Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons – the most molecularly distinct subtype – possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

9The dorsal raphe nucleus (DR) contains the largest brain population of Pet1-10 lineage neurons, a predominantly serotonergic group distributed throughout 11 multiple DR subdomains. These neurons collectively regulate diverse physiology 12 and behavior and are often therapeutically targeted to treat affective disorders. 13Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is 14 vital for understanding DR functional organization, with potential to inform 15 therapeutic separability. Here we use high-throughput and DR subdomain-16 targeted single-cell transcriptomics and intersectional genetic tools to map 17 molecular and anatomical diversity of DR-Pet1 neurons. We describe up to 18 fourteen neuron subtypes, many showing biased cell body distributions across 19 the DR. We further show that P2ry1-Pet1 DR neurons -the most molecularly 20 distinct of the subtypes -possess unique efferent projections and 21 electrophysiological properties. The present data complement and extend 22 previous DR characterizations, combining intersectional genetics with multiple 23 transcriptomic modalities to achieve fine-scale molecular and anatomic 24 identification of Pet1 neuron subtypes. 25 26 27 high-resolution, single-cell transcriptomic atlas of dorsal raphe Pet1-lineage 58 neurons revealing hierarchically and spatially organized molecular subtypes, 59 each expressing unique repertoires of neurotransmitters, plasma membrane 60 receptors, ion channels, cell adhesion molecules, and other gene categories 61 important for specifying neuronal functions. 62

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