Cell-type-resolved RNA-seq reveals molecular signatures of sexual experience in Drosophila neuromodulatory neurons.

Flexible behavioral responses depend on the ability of neural circuits to adapt their physiology and output to changing social and environmental contexts. Neuromodulation is central to this flexibility, yet how specific experiences are encoded within neuromodulatory neurons remains largely unknown. Here, we show that distinct motivational outcomes drive distinct regulatory responses in three neuromodulatory neuronal populations in male Drosophila. Using cell-type-resolved RNA sequencing, we profiled serotonergic (Trh), octopaminergic/tyraminergic (Tdc2), and neuropeptide F receptor (NPFR) neurons after successful mating, sexual rejection, or social isolation. Each experience induced a unique molecular signature within each cell type. NPFR neurons showed the strongest remodeling after rejection, Tdc2 neurons preferentially represented the naïve-single state, and Trh neurons displayed balanced, experience-specific tuning. Shared differentially expressed genes were few and often oppositely regulated, indicating strong cell-type-specific transcriptional logic. Experience-dependent changes spanned multiple regulatory layers, including chromatin organization, RNA metabolism, proteostasis, metabolism, and synaptic machinery. Notably, sexual rejection engaged transcriptional programs consistent with homeostatic synaptic scaling, including coordinated upregulation of dense-core vesicle components and cAMP signaling together with reduced inhibitory tone in Tdc2 neurons, and complementary remodeling of vesicle fusion and trafficking machinery in Trh neurons. We further identified compact, population-specific rejection-associated modules that converge functionally on circadian regulation, stress responses, and metabolic pathways. Together, these findings show that motivation is encoded through distinct regulatory responses that tune neuromodulatory circuit function and support behavioral flexibility.