Fig. 3: In vivo activity of individual PB^FoxP2 neurons during CO₂ exposure.

GRIN lens was implanted above the injection sites in the PBcl of FoxP2-Cre mice injected with Cre-dependent AAV-GCaMP6s (a) and the calcium activity profiles (ΔF/F) of individual PBcl^Foxp2 neurons in response to the CO₂ were acquired. Representative Ca_i activity of three neurons (b, c), of which two neurons peaked (ΔF/F) at point d, early during the hypercapnia stimulus while activity of the neuron shown in (c) peaked in the later half of hypercapnia stimulus (shown as point e). Two cells (marked by green and yellow arrowheads whose activity profiles are also plotted in green and yellow) shown in (b) had fluorescence that peaked at about 17–19 s after exposure, before the maximal changes in respiration. A third cell (marked by a magenta arrow and activity profile) peaked roughly 50 s after the onset of the CO₂ trial (c), but CO₂ levels were still high. The ΔF/F from nine cells is also plotted during a 30 s trial of 8% CO₂ (with no awakening), shows an overall increase in Ca_i across the population, with most cells showing peaks at the time of maximal respiration (R), but with substantial variability across neurons (f). A heat map of the mean ΔF/F over 4–7 trials (during which animals exposed to 8% CO₂ did not awaken) is shown for all 28 cells in (g) (blue to red: shows low to high ΔF/F). Note that although the RR and MV summated over these trials (mean ± SEM) increased relatively smoothly (h), the activation of the PBcl^FoxP2 neurons occurred in waves, and of the 17 cells that showed three peaks, ten of them showed second and third peaks that were 17–19 s apart and were synchronous in time after CO₂ exposure, but that not every neuron participated in each wave of excitation. Data in (h) is acquired from n = 3 mice. Source data are provided as a source data file. Two-way ANOVA compared the changes in RR and MV post CO₂ to the Pre-CO₂ baseline, followed by the Holms-Sidak method for multiple comparisons, where ***P < 0.001.