Structure of Bengalese finch song (A) Example spectrogram (bird 7) depicting an entire song bout with introductory state ‘I’, repeat phrase ‘B’ and individual syllables. (B) Example transition diagram depicting a branchpoint with variable sequencing. Numbers above the arrows denote transition probabilities in percent. (C) Example spectrogram of a chunk. Chunks are defined as highly stereotyped sequences of syllables which only have a single in- and output branch and are condensed into one state in the transition diagram (see Methods). (D) Example spectrogram of a repeat phrase, summarized by capital letter ‘B’ in the transition diagram. The repeating syllable (here, syllable ‘b’) repeats a variable number of times across different instances of the repeat phrase. (E) Schematic showing recurrent pathways projecting onto motor pathway nuclei through lMAN and mMAN. Red: pallial nuclei, blue: thalamic nuclei, green: basal ganglia. Dotted line indicates suspected connection by Kubikova et al. 2007. Abbrev: mMAN: medial magnocellular nucleus of the anterior nidopallium, DMP: dorsomedial nucleus of the posterior thalamus, BG: basal ganglia, DLM: medial portion of the dorsolateral thalamus, lMAN: lateral magnocellular nucleus of the anterior nidopallium, RA: robust nucleus of the arcopallium. (F) Example of a transition diagram. Nodes denote chunk or syllable labels, numbers denote transition probabilities (in percent, % symbol omitted for clarity), d1/d2, g1/g2 denote different states of syllables d and g respectively based on different sequential contexts, capital letters denote repeat phrases. Edges at each node may not sum to 100% because branches smaller than 5% are omitted for clarity.

Transition entropy increased after bilateral mMAN lesions (A) Example spectrogram (bird 3) pre lesion (above) and post lesion (below). ‘I’ denotes introductory state, and ‘fe2’ and ‘ah’ denote chunks, shown as single nodes in the transition diagram. (B) Pre lesion transition diagram, as in 1F. Note that ‘fe2CB’ is also a chunk before the lesions but is shown as three separate nodes in order to align with the post lesion diagram in C. (C) Post lesion transition diagram. Arrows in red mark example nodes with relatively high increase in transition entropy, including the introduction of new branches after mMAN lesions. (D) Total transition entropy for 7 birds before and after mMAN lesions (* p<0.05, n=7, Wilcoxon signed-rank test). Example bird from A-C is shown as darker line.

Chunks became more variable after bilateral mMAN lesions. (A) Example spectrogram (bird 2) before and after bilateral mMAN lesions. Atypical chunk sequences are highlighted in red. (B,C) Transitions following the first ‘c’ of the ‘ccllfk’ chunk from (A) before and after mMAN lesions. Different column colors represent different syllables. (D) Chunk consistency before and after bilateral mMAN lesions (** p<0.01, n=23, Wilcoxon signed rank test). Example bird is shown as darker line.

The number of syllables per repeat phrase (repeat number) became more variable after bilateral mMAN lesions. (A) Example spectrogram (bird 6) highlighting one repeat syllable before (blue) and after (red) mMAN lesions. (B) Repeat numbers for two additional example birds before (blue) and after (red) mMAN lesions. (C) Average repeat numbers before and after mMAN lesions for all repeat phrases. (D) Coefficient of variation for distribution of repeat numbers before and after mMAN lesions for all repeat phrases. Example birds from B are shown as darker lines.

Image of calcitonin gene-related peptide (CGRP)-stained frontal section (left) control and (right) bird 5. CGRP labels cells in both lMAN (seen in black to the left of the lesion) and mMAN (blue, intact; red, completely destroyed).

