CO₂-stimulated breathing and RTN neuron activation is decreased in GPR4(H81F) mice on a Phox2b-GFP (Jx99) background. A, CO₂-stimulated V_E (left), V_T (middle), and fR (right) in H81F-Jx99 mice compared with WT Jx99 mice (mean ± SEM, N = 9 and 9, P54–P165). Two-way RM ANOVA: minute ventilation, F_(1,16)= 5.801; p= 0.0284 for genotype and F_(4,64)= 9.152; p < 0.0001 for CO₂ exposure × genotype; frequency, F_(1,16)= 31.81; p < 0.0001 for genotype and F_(4,64)= 12.77; p < 0.0001 for CO₂ exposure × genotype; tidal volume, F_(1,16)= 0.2631, p = 0.615 for genotype and F_(4,64)= 2.259; p = 0.0724 for CO₂ exposure × genotype. B, Distribution of Fos-immunoreactive, GFP-labeled neurons and total number of GFP-expressing neurons (C) throughout the rostrocaudal extent of the RTN in either WT Jx99 or H81F-Jx99 mice after exposure in vivo to 12% CO₂ (mean ± SEM, N = 9 and 9, P54–P241; unpaired t test). D, Representative images of brainstem sections from CO₂-exposed WT Jx99 or H81F-Jx99 mice immunostained for Fos and tryptophan hydroxylase (TPH) in the PPy raphe and RPa. Activated serotonergic raphe neurons (Fos+/TPH+) are denoted by white arrowheads, Fos-labeled neurons that are not stained for TPH by green arrowheads, and TPH + neurons that were not activated by CO₂ (i.e., Fos-negative) by magenta arrowheads. E, The percentage of serotonergic neurons activated by CO₂ within the indicated raphe nuclei from WT Jx99 or H81F-Jx99 mice (N = 3 and 4, P63–P135; F_(1,5)= 3.069; p = 0.1402 for genotype, by two-way ANOVA).