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. Author manuscript; available in PMC: 2016 Sep 2.
Published in final edited form as: Neuron. 2015 Sep 2;87(5):946–961. doi: 10.1016/j.neuron.2015.08.001

Figure 2. TASK-2 and GPR4 are proton detectors in RTN neurons required for CO2 stimulation of breathing.

Figure 2

(A) Schematic of RTN neuron showing ionic mechanisms for intrinsic pH sensitivity and transmitter modulation. (B) Firing rate histogram from GFP-expressing, dissociated RTN neuron. (C) Left: Staining for β-galactosidase (β-gal; from the TASK-2 locus) in embryo whole mounts from the indicated genotypes; arrowheads indicate RTN region. Right: β-gal staining for TASK-2 (upper) and GFP and TH (lower) in Phox2b::GFP;TASK-2+/− mice; white arrowheads indicate Phox2b-expressing RTN neurons that also express TASK-2. (D) Averaged firing rates at different bath pH for RTN neurons from TASK-2+/+ and TASK-2−/− mice. (E) GPR4 and Phox2b expression detected by in situ hybridization in transverse mouse brainstem section. (F) Left: Respiratory flow recording from GPR4+/+ and GPR4−/− mice with increased inspired CO2 concentrations (balance O2). Right: Lentiviral-mediated, PRSx8-driven re-expression in the RTN of GPR4, but not a non-functional mutant GPR4(R117A), fully rescued ventilatory response to CO2 in GPR4-deleted mice. Shaded areas are 95% confidence intervals for GPR4+/+ (blue) or GPR4−/− mice before lentiviral injection (pink). (G) Multiplex in situ hybridization illustrates differential, but overlapping, expression of GPR4 and TASK-2 in Phox2b-expressing RTN neurons; TASK-2-expressing Phox2b+ neurons without GPR4 are indicated (asterisks). (H) Percent of pH-sensitive and pH-insensitive RTN neurons recorded from mice of the indicated genotypes. (I) Ventilation during incremental CO2 challenge for the indicated genotypes. †, all controls (TASK-2+/+, light blue; GPR4+/+, light pink; and TASK-2+/+:GPR4+/+, light green) greater than single (TASK-2−/−, blue; and GPR4−/−, red) or double knockouts (TASK-2−/−:GPR4−/−, green); *, both single knockouts greater than double knockouts. Panel B adapted from (Wang et al., 2013b); panel C (left) from (Gestreau et al., 2010); panels C (right) & D from (Wang et al., 2013a), and panels E–I from (Kumar et al., 2015).