The nervous system uses specialized circuits to maintain homeostasis, yet how the brain senses internal states to regulate autonomic processes remains elusive. CerebroSpinal Fluid-contacting Neurons (CSF-cNs) are a distinct medullospinal population around the central canal, poised as internal sensors. In zebrafish, they detect spinal curvature and CSF changes, but their role in tetrapod circuitry remains unclear. Here, we elucidate CSF-cN connectivity in the mouse brainstem. Using Pkd2l1Cre:Tomato mice to label CSF-cN cell bodies and axons, we identified they project to the caudal medial brainstem, wrapping the hypoglossal nucleus. We found CSF-cNs form profuse GABAergic synapses onto dorsal neurons of the Roller and the Intermedius nucleus of the Medulla (InM). Optogenetic stimulation of CSF-cNs evoked inhibitory postsynaptic currents in Roller and InM neurons, confirming functional synapsis. Molecular and transcriptomic analysis revealed target cells are a heterogeneous population of GABAergic Dbx1-derived neurons that express the transcription factor Otp and Ebf2. Here, we identified the connectivity map of CSF-cNs in the mammalian brainstem and a novel circuit regulating autonomic functions. We propose that CSF-cNs modulate breathing activity through Roller and InM in response to CSF homeostasis, tuning motor hypoglossal activity.
