“…In accordance with methods approved by the Institutional Animal Care and Use Committee, neonate Sprague-Dawley rat pups (P0–P4) were anesthetized with isoflurane, and, according to methods described elsewhere (Barnes et al, 2007; Mellen, 2008), the neuraxis was isolated, in chilled aCSF made up of (in mM) 128.0 NaCl, 3.0 KCl, 1.5 CaCl 2 , 1.0 MgSO 4 , 21.0 NaHCO 3 , 0.5 NaH 2 PO 4 , and 30.0 glucose, equilibrated with 95% O 2 -5% CO 2 , and a thick sagittal slab was cut (Mellen, 2008), exposing respiratory networks at the surface, with the highest concentration of respiratory neurons ventral, dorsal, and caudal to the facial nucleus (VIIn), and approximately 500 µm caudally in the pre-Bötzinger Complex (preBötC). The preparation was then incubated for 2 hours in an aerated solution containing the high affinity cell-permeant Ca 2+ indicator fluo-4 AM (50 µg, K d = 350 nM; Invitrogen), or the lower affinity fluo-8L (50 µg, K d = 1.86 µM, ABD Bioquest), solubilized in 25 µL of the surfactant pluronic F-127 (2g/10 ml DMSO; Invitrogen), and diluted in 750 µL aCSF for a final concentration of 60 µM.…”