Scientific). Hippocampi were mechanically-dissociated in growth medium using trituration in 1 mL pipettes. Neurons were pelleted by centrifugation (150 × g, for 5 min at 4 °C), and plated onto 8-mm glass coverslips pre-coated with 0.0005% poly-L-lysine (Sigma-Aldrich) in 48-well plates. Neurons were maintained in culture at 37 °C/5%CO2, and half of the medium was replaced with fresh growth medium every 3–4 d. After 10 d in culture, neurons were infected with control or GIRK expression viruses; electrophysiological analysis or immunofluorescence microscopy was conducted 4–5 d after infection.
