Coefficient of variation was calculated for each interval and averaged over the intervals in the bird’s song. We compared both temporal and pitch variability before and after lesion. For prelesion, we used songs produced in the mornings up to 2 days

preceding the surgery, grouped into a single catch trial block to increase our sample size. For postlesion, we analyzed morning songs for up to 2 days, at different times after surgery to parse acute (1–3 days postlesion) and persistent (3+ days postlesion) effects (Figures 3D and S4). For tCAF and pCAF, respectively, we computed learning rates as the difference in the average pitch or duration in a.m. catch trials on the first and last day of CAF, divided by the number of intervening days. We did the same for p.m. catch trials and the overall learning Alectinib rate was then averaged across a.m. and p.m. ATR inhibitor catch trials for the whole drive up and down (sign inverted) for each bird to obtain a more robust estimate of the learning. For a small number of birds that did not sing during either a.m. or p.m. catch trial blocks, we computed learning rate from the remaining block only (e.g., a.m. only). Comparing the same time periods in the day allowed us to rule out circadian effects. Estimates of pitch and duration were computed as described above. In addition, we corrected duration estimates for global

tempo and changes during directed singing (Stepanek and Doupe, 2010), estimated as the average change in the duration of nontarget intervals during directed songs compared to undirected songs immediately before presentation of females. Reversion was calculated as the difference between the pitch or duration estimate just prior to presentation of the female (undirected p.m., see Figure 4A) and during directed singing p.m. and normalized to the total change in pitch or duration during the 4–7 hr of CAF. Songs during catch trial blocks were segmented and a song template created as

described in Supplemental Experimental Procedures. Starts and ends of intervals (syllables and gaps) were extracted for each rendition and linearly warped to the template. The warping path was time shifted by 35 ms to account for the lag between HVC and sound output (Figure S6) and then applied to the band-pass filtered HVC voltage trace (0.3–6 kHz, zero-phase, 2-pole Butterworth). The squared voltage was averaged across all renditions in the block and smoothed with a 5 ms boxcar window to generate the mean neural power trace. Spectrograms warped to the common template were similarly averaged to generate a mean spectrogram for the block. The average warping paths across the renditions were then applied to the mean spectrogram and neural trace to remove any template specific effects.