Serotonergic Neurons Mediate Operant Conditioning in Drosophila Larvae

Abstract Observed across species, operant conditioning facilitates learned associations between behaviours and outcomes, biasing future action selection to maximise reward and avoid punishment. To elucidate the underlying neural mechanisms, we built a high-throughput tracker for Drosophila melanogaster larvae, combining real-time behaviour detection with closed-loop optogenetic and thermogenetic stimulation capabilities. We demonstrate operant conditioning in Drosophila larvae by inducing a bend direction preference through optogenetic activation of reward-encoding serotonergic neurons. Specifically, we establish that the ventral nerve cord is necessary for this memory formation. Our results extend the role of serotonergic neurons for learning in insects as well as the existence of learning circuits outside the mushroom body. This work supports future studies on the function of serotonin and the mechanisms underlying operant conditioning at both circuit and cellular levels.