B1522

Cochlear basilar membrane culture was performed as previously described [10 (link)]. Briefly, anesthetized rat pups were decapitated, the cochlea was separated, and placed in Hank’s balanced salt solution under a dissecting microscope. After exposing the membranous labyrinth, the basilar membrane was quickly dissected and transferred to collagen gel-coated culture dishes with Basal Medium Eagle (BME; Sigma-Aldrich, B1522) containing 10 mg/mL bovine serum albumin, 1% serum-free supplement (Sigma-Aldrich, I1884), 2 mM glutamine (Sigma-Aldrich, G6392), 120 mg/mL glucose, and 100 IU/mL penicillin G. The cultures were maintained under 5% (v/v) CO₂ at 37°C in a humidified incubator. After overnight incubation, the cultures were treated with different concentrations of nicotine for 48 h. Nicotine solution was prepared from liquid nicotine (N3876; Sigma-Aldrich, St. Louis, MO, USA) and diluted in culture medium to achieve final concentrations. This solution was prepared daily as needed and the original liquid was kept away from air and light at 4°C

Hippocampi were dissected from three to six P0-P1 mice and digested in ~20 units of papain (LK003176, Worthington) and ~200 units of deoxyribonuclease (LK003172, Worthington) dissolved to basal medium eagle solution (B1522, Sigma) at 37 °C for 25 min. Tissues were washed with BME and triturated to release cells. Cells were centrifuged at 300 × g for 4 min and plated on polylysine-coated glass coverslip at 3.5 × 10⁵ density in basal medium eagle solution containing B27 supplement (17504044, Gibco), N-2 supplement (17502048, Gibco), 0.5% penicillin/streptomycin (15140148, Gibco), 5% fetal bovine serum, 5% horse serum (260500, Gibco), and GlutaMax (350500, Gibco). The media was replaced after 5 h with the same solution containing 0% serum and 2.5 µM cytosine β-D-arabinofuranoside hydrochloride (C6645, Sigma). Hippocampal neurons were cultured for 4 to 14 d prior to conducting imaging and electrophysiology experiments.