Fig. 2: In vivo Ca_i imaging of PBcl^FoxP2 neurons by fiber photometry during exposure to CO₂.

Adeno-associated virus (AAV) expressing Cre-dependent GCaMP6s was injected into the lateral PB of FoxP2-Cre mice (a), resulting in eutopic GCaMP6s expression (green) in the PB^FoxP2 neurons (red) (b). The track of the implanted optical fiber just dorsal to GCaMP-expressing PBcl^FoxP2 neurons is outlined in (b) and allowed us to record their calcium activity as shown in the schematic in (c). A representative fiber photometry recording d shows the activity profile of the PBcl^FoxP2 neurons (ΔF/F), respiration (R) with simultaneous EEG/EMG signals, and the respiratory rate (RR) and minute ventilation (MV = RR × tidal volume, V_T), both of which increased significantly in each CO₂ trial. The control UV signal (isosbestic at 405 nm) was also recorded. Note that the GCaMP signal increased slowly during CO2 exposure, but more sharply as the animal woke up (abrupt change in EMG and EEG about 25 s after CO₂ onset, marked by red arrow). The ΔF/F from 59 trials from n = 5 mice is depicted in e as a heat map illustrating activity 15 s before and after the onset of CO₂ exposure. The graphs below show the mean ± SEM ΔF/F normalized to the pre-CO₂ values (f), %CO₂ in the plethysmograph (g), RR (h), and tidal volume (V_T, i) across the same trials. Two-way ANOVA compared the changes in ΔF/F, RR, and V_T during CO₂ exposure (f–i) compared to the pre-CO₂ baseline, followed by the Holms-Sidak method for multiple comparisons, where ^***P < 0.001; ^**P < 0.01, and ^*P < 0.05. Scale in b 200 µm (lower left) and 20 µm (lower right). Source data are provided as a source data file. Scale in b top right = 400 µm, bottom left = 200 µm; and bottom right = 40 µm. PBcl central lateral PB subnucleus, PBel external lateral PB subnucleus, scp superior cerebellar peduncle.