Motor Neurons Decode Cholinergic Inputs via Spatially Distinct nAChR Subunits to Drive Locomotion in Drosophila larvae

Neural circuits consist of neurons that differ not only in their neurotransmitter identities but also in the types and subcellular localization of neurotransmitter receptors (NRs) they express. This receptor diversity enables distinct responses to the same neurotransmitter, highlighting the need to understand NR distribution and function to fully interpret circuit logic. Here, we focus on nicotinic acetylcholine receptors (nAChRs), the primary mediators of fast excitatory transmission in the Drosophila central nervous system (CNS). Functional nAChRs are pentamers assembled from a pool of 10 subunits (1-7, {beta}1-{beta}3), yet their in vivo expression and function remain poorly defined. We used T2A-Gal4 lines and endogenous protein tagging to examine nAChR expression in larval motor neurons (MNs) and identified eight subunits (1-3, 5-7, {beta}1, {beta}2) expressed in these cells. MN-specific knockdown of individual subunits caused distinct locomotor defects, indicating their functional importance. Co-localization analysis revealed some subunit pairs are frequently co-expressed at the same synapses, while others localize to distinct subcellular domains. Supporting this, double knockdown of co-localized subunits did not worsen locomotor phenotypes compared to single knockdowns, whereas knockdown of non-co-localized subunit pairs produced additive defects. These results suggest that different nAChR subtypes are strategically positioned in discrete synaptic domains within single MNs, where they serve non-redundant roles. Our findings provide new insight into the spatial organization and functional diversity of nAChRs in motor circuits that drive locomotion. Significance StatementMotor circuits rely on precise neurotransmitter signaling, yet the diversity and subcellular organization of neurotransmitter receptors remain poorly understood. Using Drosophila larvae, we show that motor neurons express multiple nicotinic acetylcholine receptor (nAChR) subunits, which localize to distinct synaptic domains and play non-redundant roles in locomotion. These findings reveal a previously underappreciated level of receptor compartmentalization within single neurons and demonstrate that spatially organized nAChRs are essential for coordinated movement. By integrating genetic, imaging, and behavioral approaches, our work provides a new framework for understanding how receptor diversity shapes motor output and highlights the importance of mapping receptor localization to decode circuit function in both health and disease.