3 4 5 A cellular and regulatory map of the cholinergic nervous system 6 of C . elegans 7 8 9

23 24 Nervous system maps are of critical importance for understanding how nervous 25 systemsdevelop and function. We systematically map here all cholinergic neuron types in the 26 male and hermaphrodite C.elegans nervous system. We find that acetylcholine is the most 27 broadly used neurotransmitter and we analyze its usage relative to other neurotransmitters 28 within the context of the entire connectome and within specific network motifs embedded in 29 the connectome. We reveal several dynamic aspects of cholinergic neurotransmitter identity, 30 including a sexually dimorphic glutamatergic to cholinergic neurotransmitter switch in a sex31 shared interneuron. An expression pattern analysis of ACh-gated anion channels furthermore 32 suggests that ACh may also operate very broadly as an inhibitory neurotransmitter. As a first 33 application of this comprehensive neurotransmitter map, we identify transcriptional control 34 mechanisms that control cholinergic neurotransmitter identity and cholinergic circuit 35 assembly. 36