Motor neurons integrate cholinergic inputs through spatial organization of diverse nicotinic receptors

Neural circuit function depends not only on synaptic connectivity but also on the molecular composition and subcellular organization of neurotransmitter receptors. Here, we examine the expression, localization, and functional relevance of nicotinic acetylcholine receptors (nAChRs), the primary mediators of fast excitatory transmission in the Drosophila central nervous system. Functional nAChRs are pentamers assembled from ten subunits (α1–α7, β1–β3), yet how this diversity is deployed within defined circuits remains poorly understood. Using T2A-Gal4 reporters and endogenous protein tagging, we identify eight nAChR subunits (α1–α3, α5–α7, β1, β2) expressed in larval motor neurons (MNs). MN-specific knockdown of individual subunits produces impairments in crawling, peristaltic timing, and protopodium dynamics, demonstrating that multiple nAChR subtypes contribute to motor output. Co-localization analyses reveal a wide range of spatial relationships, identifying subunit pairs with high, intermediate, and low overlap within MN dendritic and postsynaptic domains. Across subunit combinations, spatial organization correlates with pair-specific functional interactions: spatially segregated pairs tend to produce stronger locomotor defects when knocked down together, suggesting largely non-redundant contributions to cholinergic excitation of MNs, whereas highly co-localized pairs often show limited additional impairment. Notably, some co-localized pairs also exhibit additive effects, indicating that spatial proximity alone does not fully predict functional interaction. Dual knockdown of selected subunit pairs also reduces muscle contraction amplitude, linking receptor organization to motor output at the effector level. Together, these results indicate that motor neurons deploy multiple nAChR populations whose spatial arrangement shapes how cholinergic inputs contribute to locomotor output.