NCM inactivation during tutor song exposure disrupts song imitation and rhythmicity. (A) Representative sonograms of subjects in each experimental group at 120 dph (left TTX injected (N = 6), right TTX injected (N = 7), and surgical controls (N = 8; birds receiving anesthesia and handling exposure) and their respective tutors. In the pupils’ songs, normal syllables are indicated with blue bars, and isolate-like syllables with red bars. Note the abnormal duration of inter-syllable gaps between isolate-like syllables. Scale bar represents 200 ms. (B) Mean scores (± SEM) of song imitation (TUT-NOV) for each experimental group. There was a significant difference in mean song imitation scores between the treatment groups (F(2,19) = 26.67, p < 0.001). Post-hoc comparisons with Bonferroni correction revealed a significant difference between left-TTX inactivated and control group (t(19) = 6.718, p < 0.001) as well as right-TTX inactivated and control group (t(19) = 5.419, p < 0.001). (C) Rhythm spectra (frequency on x-axis) for a full day of singing (on y-axis) for subjects in each experimental group at 120 dph and their respective tutors. The rhythm spectra shown represent the songs of the same birds as presented in the sonograms from (A). Clearly delineated high-power (red) bands are indicative of crystallized adult song with highly stereotyped motif durations (the fundamental frequency of the rhythm spectrum) and consistent intervals between syllable onsets (bands at higher frequencies). Although pupils do not necessarily copy the rhythm from their tutors, rhythm spectra of birds singing isolate song are less modulated (appearing more washed-out) than those from birds singing normal, crystallized song (characterized by clearly delineated bands at higher frequencies). (D) Modulation of rhythm spectra for all experimental groups. Mean RMS residuals of fitted rhythm spectra are expressed as a percentage of the normal adult songs from their tutors (± SEM; left-: N = 6, right-: N = 7, control: N = 8, adult: N = 4).