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

Flexible behavioral responses rely on the ability of neural circuits to adapt their physiology and output to changing social and environmental contexts. Neuromodulation plays a central role in this flexibility, dynamically tuning neuronal activity and gene expression to align behavior with experience and internal states. Yet how specific experiences and motivational conditions are encoded within neuromodulatory neurons remains underexplored. Here, we show that distinct motivational outcomes drive discrete, largely non-overlapping transcriptional programs across three neuromodulatory neuronal populations in male Drosophila brains. Using cell-type-resolved RNA sequencing, we profiled the transcriptomes of serotonergic (Trh), octopaminergic/tyraminergic (Tdc2), and neuropeptide F receptor (NPFR) neurons under three conditions: successful mating, sexual rejection, and the absence of social or sexual interaction. Each experience induced a unique “molecular engram” within specific neuron types. NPFR neurons exhibited the strongest transcriptional remodeling following rejection, Tdc2 neurons preferentially represented the naïve-single state, and Trh neurons displayed balanced, experience-specific tuning. Shared differentially expressed genes across neuronal classes were few and often were oppositely regulated, revealing divergent circuit-specific logic. Experience triggered multilayered reprogramming across chromatin organization, RNA metabolism, translation, proteostasis and synaptic machinery, with selective recalibration of vesicle trafficking, and neuroplasticity. We further identified a compact, cross-circuit “rejection core” enriched for circadian, stress, metabolic, neuropeptidergic, and synaptic components. Together, these findings demonstrate how neuromodulatory circuits translate social experience into coordinated molecular and synaptic adaptations that enable behavioral flexibility.