5-HT7 receptors expressed in the mouse parafacial region are not required for respiratory chemosensitivity

A brainstem homeostatic system senses CO₂ /H⁺ to regulate ventilation, blood gases and acid-base balance. Neurons of the retrotrapezoid nucleus (RTN) and medullary raphe are both implicated in this mechanism as respiratory chemosensors, but recent pharmacological work suggested that the CO₂ /H⁺ sensitivity of RTN neurons is mediated indirectly, by raphe-derived serotonin acting on 5-HT7 receptors. To investigate this further, we characterized Htr7 transcript expression in phenotypically identified RTN neurons using multiplex single cell qRT-PCR and RNAscope. Although present in multiple neurons in the parafacial region of the ventrolateral medulla, Htr7 expression was undetectable in most RTN neurons (Nmb⁺ /Phox2b⁺ ) concentrated in the densely packed cell group ventrolateral to the facial nucleus. Where detected, Htr7 expression was modest and often associated with RTN neurons that extend dorsolaterally to partially encircle the facial nucleus. These dorsolateral Nmb⁺ /Htr7⁺ neurons tended to express Nmb at high levels and the intrinsic RTN proton detectors Gpr4 and Kcnk5 at low levels. In mouse brainstem slices, CO₂ -stimulated firing in RTN neurons was mostly unaffected by a 5-HT7 receptor antagonist, SB269970 (n = 11/13). At the whole animal level, microinjection of SB269970 into the RTN of conscious mice blocked respiratory stimulation by co-injected LP-44, a 5-HT7 receptor agonist, but had no effect on CO₂ -stimulated breathing in those same mice. We conclude that Htr7 is expressed by a minor subset of RTN neurons with a molecular profile distinct from the established chemoreceptors and that 5-HT7 receptors have negligible effects on CO₂ -evoked firing activity in RTN neurons or on CO₂ -stimulated breathing in mice. KEY POINTS: Neurons of the retrotrapezoid nucleus (RTN) are intrinsic CO₂ /H⁺ chemosensors and serve as an integrative excitatory hub for control of breathing. Serotonin can activate RTN neurons, in part via 5-HT7 receptors, and those effects have been implicated in conferring an indirect CO₂  sensitivity. Multiple single cell molecular approaches revealed low levels of 5-HT7 receptor transcript expression restricted to a limited population of RTN neurons. Pharmacological experiments showed that 5-HT7 receptors in RTN are not required for CO₂ /H⁺ -stimulation of RTN neuronal activity or CO₂ -stimulated breathing. These data do not support a role for 5-HT7 receptors in respiratory chemosensitivity mediated by RTN neurons.