Transition entropy after mMAN lesions remains elevated over several days. Transition entropy did not change in a consistent way after recovery from the lesion for the followed time period. Numbers near the datapoints indicate number of song bouts recorded and analyzed for that day. While the persistence of the effects we observed is longer than transient effects such as those following Nif lesion in zebra finches (∼ 2 days by Otchy et al., 2015) we cannot rule out either recovery or further deterioration following lesions on much longer time scales, such as those reported by Kubikova et al., 2007 (X lesions, 6 months) (Kubikova, Turner and Jarvis, 2007). Additional non-connected datapoints at 14, 19, 33 and 33 days post lesion were gathered after additional behavior manipulation and were added here to analyze specifically the persistence of effects after the lesion. For three of these birds, transition entropy remained elevated above the baseline values for 14, 33 and 33 days, respectively.

Average spectrograms of 200 instances of all syllable types for all birds before and after mMAN lesions. Letters over the spectrograms indicate syllable labels and symbols over the spectrograms correspond to the symbols used in Fig. 2 – figure supplement 4.

Syllable similarity calculated using Sound Analysis Pro (SAP). ‘Self Similarity’ = Similarity comparison of syllables before mMAN lesions to syllables of the same type, taken from two separate control recordings before the lesions, ‘Pre vs Post’ = Similarity comparison of the same syllable types before and after mMAN lesions, ‘Cross Similarity’ = Similarity comparison of each syllable type to other syllable types. For Birds 1-2 and 4-7, ‘Self Similarity’ was not significantly different from ‘Pre vs Post’ Similarity (p>0.05, Wilcoxon sign rank test), while for Bird 3, there was a significant difference (p = 0.03, Wilcoxon sign rank test). For all birds ‘Pre vs Post’ was significantly different from ‘Cross Similarity’ (p<0.05, Wilcoxon sign rank test). On average, ‘Pre vs Post’ was 4.8 % less than ‘Self Similarity’ (range 0.2%-14%) while ‘Cross Similarity’ was 40% less than ‘Self Similarity’ (range 20.2%-56.3%). These measures confirm the qualitative impression from Figure 2- figure supplement 1 that for most birds and syllables there were no greater changes to syllable structure following lesions than was present across control recordings, and that pre-post similarity remained higher than cross-similarity, i.e. syllables remained clearly identifiable.

(A) CV of fundamental frequency (FF) of select syllables before and after mMAN lesions. In the Bengalese finch and zebra finch, lesions of lMAN, which sits immediately lateral to mMAN, cause a consistent reduction in the coefficient of variation (CV) of fundamental frequency across repeated renditions of a given syllable (Sakata, Hampton, Brainard 2008, Andalman, Fee 2009, Warren et al. 2011). We therefore supposed that unintended damage to lMAN or its projections to RA might have resulted in a reduction in the CV of syllables following mMAN lesions. Instead we saw a modest increase in the CV of fundamental frequency (p<0.05, Wilcoxon sign rank test; mean across birds of +20%; range -19 to +43%). These data suggest that it is unlikely that changes to syllable structure might have arisen due to accidental damage to lMAN. (B) Percent change in mean fundamental frequency after mMAN lesions vs mean fundamental frequency before mMAN lesions.

Selected acoustic features for all syllables in all birds before and after mMAN lesions. Different colors represent different syllable types per bird. ‘entS’ = Entropy of spectral envelope, ‘meanT’ = Temporal centroid for temporal envelope, ‘F1’ = First formant, ‘F2’= Second formant, ‘F3’ = Third formant. Acoustic features generally showed little change between pre and post lesion songs. They highlight as relative outliers the same individual examples that stand out in the average spectrograms in Figure 2 – figure supplement 1.

Change in transition entropy for branchpoints (A) Transition entropy for all individual branchpoints after mMAN lesions. (B) Branchpoints with low history dependence before mMAN lesions showed a nonsignificant trend towards higher changes in transition entropy.

Change in transition entropy for transitions within chunks vs. branch points (A) Duration of gaps within chunks before mMAN lesions was not significantly correlated with entropy change at the corresponding transition (p> 0.05, Wilcoxon rank sum test). (B) Change in transition entropy was not significantly different for transitions within chunks and at branchpoints (p> 0.05, Wilcoxon rank sum test).