Song practice promotes acute vocal variability at a key stage of sensorimotor learning

Abstract

Background: Trial by trial variability during motor learning is a feature encoded by the basal ganglia of both humans and songbirds, and is important for reinforcement of optimal motor patterns, including those that produce speech and birdsong. Given the many parallels between these behaviors, songbirds provide a useful model to investigate neural mechanisms underlying vocal learning. In juvenile and adult male zebra finches, endogenous levels of FoxP2, a molecule critical for language, decrease two hours after morning song onset within area X, part of the basal ganglia-forebrain pathway dedicated to song. In juveniles, experimental 'knockdown' of area X FoxP2 results in abnormally variable song in adulthood. These findings motivated our hypothesis that low FoxP2 levels increase vocal variability, enabling vocal motor exploration in normal birds.

Methodology/principal findings: After two hours in either singing or non-singing conditions (previously shown to produce differential area X FoxP2 levels), phonological and sequential features of the subsequent songs were compared across conditions in the same bird. In line with our prediction, analysis of songs sung by 75 day (75d) birds revealed that syllable structure was more variable and sequence stereotypy was reduced following two hours of continuous practice compared to these features following two hours of non-singing. Similar trends in song were observed in these birds at 65d, despite higher overall within-condition variability at this age.

Conclusions/significance: Together with previous work, these findings point to the importance of behaviorally-driven acute periods during song learning that allow for both refinement and reinforcement of motor patterns. Future work is aimed at testing the observation that not only does vocal practice influence expression of molecular networks, but that these networks then influence subsequent variability in these skills.

Figure 1. Schematic timelines for vocal learning and for experimental set up.

A) Relative time frame for zebra finch (top) versus human (bottom) vocal learning (adapted from Doupe & Kuhl, 1999 [4]). Zebra finch song learning occurs over a shorter time scale, requiring only ∼90d for song maturation versus ∼one year for uttering the first word. Sensory acquisition and sensorimotor learning in birdsong correspond to speech perception and babbling, respectively. Experiments in the present study were conducted during late sensorimotor learning at ∼65d and ∼75d. B) Following lights-on, individual male zebra finches were assigned to two categories- either 2 hours of non-singing (NS) or 2 hours of undirected singing (UD). On the following day, the bird was assigned to the other condition. At the 2 hour time-point, both groups of birds were allowed to sing uninterrupted undirected song, NS-UD or UD-UD. Analysis was conducted on songs sung during the first 20 motifs or 30 one second clips following the 2 hour time-point. Abbreviations: UD-UD: continuous undirected singing, NS-UD: 2 hours of non-singing followed by undirected singing.

Figure 2. Continuous song practice increases syllable variability at 75d.

A) Examples from one bird at 75d show spectral derivatives of syllables. Six renditions (1–6) of the same syllable are illustrated with time on the x-axis, and frequency on the y axis. In the UD-UD condition, the syllable is composed of two disjointed notes in 1,2,6, but as one continuous note in 3–5 and in all of the NS-UD renditions. B) Paired data scores for the NS-UD (filled circles) and UD-UD (open circles) conditions for each bird are represented by connected lines. The UD-UD condition had lower (*) mean syllable similarity and accuracy scores (p<0.005, p<0.001, 2-tailed paired bootstrap). C–E) Histograms for all 30 syllables representing 11 birds are shown with similarity, accuracy, and identity scores on the x-axis and counts (frequency of occurrence) on the y-axis. The dashed line shows what a fitted curve through a normal distribution would be and is provided for comparison to the actual data. The UD-UD scores (light grey bars) are more broadly distributed and shifted towards lower scores than are the NS-UD scores (dark grey bars) (2-tailed paired bootstrap for similarity, accuracy, and identity, p<0.0001).

Figure 3. Continuous song practice increases variability in several acoustic features of syllables at 75d.

A) Examples from another bird at 75d reveal more variation in Wiener entropy from rendition to rendition in the UD-UD condition. Five consecutive renditions of the same syllable are shown in both panels. Each syllable consists of 3 notes which alter their spectral appearance from rendition to rendition. Wiener entropy is more variable, as reflected in the higher CV in the UD-UD condition than in the NS-UD condition. Numbers beneath are entropy scores, with more negative values indicative of less entropy (more spectral order). Zero is maximum entropy and negative infinity is maximum order. B) Box plots show the mean scores (middle of the box), standard error (top and bottom of the box), and upper and lower 95% confidence intervals (whiskers). Data scores for the NS-UD (filled circles) and UD-UD (open circles) conditions for ∼3 syllables from each bird are represented by individual points. Mean CV scores were obtained from 25 renditions of the same syllable. The UD-UD condition had higher CV values (*; 2-tailed paired bootstrap) for amplitude (p<0.005), pitch (p<0.05), pitch goodness (p<0.005), and Wiener entropy (p<0.05). Removal of points that are greater than two standard deviations above the mean does not remove the significance.

Figure 4. Additional motif- and clip-based analyses confirm that continuous song practice increases variability.

Paired data scores for the NS-UD (filled circles) and UD-UD (open circles) conditions for each bird are represented by connected lines. A) Motif similarity (left) and accuracy (right) scores for 75d birds (n = 11) are shown by condition. The UD-UD condition had lower (*, 2-tailed paired bootstrap) similarity (p<0.0005) and accuracy scores (p<0.001) compared to the NS-UD condition. B) Similarity scores were also lower at 65d in the UD-UD condition using the clip-based analysis (p<0.05). C) Individual points represent the mean syllable accuracy score per bird (p>0.05).

Figure 5. Sequence variability increases following continuous song practice at 75d.

A) Markov chains for one bird in the two conditions illustrate the probability of syllable transitions observed using the motif-based analysis. Letters denote syllables. Line thickness corresponds to probability; thicker lines indicate greater probabilities. In the NS-UD condition, syllable E transitions to syllable C 83% (thick line) of the time whereas a thinner line represents a 16% probability that E ends the motif. By contrast, in the UD-UD condition, syllable E transitions to syllable C 50% of the time, to syllable D, 43%, and ends the motif 7%. In the NS-UD condition, syllable F occurs infrequently compared to the UD-UD condition. B) Examples of 3 consecutive motifs from the same 75d bird in the NS-UD and UD-UD conditions. Motifs occurred in the same chronological order in the selected 20 motifs analyzed (#11,12,13). Individual syllables are identified by letter. In the NS-UD condition, syllable A typically transitions to itself or to syllable E, and syllable C transitions most frequently to syllable D. By contrast, in the UD-UD condition, A also transitions to C (#13) as well. In the UD-UD condition, syllable F is observed (#11,12,13) and follows syllable D while in NS-UD, syllable D transitions to E (#12) or ends the motif (#11,13). C–D) Paired data scores for the NS-UD (filled circles) and UD-UD (open circles) conditions for each bird are represented by connected lines. At 75d, songs sung in the UD-UD condition exhibited greater variability (*, 2-tailed paired bootstrap ‘Rescaled Entropy’) compared to the NS-UD condition for both the string- (C, p<0.005) and motif-based analysis (D, p<0.05). E) Histogram reveals the percent change in stereotypy between the conditions.