(A) Ventral view of triple immunofluorescence for Islet1,2, Phox2b and tdTomato over the facial area in Atoh1FRTCre;P2b::FLPo;R26tdTomato embryos showing Phox2bon/Atoh1on cells (yellow) at E14.5 (left), E16.5 (middle) and E18.5 (right). (B) RTN activity map and right, close up of the boxed area showing Phox2bon/Atoh1on cells (red) loaded with Calcium Green-1 AM (Ca-G) (yellow). (C) Histogram showing that the burst frequencies of the global population of oscillating RTN neurons (white bars, n = 27 preparations) are not significantly different from that of the tdTomato+ (thus Phox2bon/Atoh1on) cells in Atoh1FRTCre;P2b::FLPo;R26tdTomato embryos (red bars, n = 6 preparations) at pH7.4 (7.4) and pH7.2 (7.2). (D) Superimposed traces showing spontaneous rhythmic Ca-G fluorescence changes of 16 individual Phox2bon/Atoh1on cells (red traces) and their average (black trace) at pH7.4 (top, 7.4) and pH7.2 (bottom, 7.2). Note the frequency increase in low pH. (E) Left panel, ventral view of an E14.5 brainstem preparation showing Phox2bon/Atoh1on tdTomato+ RTN neurons (red) on either side of the midline (marked by the auto-fluorescence of the holding mesh) during an electrophysiology experiment. Middle panel, close up of the boxed area showing Phox2bon/Atoh1on cells (red) and right, one biocytin-filled Phox2bon/Atoh1on cell during whole-cell patch-clamp recording. (F) Top row, trace of the membrane potential trajectory of the biocytin-labeled cell in E showing spontaneous burst discharges of action potentials (indicated by *) at pH7.4 (7.4); bottom row, same trace at pH7.2 (7.2). Note the increased frequency of bursts at low pH. (G) Close up of a burst discharge (top) that can be curtailed by negative current pulses (bottom) applied 10 ms (red traces), 50 ms (blue traces) or 100 ms (green traces) after onset of the first action potential of the burst. (H) Photostimulation of Phox2bon/Atoh1on cells expressing ChR2 at E15.5 evokes C4 motor responses. Left panel, ΔF/F changes in the RTN and adjacent nVII (dotted line) following photostimulation of the RTN region. Right, at top, integrated activity of the C4 root showing spontaneous activity and bursts of activity evoked by light pulses (blue bars) triggered 4 s after a spontaneous burst. Bottom left, close up of a set of 10 superimposed spontaneous (Spont.) C4 bouts of activity and right, a corresponding set of light-evoked (Light-evoked) C4 responses, synchronized on the light pulse (blue bar), showing their variable latencies.