Analyzing the neuroglial brainstem circuits for respiratory chemosensitivity in freely moving mice

The regulated excretion of CO ^₂ during breathing is a key life-preserving homeostatic mechanism. In the rostral medulla oblongata, neurons in two nuclei -the retrotrapezoid nucleus (RTN) and the rostral medullary Raphe -have been proposed as central CO ₂ chemosensors that mediate adaptive changes in breathing. One such adaptive change is active expiration, thought to be controlled by the lateral parafacial region (pF _L ). Here we use promoter-driven expression of GCaMP6 and head-mounted mini-microscopes to image the Ca ²⁺ activity in neurons and glia of RTN, Raphe and pF _L in awake adult mice during inspiration of elevated CO ₂ to discriminate their roles in the adaptive response to hypercapnia. While there were multiple types of neuronal response to hypercapnia in both the RTN and the Raphe, a substantial proportion of neurons in the Raphe encoded the inspired level of CO ₂ . By contrast, such responses were very rare in the RTN and the responses of RTN neurons suggest that this nucleus plays an integrative rather than a direct sensing role in the chemosensory reflex. pF _L neurons were reliably activated by hypercapnia and their activity preceded active expiration suggesting a key causal link to this aspect of breathing. Our analysis considerably revises understanding of chemosensory control in the adult awake mouse and paves the way to understanding how breathing is coordinated with complex non-ventilatory behaviours such as swallowing and vocalisation.