Figure 6. Anatomical, behavioral, and physiological characterization of dsx+ pC1 neurons in auditory sensation.

(A–D) Co-registration of vPN1(green) and pC1(magenta) neurons onto the standard brain. Genotype for labeling vPN1 neurons: LexAop2-FLP/+; fru^LexA, UAS>stop>myr::GFP/R72E10-GAL4. Genotype for labeling vPN1 neurons: R71G01-LexA/UAS>stop>myr::GFP; dsx^GAL4, LexAop2-FLP/+. vPN1 processes (B) and pC1 (C) processes overlap in the region of lateral protocerebral complex (LPC). (D) Merge of (B) and (C). Scale bars, (A) 100 μm and (D) 20 μm. (E) Song-induced chaining response was impaired by inactivation of pC1 neurons. Genotypes: UAS>stop>TNTⁱⁿ/71G01-LexA; dsx^GAL4, LexAop2-FLP/+ (TNTⁱⁿ), UAS>stop>TNT/+; dsx^GAL4, LexAop2-FLP/+ (no LexA), UAS>stop>TNT/71G01-LexA; dsx^GAL4, LexAop2-FLP/+ (TNT). n = 16 for all the conditions. *p < 0.0001 when comparing TNT group to both controls at 80 dB, Wilcoxon rank-sum test. Bottom, heat map analysis of chaining events for individual groups of flies. (F) Diagram of pC1 neurons labeled with dsx^GAL4 driving expression of GCaMP6. Neurites innervating the LPC are circled. (G) Calcium responses of pC1 neurons responding to different IPIs, sine song, and white noise at 80 dB. Pulse song stimuli consist of a train of 40 pulses. Black lines indicate mean values, while gray areas indicate SEM. Song stimulus durations are indicated as red bars. (H) Peak ∆F/F values of pC1 neurons stimulated with pulse song (35-ms IPI, 40 pulses), sine song (140 Hz, 1.4 s), and white noise (1.4 s) at 80 dB. *p < 0.01, Wilcoxon rank-sum test. No significance was observed for sine vs white noise. n = 12 for all the groups. (I) Peak ∆F/F values of pC1 neurons stimulated with pulse song (35-ms IPI, 40 pulses) from 60 dB to 90 dB. pC1 neurons are only sensitive to pulse song stimuli above 80 dB. n = 9 trials for each sound level. (J) Normalized calcium traces of pC1 neurons at different IPIs. Each ∆F/F was normalized by the maximum ∆F/F. (K) Peak ∆F/F of pC1 neurons stimulated with different IPIs at 80 dB (40 pulses). n = 12 for all groups. *p < 0.01, Wilcoxon signed-rank test. (L) Comparison between the calcium response (peak ∆F/F) of vPN1 neurons and chaining responses to different IPIs shown in Figure 3E (R = 0.67; p < 0.017, permutation test). Both calcium responses and chaining responses are normalized to their respective maximum responses. (M) Comparison between the calcium response (peak ∆F/F) of pC1 neurons and chaining responses to different IPIs shown in Figure 3E (R = 0.89; p < 0.001, permutation test). Both calcium responses and chaining responses are normalized to their respective maximum responses. (N) Comparison between normalized vPN1 responses and pC1 responses. Each colored square represents a different IPI indicated by the heat map. Error bars represent SEM in all panels.

DOI: http://dx.doi.org/10.7554/eLife.08477.013

Figure 6—figure supplement 1. Raster plots of pC1 neurons in individual flies.

(A) Raster plots of ∆F/F during presentation of different song stimuli. Each row represents calcium responses from one male fly. (B) Raster plots of peak ∆F/F during presentation of different song stimuli. Each row represents calcium responses from one male fly while each column represents a different IPI. All the responses from an individual fly are normalized by the maximum response from that fly.

DOI: http://dx.doi.org/10.7554/eLife.08477.014

Figure 6—figure supplement 2. Transfer function between vPN1 and pC1 responses to IPIs of the pulse song.

(A) Normalized peak ∆F/Fs for the calcium responses of vPN1 (green) and pC1 (yellow) neurons to different IPIs. The tuning curve of aPN1 neurons (gray) is reported previously (Vaughan et al., 2014) and superimposed for comparison. *p < 0.05 between vPN1 and pC1 responses, One-sided Wilcoxon rank-sum test. (B) A transfer function was calculated by dividing pC1 responses by vPN1 responses. This transfer function resembles a band-pass filter. The SEM of the transfer function was estimated using standard error propagation.

DOI: http://dx.doi.org/10.7554/eLife.08477.015

Figure 6—figure supplement 3. Comparison of synthetic courtship song and natural courtship song with a particle-velocity microphone.

(A) Synthetic pulse song (top) and sine song (bottom) measured by a particle-velocity microphone. Pulse song train at 80 dB sound pressure level corresponds to particle velocity of ∼2 mm/s, while sine song at 80 dB corresponds to ∼0.35 mm/s. (B) Natural courtship song recorded by a particle-velocity microphone located ∼2 mm above a pair of male and female Canton S flies. Pulse songs from a courting male can reach up to ∼7.5 mm/s, and sine songs can reach up to ∼1 mm/s in this setup.

DOI: http://dx.doi.org/10.7554/eLife.08477.016