Audition-independent vocal crystallization associated with intrinsic developmental gene expression dynamics

Abstract

Complex learned behavior is influenced throughout development by both genetic and environmental factors. Birdsong, like human speech, is a complex vocal behavior acquired through sensorimotor learning and is based on coordinated auditory input and vocal output to mimic tutor song. Song is primarily learned during a specific developmental stage called the critical period. Although auditory input is crucial for acquiring complex vocal patterns, its exact role in neural circuit maturation for vocal learning and production is not well understood. Using audition-deprived songbirds, we examined whether auditory experience affects developmental gene expression in the major elements of neural circuits that mediate vocal learning and production. Compared with intact zebra finches, early-deafened zebra finches showed excessively delayed vocal development, but their songs eventually crystallized. In contrast to the different rates of song development between the intact and deafened birds, developmental gene expression in the motor circuit is conserved in an age-dependent manner from the juvenile stage until the older adult stage, even in the deafened birds, which indicates the audition-independent robustness of gene expression dynamics during development. Furthermore, even after adult deafening, which degrades crystallized song, the deteriorated songs ultimately restabilized at the same point when the early-deafened birds stabilized their songs. These results indicate a genetic program-associated inevitable termination of vocal plasticity that results in audition-independent vocal crystallization.

Keywords: critical period; deaf; motor pattern generation; sensorimotor learning; songbird; species specificity.

Figure 1.

Comparison of song development and stabilization in intact, socially isolated, and early-deafened zebra finches. A, B, Examples of song development and syllable scatter plots (duration vs mean FM) in an intact (A) and a socially isolated (B) bird. Color shadings (blue and green) highlight stable song motifs. The intact and socially isolated birds exhibited song stability at approximately dph 110. The crystallized song pattern of the socially isolated bird is similar to that of the intact bird, except for a prolonged and variable syllable (green bracket). C, Examples of song development and syllable scatter plots (duration vs mean FM) in two early-deafened zebra finches. Orange shading highlights stable song motifs. D, Normalized K–L distance calculated from the last syllable scatter plot representing crystallized/stabilized song patterns. The values of K–L distance at dph 45–59 are normalized as 1.0. A value near 0 indicates a crystallized/stabilized song pattern. Mean ± SEM (intact, n = 4; socially isolated, n = 5; early-deafened, n = 6; Student's t test with Bonferroni's correction, **p < 0.01).

Figure 2.

Individual differences in stabilized song patterns in early-deafened zebra finches. A, Zebra finches deafened at dph 18–22 developed unique song patterns with a characteristic motif-like structure (colored shadings) at the old adult stage (older than dph 300). Sixteen early-deafened birds from four families are indicated. B, Average levels of circulating testosterone at three developmental stages, juvenile (dph 50–60), young (dph 105–130), and old (older than dph 330), in the intact (blue bars) and early-deafened (orange bars) birds. No significant difference was observed in serum testosterone between the two groups at any developmental stage. Each dot and bar represents individual values and mean ± SEM, respectively, of testosterone concentrations (nanograms per milliliter). Each animal number is indicated under the bar. C, Average number of song bouts per day during the three developmental periods, juvenile (dph 60–65), young (dph 100–110), and old adult (dph 440–920), in the intact (blue bars) and early-deafened (orange bars) zebra finches. Comparison of the intact and early-deafened birds indicates no significant difference at each stage (juvenile, p = 0.236; young, p = 0.352; old, p = 0.237; Student's t test). Each dot and bar represents individual values and the mean ± SEM, respectively. Each animal number is indicated under the bar.

Figure 3.

Similar induction of neural activity-induced gene expression and vocal output from the basal ganglia–forebrain circuit (AFP) before and after song crystallization/stabilization. A, Diagram of brain song circuits. Black solid lines denote connections within the posterior vocal motor circuit and black dashed lines within the basal ganglia–forebrain circuit AFP. B, Singing-driven Dusp1 mRNA expression in the AFP song nuclei, LMAN, Area X, and DLM in the intact and early-deafened zebra finches at juvenile (dph 47–59), young (dph 102–147), and old (dph 327–1715) stages. The young stage is pre-song stabilization stage for the early-deafened finches and post-stabilization for the intact finches. The old stage is post-stabilization for the early-deafened finches. White and red represent the Dusp1 mRNA signal and Nissl counterstained cells, respectively, in sagittal brain sections (right, anterior; up, dorsal). Scale bar, 1 mm. Summarized graphs show the relationship between total singing duration and Dusp1 gene expression level in LMAN, Area X, and DLM. There is little difference in this relationship between the intact and early-deafened songbirds at all three developmental stages (LMAN, p = 0.83; Area X, p = 0.36; DLM, p = 0.11; regression analysis). Each dot represents individual values; the regression line is indicated for each group (intact, blue; early-deafened, orange; juveniles, circles; young adults, triangle; old adults, diamonds). C, Example of similar vocal outputs after HVC lesion (producing AFP-driven songs) at young and old adult stages in both the intact and early-deafened zebra finches. Characteristic motif-like structures are shaded. Changes in song patterns (left) and distributions of syllable durations (right) before and after HVC lesion (black and red lines, respectively). D, Average probability density distribution of syllable duration for AFP-driven songs after HVC lesion (light blue, young intact bird at dph 115–138; blue, old intact bird at dph 660–1746; orange, young early-deafened songbird at dph 108–159; and red, old early-deafened songbird at dph 549–1037). d, Dorsal; a, anterior; p, posterior; v, ventral; Nlf, interfacial nucleus of the nidopallium; RAm/PAm, nuclei retroambiguus and paraambiguus; nXIIts, 12th nucleus, tracheosyringeal part.

