CENTRAL CANAL NEURONS PROJECT TO RESPIRATORY MODULATORY CENTERS IN THE MOUSE BRAINSTEM

The nervous system uses specialized circuits to maintain bodily functions. Understanding how the brain senses homeostatic changes is essential for regulating autonomic processes. In this context, CerebroSpinal Fluid-contacting Neurons (CSF-cNs) are a distinct medullospinal population located around the central canal, strategically poised as internal sensors. In zebrafish, they detect spinal curvature and changes in CSF composition, however, their role within tetrapods' circuitry remains unclear. Here, we aim to elucidate the connectivity and function of CSF-cNs in the mouse brainstem. We mapped CSF-cN network using Pkd2l1Cre:Tomato mice to selectively label their cell bodies and axons. We confirmed CSF-cNs presence along the brainstem central canal, extending up to the fourth ventricle and projecting to caudal medial brainstem areas. Anatomical atlas and molecular characterization led us to identify CSF-cN axons tightly wrapping the hypoglossal nucleus. Additionally, using Pkd2l1Cre:Synaptophysin-Tomato to genetically label presynaptic terminals, we found that CSF-cNs form profuse GABAergic synapses onto dorsal neurons of the Roller and Intermedius nucleus of the Medulla (InM). Specifically, these terminals innervate poorly described Dbx1-derived neurons within these nuclei. Consistently, optogenetic stimulation of CSF-cNs (Pkd2l1Cre; ChR2) evoked inhibitory postsynaptic currents in Roller and InM neurons, confirming functional synaptic connection. The hypoglossal nucleus controls tongue movements and receives respiratory rhythmic information via regulatory nuclei, potentially the Roller and InM. We propose that CSF-cNs modulate breathing activity through these nuclei in response to CSF homeostasis. Altogether, our results describe the first connectivity map of CSF-cNs in the mammalian brainstem, proposing a previously unknown circuit that regulates autonomic functions.