Forebrain projection neurons target functionally diverse respiratory control areas in the midbrain, pons and medulla oblongata

Eupnea is generated by neural circuits located in the ponto-medullary brainstem, but can be modulated by higher brain inputs which contribute to volitional control of breathing and the expression of orofacial behaviors, such as vocalization, sniffing, coughing and swallowing. Surprisingly, the anatomical organization of descending inputs that connect the forebrain with the brainstem respiratory network remains poorly defined. We hypothesized that descending forebrain projections target multiple distributed respiratory control nuclei across the neuraxis. To test our hypothesis, we made discrete unilateral microinjections of the retrograde tracer Cholera toxin subunit B (CT-B) in the midbrain periaqueductal gray (PAG), the pontine Kölliker-Fuse nucleus (KFn), the medullary Bötzinger complex (BötC), pre-Bötzinger complex (pre-BötC) or caudal midline raphé nuclei. We quantified the regional distribution of retrogradely-labeled neurons in the forebrain 12-14 days post-injection. Overall, our data reveals that descending inputs from cortical areas predominantly target the PAG and KFn. Differential forebrain regions innervating the PAG (prefrontal, cingulate cortices, and lateral septum) and KFn (rhinal, piriform, and somatosensory cortices) imply that volitional motor commands for vocalization are specifically relayed via the PAG, while the KFn may receive commands to coordinate breathing with other orofacial behaviors (e.g. sniffing, swallowing). Additionally, we observed that the limbic or autonomic (interoceptive) systems are connected to broadly distributed downstream bulbar respiratory networks. Collectively, these data provide a neural substrate to explain how volitional, state-dependent, and emotional modulation of breathing is regulated by the forebrain.