Isolation and Culture of Rabbit Sympathetic Neurons

The method used followed the same procedures as those described by Schofield and Ikeda (1988) (link) and our previous studies (Li et al., 2010 (link); Cheng et al., 2016 (link)). Briefly, the rabbits were anesthetized, and the SCGs were removed and placed in the incubation solution (comprising NaCl 130 mmol/L, KCl 5 mmol/L, glucose 10 mmol/L, HEPES 10 mmol/L, NaH₂PO₄ 1.5 mmol/L, NaHCO₃ 25 mmol/L, MgCl₂ 1 mmol/L, and CaCl₂ 2 mmol/L, aerated with 95% O₂ + 5% CO₂ for 30 min, and adjusted to pH 7.3). Subsequently, SCG slices were cut and incubated for 30 min in the incubation solution (continuously bubbled with 95% O₂ + 5% CO₂). Afterward, SCG pieces were digested in another incubation solution (4 ml, adding 1.7–1.8 g/L collagenase type II (Worthington, United States), 0.6–0.7 g/L pronase E (Roche, Switzerland), and 7.0–8.0 g/L bovine serum albumin (Roche, Switzerland), 95% O₂ + 5% CO₂ for 50–60 min, 37°C). After digestion, SCG pieces were washed with the incubation solution and neurons were dispersed gently by glass tubes with small calibers. The neuron suspension was transferred into a culture dish for the following experiments.