Localization of muscarinic acetylcholine receptor dependent rhythm generating modules in the Drosophila larval locomotor network

Mechanisms of rhythm generation h­­ave been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. Here we explored how muscarinic acetylcholine receptor (mAChR) modulated rhythm generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. We measured fictive motor patterns in isolated CNS preparations using a combination of Ca²⁺ imaging and electrophysiology while manipulating mAChR signalling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca²⁺ levels and abolished rhythmic activity. Overall, these results suggest that mAChR signalling plays a role in enabling rhythm generation at multiple sites in the larval CNS. This work furthers our understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm generating networks in an emerging genetically tractable locomotor system.