Synergistic mechanisms of medullary cholinergic-serotonergic pathway interactions in regulating neuronal excitability and locomotor activities

Medullary serotonergic (5-HT) neurons play a pivotal role in locomotor initiation. While these neurons receive cholinergic innervation, the functional consequences and underlying mechanisms remain poorly understood. Using ePet-EYFP transgenic mice (P3–P6) and multidisciplinary approaches, we elucidated the mechanisms by which cholinergic signaling regulated medullary 5-HT neuronal activity and locomotor output. Key findings included: (1) acetylcholine (ACh) elicited location-dependent excitatory, inhibitory, or neutral responses in 5-HT neurons, recapitulated by muscarine and abolished by atropine, indicating muscarinic receptor (mAChR)-mediated regulation. (2) 5-HT neurons in parapyramidal region exhibited ACh-induced excitation, whereas those in midline raphe nuclei displayed inhibition. (3) ACh-enhanced excitability was mediated via M3 receptors, whereas ACh-induced suppression depended on M2/M4 receptor activation. (4) In vitro bath application of muscarine to medullary region induced triphasic modulation of fictive locomotion. Blockade of M1/M5 receptors did not affect locomotion, whereas M3 receptor antagonism reduced gait velocity and perturbated locomotor rhythm. Conversely, M2/M4 receptor antagonism increased stepping frequency without altering locomotor pattern. This study provided the first mechanistic dissection of how medullary cholinergic-serotonergic interactions regulated neuronal excitability and locomotion, uncovering distinct mAChR subtype contributions to motor control.