An octopamine-glutamate cotransmitting neuron gates aggressive escalation through layered inhibition in Drosophila.

Aggression is an evolutionarily conserved behavior essential for survival and reproduction, yet escalation to high-intensity forms entails substantial metabolic costs and injury risks, necessitating precise neural control. Using Drosophila melanogaster, we uncovered a multilayered inhibitory circuit that constrains aggressive escalation. This circuit involves a cotransmitting octopamine-glutamate ventral paired medial 4 (VPM4) neuron and its downstream GABAergic target, MBON-11. Neurotransmitter-specific manipulations reveal that octopamine and glutamate release from VPM4 is independently regulated by presynaptic OAα2R and mGluR receptors, providing transmitter-specific feedback. Postsynaptically, glutamate inhibits the approach-promoting MBON-11 neuron via GluClα receptors, restraining transitions to high-intensity aggression and supporting a role in approach/avoidance behaviors. Furthermore, the Rdl GABAergic receptor within MBON-11 neurons provides rapid inhibitory feedback, creating an additional layer of regulation. Together, these findings reveal a circuit architecture in which cotransmission and inhibitory feedback loops form a layered inhibition mechanism that continuously constrains escalation, indicating that aggression intensity is actively regulated to align behavior with context and cost-benefit trade-offs.