Wan-Chun Liu

When humans speak, we use rhythmic hand and head gestures that are closely coordinated with the temporal structure of speech to emphasize words and phrases. Similarly, human singing is often accompanied by rhythmic body movements that are aligned with the timing and prosodic structure of the vocal sequence. This rhythmic synchronization of bodily gesture with vocal production is thought to be a shared feature of vocal-learning species. How do these two sensorimotor systems develop, coordinate, and synchronize with precise timing to support multimodal communication? The mechanisms underlying this rhythmic entrainment remain poorly understood, and no established animal model to date captures the human combination of speech and co-speech gesture. Here, we show that a vocal-learning songbird, the zebra finch, has evolved song-entrained head gestures. These co-song gestures rhythmically align with acoustically complex song syllables and are developed and produced independently of other innate, stereotyped, song-entangled courtship displays. Even when the song remains largely the same, co-song gestures can be dynamically modulated in rhythm, form, and/or extent across different social contexts. The rhythmic alignment requires auditory feedback, is under the control of a premotor song nucleus, and gradually develops during the sensitive period of vocal learning. Females respond differently when songs and co-song gestures are misaligned, suggesting a social function. This dynamic modulation of co-song gestures provides a behavioral window into brain and cognitive states, making the zebra finch a promising model for understanding the mechanisms underlying rhythmic synchronization of multi-sensorimotor systems and the origin and evolution of co-speech gestures in humans.

The evolution and development of complex, learned motor skills are thought to be closely associated with other locomotor movement and cognitive functions. However, it remains largely unknown how different neuromuscular programs may interconnect during the protracted developmental process. Here we use a songbird to examine the behavioral and neural substrates between the development of locomotor movement and vocal-motor learning. Juvenile songbirds escalate their locomotor activity during the sensitive period for vocal learning, followed by a surge of vocal practice. Individual variability of locomotor production is positively correlated with precision of tutor imitation and duration of multi-syllable sequences. Manipulation of juvenile locomotion significantly impacts the precision of vocal imitation and neural plasticity. The locomotor program developed during the sensitive period of vocal learning may enrich the neural substrates that promote the subsequent development of vocal learning.

Songbirds are one of the few animal taxa that possess vocal learning abilities. Different species of songbirds exhibit species-specific learning programs during song acquisition. Songbirds with open-ended vocal learning capacity, such as the canary, modify their songs during adulthood. Nevertheless, the neural molecular mechanisms underlying open-ended vocal learning are not fully understood. We investigated the singing-driven expression of neural activity-dependent genes (Arc,Egr1,c-fos,Nr4a1,Sik1,Dusp6, andGadd45β) in the canary to examine a potential relationship between the gene expression level and the degree of seasonal vocal plasticity at different ages. The expression of these genes was differently regulated throughout the critical period of vocal learning in the zebra finch, a closed-ended song learner. In the canary, the neural activity-dependent genes were induced by singing in the song nuclei throughout the year. However, in the vocal motor nucleus, the robust nucleus of the arcopallium (RA), all genes were regulated with a higher induction rate by singing in the fall than in the spring. The singing-driven expression of these genes showed a similar induction rate in the fall between the first year juvenile and the second year adult canaries, suggesting a seasonal, not age-dependent, regulation of the neural activity-dependent genes. By measuring seasonal vocal plasticity and singing-driven gene expression, we found that in RA, the induction intensity of the neural activity-dependent genes was correlated with the state of vocal plasticity. These results demonstrate a correlation between vocal plasticity and the singing-driven expression of neural activity-dependent genes in RA through song development, regardless of whether a songbird species possesses an open- or closed-ended vocal learning capacity.

In the vocal learning model, the juvenile first memorizes a model sound, and the imprinted memory gradually converts into vocal-motor output during the sensorimotor integration. However, early acquired memory may not precisely represent the fine structures of a model sound. How do juveniles ensure precise model imitation? Here we show that juvenile songbirds develop an auditory learning program by actively and attentively engaging with tutor’s singing during the sensorimotor phase. The listening/approaching behavior requires previously acquired model memory and the individual variability of approaching behavior correlates with the precision of tutor song imitation. Moreover, it is modulated by dopamine and associated with forebrain regions for sensory processing. Overall, precise vocal learning may involve two steps of auditory processing: a passive imprinting of model memory occurs during the early sensory period; the previously acquired memory then guides an active and selective engagement of the re-exposed model to fine tune model imitation.

Previous studies have revealed diel patterns in the songs of Chipping Sparrows (Spizella passerina ), with songs shorter in duration before dawn than after. However, the extent to which this phenomenon generalizes to the full geographic range of these sparrows is unclear, as is the question of whether citizen‐science data can be used to detect diel patterns in song. We analyzed all available songs of Chipping Sparrows from the Macaulay Library and xeno‐canto databases and compared the distributions of song features of recordings made at different times of day. We show that, across their entire geographic range in North and Central America, Chipping Sparrows sing shorter songs before sunrise (dawn song) than after sunrise (day song). Furthermore, we show that Chipping Sparrows shorten their songs by singing fewer syllables, not by singing faster: the number of syllables per song accounts for the observed difference in duration, not the syllable nor the intersyllable duration. Our results demonstrate that recordings from public repositories can be used to determine whether daily song patterns exist in species even in the absence of prior fieldwork, and we further propose that citizen‐science recordings can be used to inform cross‐species hypotheses and facilitate future studies to determine whether diel patterns in song are associated with differences in social behavior.

Transgenesis involves the insertion of an exogenous gene into an animal’s genome, which allows the identification of the expressed phenotypes in brain function or behavior. Lentiviral-mediated transgenesis offers unique transduction potency making it possible to deliver and stably integrate transgenes into a wide variety of dividing and nondividing cells. The ability to establish long-term expression of such transgenes allows their use for transgenesis which is especially useful in organisms lacking quality pluripotent stem cell lines and which is otherwise difficult to produce via traditional pronuclear microinjection, such as songbirds. Here we describe a protocol to generate the transgenic songbird, the zebra finch, by producing and inserting lentiviral-mediated transgene into the blastoderm of freshly laid eggs. This protocol includes procedures for production of lentiviral vectors, injection of a virus into zebra finch embryos, and postinjection care. The implementation of the songbird transgenic approach provides a leap toward basic and translational neuroscience that uses an animal model for speech and language and their pathologies. Additionally, the highly quantifiable song behavior, combined with a well-characterized song circuitry, offers an exciting opportunity to develop therapeutic strategies for neurological disorders.