Extended Data Fig. 1: Driver lines for inducing courtship song.

a, Expression patterns of the dPR1 split-Gal4 (left) and the TN1A-2 split-Gal4 (right) in the brain. b, A single neuron visualized with stochastic labeling using the dPR1 split-Gal4 line. c, The dsx gene is expressed in the neurons labeled with the dPR1 split-Gal4 line. d, Cell body expression pattern of CsChrimson in the TN1A-2 split-Gal4 line. Arrows indicate the ___location of cell bodies. e, Examples of single TN1A-2 neurons visualized with stochastic labeling using the TN1A-2 split-Gal4. Arrows indicate the cell bodies. f, Example of the sound induced by optogenetic activation of dPR1 in the single fly optogenetic activation experiment. The shaded area represents the period during which LED stimulation was applied. g, The mean proportions of pulse (left) and sine (right) song during the stimulation period. Each dot represents a fly. Lines represent mean ± s.d. across flies (n = 12 flies). h,i, Same as f,g but for TN1A-2 (n = 12 flies). j, Results for control flies in the single fly optogenetic activation experiment. The mean proportions of pulse and sine song during the stimulation period. Lines represent mean ± s.d. across flies (n = 11 flies for UAS-CsChrimson; n = 12 flies for dPR1 split-Gal4; n = 12 flies for TN1A-2 split-Gal4). k, Expression pattern of the song driver, which was used to induce pulse and sine song with optogenetic activation. Data were from a male fly. Similar expression patterns were observed in three flies. Scale bars, 50 μm. l, Expression pattern of the song driver at an anterior-dorsal part of the central brain of a male fly. The arrow indicates the commissural fibers of P1 neurons. m, Flies expressing CsChrimson under the control of the song driver were attached to the recording plate and optogenetically stimulated with a red laser. For the decapitated group, the head was removed after attachment to the recording plate. No thoracic dissection was made on these flies. The graphs show the time course of the proportions of pulse and sine song during laser stimulation at an irradiance of 12.2 μW/mm². Data are represented as mean ± s.e.m. across flies (n = 4 flies for the intact group; n = 6 flies for the decapitated group). n, The mean proportions of pulse (left) and sine (right) song during the stimulation period for the intact group. Each dot represents a fly. Lines represent mean ± s.d. across flies (n = 4 flies). o, The distributions of the durations of pulse and sine song bouts. Dark grey bars, the songs induced by optogenetic activation with the song driver (n = 4 intact flies). White bars, the songs in a group of control flies in the optogenetic activation experiment shown in j (w¹¹¹⁸ crossed to UAS-CsChrimson; n = 11 flies). p, Same as n but for the decapitated group (n = 6 flies). q, Flies expressing CsChrimson under the control of the split-Gal4 line for pIP10 were attached to the recording plate, decapitated, and provided with optogenetic stimulation. No dissection was made on the thorax. Graphs show time courses of the proportions of pulse and sine song during laser stimulation at an irradiance of 12.2 μW/mm². Data are represented as mean ± s.e.m. across flies (n = 5 flies). r, Same as p but for pIP10 (n = 5 flies). LED, light-emitting diode.