Figure 4.

Developmental regulation of singing-induced Egr1 expression in the motor circuit among intact and early-deafened zebra finches. In contrast to HVC, induction of Egr1 mRNA expression is differentially regulated through development in RA, i.e., higher in the juvenile (dph 47–59) than in the young (dph 102–147) and old (dph 327–1715) adult stages for both the intact and early-deafened birds (HVC, p = 0.55; RA, *p = 0.0232 for juvenile vs young; **p = 0.0017 for juvenile vs old, regression analysis). However, there is no significant difference in the induction rate of Egr1 among the developmental stage-matched intact and early-deafened groups in HVC and RA (juvenile, p = 0.6024; young, p = 0.7451; old, p = 0.8153 for the intact vs early-deafened birds in RA, regression analysis). Each dot represents individual values; the regression line is indicated for each group (intact, blue; early-deafened, orange; juveniles, circles; young adults, triangle; old adults, diamonds). White and red represent Egr1 mRNA signal and Nissl counterstained cells, respectively, in sagittal brain section (right, anterior; up, dorsal). Scale bar, 1 mm.

Figure 5.

Gene expression dynamics in the motor circuit nuclei HVC and RA during development in intact and early-deafened zebra finches. A, Left, heat maps; expression levels of 3362 and 1466 gene probes in HVC and RA, respectively, with significant differences across developmental stages in the intact zebra finches (ANOVA, p < 0.05). The greater the absolute value of the Z score, the greater the deprivation of the expression of the gene from the mean, which indicates the significance of change in expression. Expression of the majority of these gene probes (2959 in HVC and 1357 in RA, white vertical bars) were not significantly different at the aged-matched developmental stages between the intact and early-deafened birds (ANOVA, p < 0.05), indicating that the gene expression dynamics of the early-deafened songbirds qualitatively has a similar trend to those of the intact songbirds through developmental stages. Middle column, Differences in average gene expression level between the intact and deafened birds at age-matched stages. Right columns, CV of gene expression level for each gene probe at each developmental stage in the intact and early-deafened birds. B, mRNA expression levels of the differentially regulated transcription factors Sox4, Hey1, Znf238, Neurod6, and Mxi1 in HVC and RA at three developmental stages for the intact and early-deafened songbirds (juvenile, dph 47–59; young, dph 104–146; and old adult, dph 332–1715). White and red represent mRNA signal and Nissl counterstained cells, respectively, in the sagittal brain section (right, anterior; up, dorsal). Scale bar, 1 mm. In the intact birds, all genes examined were differentially regulated during development (*p < 0.05 after Bonferroni's correction). In contrast, Znf238, Neurod6, and Mxi1 expression levels were not significantly different between stages in the early-deafened songbirds. Summarized bar graphs show the average gene expression level in each group (each animal number is indicated by inside bars). Each dot represents individual values (intact, blue; early-deafened, orange). C, Gene expression level (left) and CV of gene expression (right) in HVC and RA at juvenile, young, and old adult stages in the intact (blue) and early-deafened (orange) birds, using the same set of gene probes as shown in A. Each box illustrates median and interquartile range. D, Gene expression dynamics in HVC and RA calculated by PCA using the same set of gene probes as shown in A. Beige shading indicates age-matched intact (light blue, blue, and dark blue) and early-deafened (yellow, orange, and red) groups. The darker color indicates the older group.

Figure 6.

Gene coexpression network analysis reveals strong correlations between gene modules and age-related factors but not with auditory input. Top, Dendrogram produced by average linkage hierarchical clustering of gene probes identifies seven and nine modules of coexpressed genes in HVC and RA, respectively. The red lines in the dendrograms indicate the height at which the tree was cut. Colored blocks denote individual gene modules. Bottom, Correlation between gene modules and age-, auditory input-, temporal regulation-, and syllable phonology-related parameters. Colored bands indicate positive (green) and negative (red) correlations. For each module (columns), heat maps show correlations to measured parameters (rows). Asterisks in each cell indicate significant trait relationships (p < 0.05, Student's asymptotic p values), for example, all seven gene modules in HVC and four gene modules (modules 1, 2, 3, and 7) in RA significantly correlate with the age-related parameters. Bar plots show average strength of module correlations to measured parameters (mean ± 95% confidence intervals).

Figure 7.

Zebra finches deprived of auditory input after song crystallization restabilize their song at the old adult stage after deafening-induced song deterioration. A, Spectrograms of two example birds show song before and after adult deafening. Songs restabilize without auditory input at the old adult stage (orange shading in the bottom panels), i.e., the same period when early-deafened birds stabilize their song. Blue shading indicates that the first crystallized songs developed at dph 100–150, with audition appearing before adult deafening. B, Change of motif consistency at each experimental stage before and after adult deafening (blue and orange spots, respectively; n = 4, mean ± SD). Motif consistency was calculated as the song similarity among 20 song motifs that were randomly selected at each developmental time point in each bird. C, mRNA expression levels of the developmentally regulated transcription factors Sox4, Hey1, Znf238, Neurod6, and Mxi1 in HVC and RA at two developmental stages for the intact, early-deafened, and young adult-deafened songbirds (young, dph 104–161; old adult, dph 1158–1551). Young adult-deafened operations were performed at dph 92–144. No significant difference was observed for any gene among the three groups at both stages (ANOVA, p > 0.05). Bar graphs show average the gene expression level in each group (each animal number is indicated by inside bars). Each dot represents individual value (intact, blue; early-deafened, orange; and young adult-deafened, black).