Calcium imaging of the preBötC

For calcium imaging, the preBötC was stained by Oregon Green 488 BAPTA-1 AM (OGB-1, Thermo Fisher Scientific Inc., Waltham, MA, United States) as described in detail previously (Winter et al., 2009 (link)). Briefly, 50 μg of OGB-1 was dissolved in 40 μl of DMSO containing 20% Pluronic F-127 (Thermo Fisher Scientific Inc., Waltham, MA, United States), and stored at -20°C in 4 μl aliquots before use. One aliquot of this stock solution was dissolved in 16 μl of an extracellular solution containing the following (in mM): 150 NaCl, 2.5 KCl, 10 HEPES, pH 7.4) to prepare 200 μM of OGB-1 at a final concentration. The OGB-1 solution was injected into the preBötC at the depth of 50–100 μm from the rostral surface of the slice for 10 min under 0.7 bar, followed by a perfusion for >40 min at 28°C to washout excess OGB-1.