For in situ hybridization, embryos were collected, rinsed, and freshly embedded for serial cryosectioning (20 μm). Sections were air-dried for 30 min prior to storage (−20°C). Probes were amplified from reverse-transcribed cDNA collected from embryonic C57/B6 brainstem by using primers from the Allen Brain Atlas (http://www.brain-map.org). Atoh1Phox2bCKO (Phox2bCre; Atoh1flox/LacZ) mice and their littermates (WT) were delivered by cesarean section on embryonic day 18.5 from anesthetized (ketamine/xylazine mixture)

timed-pregnant animals. Standard brainstem-spinal (en bloc) preparations with an anterior transection near diencephalon-midbrain junction were made from them, which rostrally included cerebellum, pons, and medulla, find more selleck inhibitor and extended caudally up to the sacral region of the spinal cord, while submerged in cold (4°C) artificial cerebral spinal fluid (aCSF: 124 mM NaCl, 3 mM KCl, 1.5 mM

CaCl2, 1 mM MgSO4, 25 mM NaHCO3, 0.5 mM NaH2PO4, 30 mM D-Glucose (all Sigma, St. Louis, MO) equilibrated with 95% O2 and 5% CO2 to pH = 7.4). The preparations were transferred into a partitioned recording chamber with a rostral (∼2 ml) and a caudal (∼4 ml) spinal cord compartment that were gravity fed by separate reservoirs of heated (25°C–26°C) and aerated (95% O2 and 5% CO2) aCSF at a rate of 3–4 ml/min. These compartments were rendered mutually impervious by plugging the passage connecting the two compartments with paraffin wax. The preparations were allowed to stabilize in the chamber for ∼30 min in circulating (rate 3–4 ml/min) aerated aCSF (25°C–26°C). Extracellular electrophysiological recording were made

from C2–C6 ventral spinal motor roots using a suction electrode. The recordings were amplified using a low-noise differential amplifier (Grass Instruments) with band pass filtering (0.3–3 kHz). The signals were acquired at a rate of 4 kHz and digitized using an analog to digital converter (AD instruments, Colorado Springs, CO). Signal processing that included digital filtration (high-pass, cutoff frequency = 0.3 Hz) and integration over time (absolute value with a 100 ms decay time constant) was done using LabChart 7 Pro software Sodium butyrate (version 7.2.4, AD Instruments). After recording baseline activity, 1 μM Substance P (SP) was added to the rostral brainstem compartment. Peak times of the integrated bursts were determined and respiratory frequencies (bursts/min) were calculated. For statistical comparisons the fictive respiratory frequencies during baseline and SP application of all animals were expressed as percent normalized frequency using the mean baseline cervical burst frequency of WT (cervical burst frequency / mean baseline cervical burst frequency in wild-type mice × 100). The percent normalized frequencies from WT and Atoh1Phox2bCKO mice during baseline and application of SP were compared using independent samples t- and paired t test, respectively.