23Innate behaviors involve both reflexive motor programs and internal states. In 24 Drosophila, optogenetic activation of male-specific P1 interneurons triggers courtship song, 25 as well as a persistent behavioral state that prolongs courtship and enhances aggressiveness. 26 Here we identify pCd neurons as persistently activated by repeated P1 stimulation. pCd 27 neurons are required for P1-evoked persistent courtship and aggression, as well as for 28 normal social behavior. Activation of pCd neurons alone is inefficacious, but enhances and 29 prolongs courtship or aggression promoted by female cues. Transient female exposure 30 induced persistent increases in male aggressiveness, an effect suppressed by transiently 31 silencing pCd neurons. Transient silencing of pCd also disrupted P1-induced persistent 32 physiological activity, implying a requisite role in persistence. Finally, P1 activation of pCd 33 neurons enhanced their responsiveness to cVA, an aggression-promoting pheromone. Thus, 34 pCd neurons function within a circuit that integrates P1 input, to promote a persistent 35 internal state that enhances multiple social behaviors. 36 Fig. 1b) sensitized PPF1 neurons (ED Fig. 3).87 Gal4 line R41A01 labels a cell cluster called pCd, previously reported to play an important 88 role in female sexual receptivity 27 . Analysis of marker expression indicated that pCd cells are 89 5 cholinergic neurons that express both Fru and Dsx (ED Fig. 2f-i), two sex-determination factors 90that label neurons involved in male courtship and aggression 3,8,[28][29][30][31] . pCd neurons project densely 91 to the superior-medial protocerebrum (SMP), while extending an additional long fiber bundle 92 ventrally to innervate the dorsal region of the subesophageal zone (SEZ; Fig. 1d). Double labeling 93 of pCd neurons with somatodendritic (Denmark-RFP 32 ) and pre-synaptic (Syt-GFP 33 ) markers 94 revealed that their SMP projections are mostly dendritic, while their pre-synaptic terminals are 95 located in the SMP and the SEZ (ED Fig. 4d-f). Registration of P1 pre-synaptic labeling with pCd 96 somatodendritic labeling in a common brain template failed to reveal clear overlap (ED Fig. 4g-i), 97 and application of the GFP Reconstitution Across Synaptic Partner (GRASP 34 ) technique failed to 98 detect close proximity between P1 and pCd neurons (ED Fig. 4j-r), suggesting that functional 99 connectivity between these cells is unlikely to be monosynaptic. 100 pCd neuronal activity is required for P1-induced persistent social behaviors 101To test whether P1-evoked persistent social behaviors require pCd activity, we silenced the 102 latter using R41A01-LexA>LexAop-Kir2.1 while activating P1 a -split GAL4 neurons using UAS-103 Chrimson. In solitary males ( Fig. 2a), silencing pCd neurons dramatically reduced persistent wing 104 extension evoked by Chrimson activation of P1 cells ( Fig. 2b vs. 2c, green shading; 2d).
105Importantly, time-locked wing-extension during photostimulation was unaffected ( Fig. 2b-d, gray 106 shading). Pe...