We confirmed this result in LMAN-lesioned birds, with learning rates in pCAF going from 13.3 ± 5.9 Hz/day before lesions to 0.7 ± 1.1 Hz/day after lesions (p = 6.7 × 10−4; n = 4 birds, 3 of which also tested for tCAF; p = 0.11 when comparing LMAN-lesioned birds in pCAF to normal drift; Figure 4D). In the temporal domain,

however, LMAN-lesioned birds retained the ability to Dorsomorphin learn, albeit at a reduced rate compared to prelesion (Figure 4E; prelesion: 2.8 ± 1.6 ms/day, postlesion: 0.9 ± 0.6 ms/day, p = 0.003 when comparing LMAN-lesioned birds in tCAF to normal drift). Mean reduction in the learning rate within a bird was 60.7% ± 29.4% (n = 8 birds, p = 6.3 × 10−4). Since LMAN is known to induce vocal exploration in both the temporal and

spectral domains (Thompson et al., 2011), we wondered whether the decreased learning rates in tCAF following lesions could be explained by a reduction in temporal variability. Consistent with this, we found that variability in the duration of song elements (CV of syllable and intersyllable gaps [Glaze and Troyer, 2013], see Experimental Procedures) decreased within a bird by, on average, 38%, from 3.3% ± 1.2% to 2.1% ± 1.1% (Figure 4F; p = 4.8 × 10−4). These Akt inhibitor results suggest that LMAN contributes to temporal learning by inducing variability in song timing. The process of converting information derived from this variability into improved motor timing, however, is probably implemented outside the AFP, as this process does not require an intact Area X or LMAN. Given the architecture of the song circuit, and the assumed role of the basal ganglia in reinforcement learning, an obvious candidate for driving temporal learning is the only other known song-related basal ganglia-thalamo-cortical circuit—a parallel circuit to the AFP that includes a basal ganglia-like structure medial to traditionally defined Area X (mArea X) (Kubikova et al., 2007), the thalamic nucleus DMP and the medial part

of MAN (MMAN) (Figure 5A). Whereas the AFP projects directly to RA, which encodes spectral features (Sober et al., 2008), MMAN outputs directly to HVC and could, in analogy to its lateral counterpart (LMAN), provide the instructive signal for altering neural dynamics in HVC and thus temporal structure of song. To test this, we lesioned MMAN bilaterally (Tables S1 and S2 and Figure S5C), during comparing learning rates in our tCAF paradigm before and after lesions. We saw no significant change in the capacity of birds to shift the duration of targeted song segments after MMAN lesions (Figure 5B; prelesion: 3.4 ± 1.8 ms/day, postlesion: 3.0 ± 1.3 ms/day, n = 3 birds, p = 0.34). Neither did MMAN lesions influence variability (CV) in temporal (prelesion: 2.6% ± 0.6%, postlesion: 2.6% ± 0.4%) or spectral (prelesion: 3.0% ± 0.9%, postlesion: 2.8% ± 0.5%) features of song (Figure 5C; p = 0.94 and 0.53, respectively), leaving its role in song learning, if any, to be elucidated.