We determined the cellular substrate for male courtship behavior by quasinatural and artificial stimulation of brain neurons. Activation of fruitless (fru)-expressing neurons via stimulation of thermosensitive dTrpA1 channels induced an entire series of courtship acts in male Drosophila placed alone without any courting target. By reducing the number of neurons expressing dTrpA1 by MARCM, we demonstrated that the initiation of courtship behavior is significantly correlated with the activation of the transmidline P1 interneurons, the descending P2b interneurons, or both, indicating that these interneurons trigger courtship. Using an experimental paradigm in which a tethered male can be stimulated to initiate courtship by touching his foreleg tarsus to a female's abdomen, we found that P1 neurites of tethered males showed a transient Ca2+ rise after tarsal stimulation with the female-associated sensory cues. These observations strongly suggest that P1 neurons are the prime components of the neural circuitry that initiates male courtship.
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