Beyond Critical Period Learning: Striatal FoxP2 Affects the Active Maintenance of Learned Vocalizations in Adulthood

Abstract

In humans, mutations in the transcription factor forkhead box P2 (FOXP2) result in language disorders associated with altered striatal structure. Like speech, birdsong is learned through social interactions during maturational critical periods, and it relies on auditory feedback during initial learning and on-going maintenance. Hearing loss causes learned vocalizations to deteriorate in adult humans and songbirds. In the adult songbird brain, most FoxP2-enriched regions (e.g., cortex, thalamus) show a static expression level, but in the striatal song control nucleus, area X, FoxP2 is regulated by singing and social context: when juveniles and adults sing alone, its levels drop, and songs are more variable. When males sing to females, FoxP2 levels remain high, and songs are relatively stable: this "on-line" regulation implicates FoxP2 in ongoing vocal processes, but its role in the auditory-based maintenance of learned vocalization has not been examined. To test this, we overexpressed FoxP2 in both hearing and deafened adult zebra finches and assessed effects on song sung alone versus songs directed to females. In intact birds singing alone, no changes were detected between songs of males expressing FoxP2 or a GFP construct in area X, consistent with the marked stability of mature song in this species. In contrast, songs of males overexpressing FoxP2 became more variable and were less preferable to females, unlike responses to songs of GFP-expressing control males. In deafened birds, song deteriorated more rapidly following FoxP2 overexpression relative to GFP controls. Together, these experiments suggest that behavior-driven FoxP2 expression and auditory feedback interact to precisely maintain learned vocalizations.

Keywords: auditory feedback; basal ganglia; birdsong; sensorimotor; speech.

Figure 1.

AAV construct drives overexpression of FoxP2 in adult male zebra finches. A, Timeline of experimental manipulations. The song-dedicated striatal nucleus area X was bilaterally injected with an AAV construct (surgery A) to drive overexpression of GFP (control) or FoxP2. To remove auditory feedback in half of the birds, surgery B was performed ∼20 d following surgery A. Songs were analyzed (vertical lines) at two time points directly before each surgery and at 6, 14, 25, and 60 d after deafening (e.g., D06, D14, etc.), and on the morning of sacrifice (DOS). B, Schematic of the AAV construct used to drive expression either GFP or FoxP2 using the CAG promoter. C, Protein levels of FoxP2 appear higher in hemispheres injected with FoxP2 compared to hemispheres injected with GFP in the same bird. D, In hearing birds used for evaluating the time line of FoxP2 overexpression, RT-qPCR confirms augmented levels at 20, 35, 45, and 80 d after injection (equivalent to surgery A time point in panel A) relative to uninjected controls (U). Fold change values are normalized to the mean of the controls. E, Across all birds used for behavioral analysis, FoxP2 expression levels (ΔCt; mean ± SEM) are higher in FoxP2-injected versus GFP-injected birds (p = 0.042), on the morning of sacrifice (DOS) approximately six months after surgery A. F, ΔCt values of FoxP2 levels on DOS and time spent singing for each group shows that FoxP2-Deaf birds trend toward higher FoxP2 expression despite singing similar amounts as other groups (mean ± SEM). G, ΔΔCt values of FoxP2 levels positively correlate with dopamine markers D1R and TH, but not with D2R (H). In D–H, dots represent individual birds. Figure Contributions: Jon Heston identified the appropriate viral construct. Nancy Day performed the experiments and analyzed the data. *p < 0.05.

Figure 2.

In hearing adults, area X FoxP2 overexpression does not alter UDs relative to those of GFP control birds. A, Representative spectrograms of song bouts from two zebra finches before and after injection of an AAV that drives expression of a control GFP or FoxP2 construct. Scale bars = 500 ms. B, Mean syllable identity is unchanged following FoxP2 overexpression. C, Following surgery, UDs were similar to pre-surgery songs (PRE) except at the final six-month time point (DOS) for both GFP-injected and FoxP2-injected birds; *p < 0.05. D, Syllable sequence (syntax similarity) is not altered following injection of AAV-GFP or AAV-FoxP2. Figure Contributions: Nancy Day performed the experiments and analyzed the data.

Figure 3.

FoxP2 overexpression hastens deafening-induced song deterioration. A, Representative spectrograms show deafening-induced song deterioration in two brothers who were deafened at 180 dph (surgery B) 29 d after injection of AAV-GFP (left) or AAV-FoxP2 (right; surgery A; Fig. 1). Scale bar = 500 ms. B, At 14 d post-deafening, motif similarity is persistently altered in the AAV-FoxP2 group (two-way ANOVA with Sidak’s multiple comparisons, p = 0.0006, p = 0.0002, p < 0.0001, and p < 0.0001 at D14, 25, 60, and DOS, respectively, n = 6 birds). In comparison to AAV-GFP-deafened birds, degradation of songs by AAV-FoxP2-injected birds is accelerated by at least 10 d. Statistically significant changes to songs by AAV-GFP-deafened birds are present at DOS (two-way ANOVA with Sidak’s multiple comparisons, p = 0.0007, n = 5 birds). All motif similarity scores are normalized to motif similarity calculated between songs collected on 2 d before AAV injection (refer to Fig. 1A). ***p < 0.001, ****p < 0.0001. Figure Contributions: Nancy Day performed the experiments and analyzed the data.

Figure 4.

Spectral variability and sequencing are affected by FoxP2 overexpression in deaf birds. A, Vocal variability increased more rapidly in deafened AAV-FoxP2 birds (solid black bars) than in deafened AAV-GFP birds (solid green bars) in most song features analyzed (e.g., entropy, entropy variance, duration, pitch goodness, FM). Positive values indicate an increase in the CV of each feature relative to PRE; negative values reflect lower variability than observed in PRE. B, Syllable omission occurs faster in FoxP2-deafened animals than in GFP-deafened animals. C, Syntax similarity (syllable sequencing; normalized to PRE) is disrupted in FoxP2-deafened animals by 14 d following deafening (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.) Figure Contributions: Nancy Day performed the experiments and analyzed the data.

Figure 5.

Female conspecifics perceive alterations in social context-dependent song variability. A, Exemplar syllable with a harmonic element/stack. Only the “flat” component of the syllable (indicated by dotted lines) was analyzed to determine the CV of the FF. B, Before AAV injections, syllables are performed with less rendition-to-rendition variability during female-directed song compared to UD in both GFP and FoxP2 groups (Wilcoxon matched-pairs signed-rank test, one-tailed; AAV-GFP: p = 0.0002, n = 12 syllables, AAV-FoxP2: p = 0.0001, n = 13 syllables). Following AAV injection, the CV of FF is significantly lower for female-directed syllables in GFP-injected zebra finches (Wilcoxon matched-pairs signed-rank test, one-tailed, p = 0.017, n = 12 syllables), but not in FoxP2-injected animals (Wilcoxon matched-pairs signed-rank test, one-tailed, p = 0.064, n = 13 syllables; *p < 0.05, ***p < 0.001). C, “Bird’s eye view” schematic of the testing arena for assaying female preference. D, Female preference for FD song is reduced (Preference_POST – Preference_PRE) following FoxP2 overexpression compared to songs following GFP overexpression (two-tailed t test, p = 0.047, t = 2.34, df = 8, n = 5 male birds per group). Figure Contributions: Taylor Hobbs designed the female preference testing area. Taylor Hobbs and Nancy Day performed the female preference experiments and analyzed the data.