P.A.M.), mucida@rockefeller.edu (D.M.) 15 16 Gut-brain connections monitor the intestinal tissue and its microbial and dietary content 1 , 17 regulating both intestinal physiological functions such as nutrient absorption and motility 2-4 , and 18 brain-wired feeding behaviour 3 . It is therefore plausible that circuits exist to detect gut microbes 19 and relay this information to central nervous system (CNS) areas that, in turn, regulate gut 20 physiology 5 . We characterized the influence of the microbiota on enteric-associated neurons (EAN) 21 by combining gnotobiotic mouse models with transcriptomics, circuit-tracing methods, and 22 functional manipulation. We found that the gut microbiome modulates gut-extrinsic sympathetic 23 neurons; while microbiota depletion led to increased cFos expression, colonization of germ-free 24 mice with short-chain fatty acid-producing bacteria suppressed cFos expression in the gut 25 sympathetic ganglia. Chemogenetic manipulations, translational profiling, and anterograde tracing 26 identified a subset of distal intestine-projecting vagal neurons positioned to play an afferent role in 27 microbiota-mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal 28 tracing from the intestinal wall identified brainstem sensory nuclei activated during microbial 29 depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate 30 gastrointestinal transit. These results reveal microbiota-dependent control of gut extrinsic 31 sympathetic activation through a gut-brain circuit. 33Extrinsic enteric-associated neurons (eEAN), comprised of sensory afferents, parasympathetic and

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