Birdsong, like human speech, consists of a sequence of temporally precise movements acquired through vocal learning. The learning of such sequential vocalizations depends on the neural function of the motor cortex and basal ganglia. However, it is unknown how the connections between cortical and basal ganglia components contribute to vocal motor skill learning, as mammalian motor cortices serve multiple types of motor action and most experimentally tractable animals do not exhibit vocal learning. Here, we leveraged the zebra finch, a songbird, as an animal model to explore the function of the connectivity between cortex-like (HVC) and basal ganglia (area X), connected by HVC(X) projection neurons with temporally precise firing during singing. By specifically ablating HVC(X) neurons, juvenile zebra finches failed to copy tutored syllable acoustics and developed temporally unstable songs with less sequence consistency. In contrast, HVC(X)-ablated adults did not alter their learned song structure, but generated acoustic fluctuations and responded to auditory feedback disruption by the introduction of song deterioration, as did normal adults. These results indicate that the corticobasal ganglia input is important for learning the acoustic and temporal aspects of song structure, but not for generating vocal fluctuations that contribute to the maintenance of an already learned vocal pattern.

Acetylcholine receptors (AChRs) abound in the central nervous system of vertebrates. Muscarinic AChRs (mAChRs), a functional subclass of AChRs, mediate neuronal responses via intracellular signal transduction. They also play roles in sensorimotor coordination and motor skill learning by enhancing cortical plasticity. Learned birdsong is a complex motor skill acquired through sensorimotor coordination during a critical period. However, the functions of AChRs in the neural circuits for vocal learning and production remain largely unexplored. Here, we report the unique expression of mAChRs subunits (chrm2-5) in the song nuclei of zebra finches. The expression of excitatory subunits (chrm3 and chrm5) was downregulated in the song nuclei compared with the surrounding brain regions. In contrast, the expression of inhibitory mAChRs (chrm2 and chrm4) was upregulated in the premotor song nucleus HVC relative to the surrounding nidopallium. Chrm4 showed developmentally different expression in HVC during the critical period. Compared with chrm4, individual differences in chrm2 expression emerged in HVC early in the critical period. These individual differences in chrm2 expression persisted despite testosterone administration or auditory deprivation, which altered the timing of song stabilization. Instead, the variability in chrm2 expression in HVC correlated with parental genetics. In addition, chrm2 expression in HVC exhibited species differences and individual variability among songbird species. These results suggest that mAChRs play an underappreciated role in the development of species and individual differences in song patterns by modulating the excitability of HVC neurons, providing a potential insight into the gating of auditory responses in HVC neurons.

Learned vocalizations are important for communication in some vertebrate taxa. The neural circuitry for the learning and production of vocalizations is well known in songbirds, many of which learn songs initially during a critical period early in life. Dopamine is essential for motor learning, including song learning, and dopamine-related measures change throughout development in song-control regions such as HVC, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and the robust nucleus of the arcopallium (RA). In mammals, the neuropeptide neurotensin strongly interacts with dopamine signaling. This study investigated a potential role for the neurotensin system in song learning by examining how neurotensin (Nts) and neurotensin receptor 1 (Ntsr1) expression change throughout development. Nts and Ntsr1 mRNA expression was analyzed in song-control regions of male zebra finches in four stages of the song learning process: pre-subsong (25 days posthatch; dph), subsong (45 dph), plastic song (60 dph), and crystallized song (130 dph). Nts expression in LMAN during the subsong stage was lower compared to other time points. Ntsr1 expression was highest in HVC, Area X, and RA during the pre-subsong stage. Opposite and complementary expression patterns for the two genes in song nuclei and across the whole brain suggest distinct roles for regions that produce and receive Nts. The expression changes at crucial time points for song development are similar to changes observed in dopamine studies and suggest Nts may be involved in the process of vocal learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 671-686, 2018.

Neuronal tuning for spectral and temporal features has been studied extensively in the auditory system. In the auditory cortex, diverse combinations of spectral and temporal tuning have been found, but how specific feature tuning contributes to the perception of complex sounds remains unclear. Neurons in the avian auditory cortex are spatially organized in terms of spectral or temporal tuning widths, providing an opportunity for investigating the link between auditory tuning and perception. Here, using naturalistic conspecific vocalizations, we asked whether subregions of the auditory cortex that are tuned for broadband sounds are more important for discriminating tempo than pitch, due to the lower frequency selectivity. We found that bilateral inactivation of the broadband region impairs performance on both tempo and pitch discrimination. Our results do not support the hypothesis that the lateral, more broadband subregion of the songbird auditory cortex contributes more to processing temporal than spectral information.

Many songbirds learn to produce songs through vocal practice in early life and continue to produce many renditions of learned songs daily throughout their lifetime. While it is well-known that adult songbirds sing as part of a mating ritual, additional functions of their singing behavior are not fully understood. Here, we demonstrate that adult singing outside the reproductive contexts functions to prevent passive changes in song performance. Using a reversible behavioral manipulation, we suppressed the daily singing behavior of adult zebra finches produced in the solo context for two weeks and examined its effect on song performance. We found that singing suppression significantly decreases the pitch of song elements and both amplitude and duration of song motifs. This suggests that adult song is not acoustically stable without singing and that adult birds maintain their song performance by daily singing. Moreover, suppression-induced changes in song structure were substantially recovered within two weeks of free singing even in deafened birds. Thus, the recovery of song performance does not necessarily require auditory feedback but rather is caused predominantly by singing behavior per se (i.e., the physical act of singing). Finally, unlike the auditory feedback-dependent song plasticity previously reported, passive song changes caused by singing suppression were not significantly dependent on age. Our findings demonstrate that adult songbirds maintain song performance by preventing passive song changes through the physical act of daily singing outside the reproductive contexts throughout life. Such daily singing presumably functions as vocal training to maintain the neural and/or muscular system for song production in optimal conditions to make the best song performance in reproductive contexts.