FoxP2 cells in the lateral parabrachial area may drive respiratory responses to hypercapnia

We have previously reported that CGRP neurons in the external lateral parabrachial nucleus (PBCGRP) play a critical role in transmitting the arousal signal to cause EEG desynchronization in response to hypercapnia. A second population of neurons adjacent to the PBCGRP group, which projects to respiratory control areas in the medulla, are marked by expression of the transcription factor FoxP2. We hypothesize that while the PBCGRP neurons wake up the forebrain during apneas, the adjacent PBFoxP2 neurons may trigger sudden activation of the airway dilators and other components of the respiratory system during apnea. We first exposed mice (n=4) to 2h of 10% CO2, and tested if PBFoxP2 cells can be activated by such exposure, by double- immunostaining the brain tissue for both FoxP2 and cFos. We found that PBFoxP2 neurons showed cFos activation during CO2 exposure, but that none of them also stained for CGRP. Second, we investigated the response of either optogenetic inhibition or activation of PBFoxP2 neurons on respiration. We injected FoxP2Cre mice, that express Cre-recombinase enzyme in all the FoxP2 cells, bilaterally in the PB with adeno-associated virus containing the gene for either channel rhodopsin2 (AAV-FLEX-ChR2-mCherry; n=3, for neuronal activation) or archaerhodopsin T TP009 (AAV-FLEX-ArchT-GFP ArchT; n=2, for neuronal inhibition) in a Cre-inducible FLEX cassette that expressed either ChR2 or ArchT only in the PBFoxP2 cells. These mice were prepared for sleep recordings and were implanted with bilateral optic fiber targeting the PB. Blue laser (473nm) light targeted to the PB, activated ChR2 expressing neurons with 10ms pulses of 473nm given at 5, 10 and 20Hz, for 5s every 5 minutes, without any hypercapnia stimulus and then respiration was measured. Photo-stimulation of ChR2 expressing PBFoxP2 at 10Hz for 5s significantly increased the respiratory rate (RR) (F2, 12= 6.35, P=0.013) and the tidal volume (TV) compared to the pre-stimulation period (F2, 12= 3.20, P=0.04), with no effect at 5 Hz. At 20 Hz for 5s, RR increased by 63% and VT by 62%. Animals woke-up in most trials towards the end of the 5s stimulation period. We also investigated the respiratory response to10% CO2 given for 30s every 300s, with and without photo-inhibition of the PBFoxP2 neurons with 593 nm laser light. Laser light was on for 60s beginning 20s prior and extending 10s after the CO2 stimulus. Inhibition of the PBFoxP2 cells caused 27% reduction in the ventilatory effort as calculated by the minute ventilation (MV) during the time the animals were exposed to CO2 both before (F1,11=3.68; P= 0.026) and after EEG arousal (F1,11=3.68; P<0.001), without affecting the latency of arousal from CO2. Finally, the GCaMP fiber photometry recordings suggested that the firing of the PBFoxP2 neurons as seen by the increase in GCaMP fluorescence correlates positively with respiratory rate and tidal volume (R2= 0.36; P<0.001), thus corroborating the role of PBFoxP2 neurons in regulating respiratory response to hypercapnia.