2.11 Preparation of DRG neuronal cultures
Primary cultures of DRG sensory neurons were obtained from naïve, 3–4-week-old, male C57Bl/6 and Oprm1 cKO mice weighing 12–15 g. Once the mice were anesthetized, the DRGs were carefully removed and immediately placed into fresh, ice-cold DH10 media consisting of DMEM/F-12 supplemented with 10% fetal bovine serum (no DMSO) and 1% penicillin/streptomycin (~1.5 ml per animal), washed with Hanks’ balanced salt solution (HBSS) 1–2×, and then treated with an enzyme solution containing 5 mg∙ml-1 dispase and 1 mg∙ml-1 collagenase type I in HBSS without Ca2+ and Mg2+ (Gibco). Cells were shaken slowly for >45 min at 37°C. After trituration (10–15×) and centrifugation, the cells were resuspended in DH10 media containing nerve growth factor (50 U∙ml-1; Upstate Biotechnology) and plated on freshly made poly-D-lysine- (100 µg∙ml-1; Biomedical Technologies) and laminin- (100 µg∙ml-1) coated coverslips. Cultures were then incubated at 37°C in 5% CO2 until being used in subsequent electrophysiological studies.