Leveraging the Rich Genetic Diversity of Vagal Motor Neurons to Decode Brain-to-Gut Communication The motor vagus was originally treated as a single entity – the principal arm of the parasympathetic ‘rest and digest’ response. Over time, as diverse vagal actions were uncovered, it came to be viewed as a composite of many functionally discrete motor units, which are themselves differentially regulated. Indeed, neurons originating in the dorsal motor nucleus of the vagus (DMV) control at least three different processes in the stomach (secretion of acid, contraction and relaxation), two in the pancreas (exocrine and endocrine secretion) and also contraction of the gallbladder. Likewise, these motor units are regulated by many inputs – by cephalic signals that anticipate eating to prepare the gut and body for food, and by vagal sensory neurons and gut hormones that, via vago-vagal and endocrine-vagal reflexes, coordinate digestion and assimilation of nutrients. A major impediment to understanding the neural basis for this regulation, however, is that it is not known how many functionally discrete vagal motor units actually exist, and, more importantly, there is a lack of any means for selectively mapping and manipulating the different motor units. These issues have held the field back. This Multi-PI R01 application addresses this problem by: a) using single cell RNA profiling to catalogue the different subtypes of DMV motor neurons and identify genetic markers that specify each subtype, b) by assembling or generating “gene marker”-recombinase mice that enable recombinase-dependent exploration of the DMV neuron subtypes, and then c) by utilizing these mice to determine each neuron’s respective target organ(s) and downstream enteric neuron(s), the role each DMV neuron plays in regulating GI physiology, and the ways the DMV neuron subtypes are uniquely regulated by CNS afferents, vagal afferents and hormones. The Lowell and Liberles labs are ideally suited to this effort because their knowledge and areas of technical expertise are highly relevant, and also very complementary. The Lowell lab has: a) preliminarily discovered genetically distinct subsets of vagal motor neurons (via single cell RNA sequencing), b) is converting this information into neuron subtype-specific recombinase mice, and c) has expertise in using recombinase mice and recombinase-dependent technologies to investigate neural circuits. The Liberles lab, on the other hand has: a) discovered functionally and genetically distinct vagal sensory neurons – the afferent arms of the vago- vagal reflexes, b) has extensive experience with manipulating activity of vagal fibers and assessing effects on gastrointestinal physiology, and c) has preliminarily discovered distinct subsets of the downstream enteric neurons. Combined, the Lowell and Liberles labs are well poised to deconvolute vagal motor function.