Vocal Learning Research Articles

Expression and regulation of SETBP1 in the song system of male zebra finches (Taeniopygia guttata) during singing

Rare de novo heterozygous loss-of-function SETBP1 variants lead to a neurodevelopmental disorder characterized by speech deficits, indicating a potential involvement of SETBP1 in human speech. However, the expression pattern of SETBP1 in brain regions associated with vocal learning remains poorly understood, along with the underlying molecular mechanisms linking it to vocal production. In this study, we examined SETBP1 expression in the brain of male zebra finches, a well-established model for studying vocal production learning. We demonstrated that zebra finch SETBP1 exhibits a greater number of exons and isoforms compared to its human counterpart. We characterized a SETBP1 antibody and showed that SETBP1 colocalized with FoxP1, FoxP2, and Parvalbumin in key song nuclei. Moreover, SETBP1 expression in neurons in Area X is significantly higher in zebra finches singing alone, than those singing courtship song to a female, or non-singers. Importantly, we found a distinctive neuronal protein expression of SETBP1 and FoxP2 in Area X only in zebra finches singing alone, but not in the other conditions. We demonstrated SETBP1´s regulatory role on FoxP2 promoter activity in vitro. Taken together, these findings provide compelling evidence for SETBP1 expression in brain regions to be crucial for vocal learning and its modulation by singing behavior.

A versatile pipeline to identify convergently lost ancestral conserved fragments associated with convergent evolution of vocal learning.

Molecular convergence in convergently evolved lineages provides valuable insights into the shared genetic basis of converged phenotypes. However, most methods are limited to coding regions, overlooking the potential contribution of regulatory regions. We focused on the independently evolved vocal learning ability in multiple avian lineages, and developed a whole-genome-alignment-free approach to identify genome-wide Convergently Lost Ancestral Conserved fragments (CLACs) in these lineages, encompassing noncoding regions. We discovered 2711 CLACs that are overrepresented in noncoding regions. Proximal genes of these CLACs exhibit significant enrichment in neurological pathways, including glutamate receptor signaling pathway and axon guidance pathway. Moreover, their expression is highly enriched in brain tissues associated with speech formation. Notably, several have known functions in speech and language learning, including ROBO family, SLIT2, GRIN1, and GRIN2B. Additionally, we found significantly enriched motifs in noncoding CLACs, which match binding motifs of transcriptional factors involved in neurogenesis and gene expression regulation in brain. Furthermore, we discovered 19 candidate genes that harbor CLACs in both human and multiple avian vocal learning lineages, suggesting their potential contribution to the independent evolution of vocal learning in both birds and humans.

Model of the HVC neural network as a song motor in zebra finch.

The nucleus HVC within the avian song system produces crystalized instructions which lead to precise, learned vocalization in zebra finches (Taeniopygia guttata). This paper proposes a model of the HVC neural network based on the physiological properties of individual HVC neurons, their synaptic interactions calibrated by experimental measurements, as well as the synaptic signal into this region which triggers song production. This neural network model comprises of two major neural populations in this area: neurons projecting to the nucleus RA and interneurons. Each single neuron model of HVCRA is constructed with conductance-based ion currents of fast Na+ and K+ and a leak channel, while the interneuron model includes extra transient Ca2+ current and hyperpolarization-activated inward current. The synaptic dynamics is formed with simulated delivered neurotransmitter pulses from presynaptic cells and neurotransmitter receptor opening rates of postsynaptic neurons. We show that this network model qualitatively exhibits observed electrophysiological behaviors of neurons independent or in the network, as well as the importance of bidirectional interactions between the HVCRA neuron and the HVCI neuron. We also simulate the pulse input from A11 neuron group to HVC. This signal successfully suppresses the interneuron, which leads to sequential firing of projection neurons that matches measured burst onset, duration, and spike quantities during the zebra finch motif. The result provides a biophysically based model characterizing the dynamics and functions of the HVC neural network as a song motor, and offers a reference for synaptic coupling strength in the avian brain.

Social and auditory experience shapes forebrain responsiveness in zebra finches before the sensitive period of vocal learning.

Early-life experiences with signals used in communication are instrumental in shaping an animal's social interactions. In songbirds, which use vocalizations for guiding social interactions and mate choice, recent studies show that sensory effects on development occur earlier than previously expected, even in embryos and nestlings. Here, we explored the neural dynamics underlying experience-dependent song categorization in young birds prior to the traditionally studied sensitive period of vocal learning that begins around 3 weeks post-hatch. We raised zebra finches either with their biological parents, cross-fostered by Bengalese finches beginning at embryonic day 9, or with only the non-singing mother from 2 days post-hatch. Then, 1-5 days after fledging, we conducted behavioral experiments and extracellular recordings in the auditory forebrain to test responses to zebra finch and Bengalese finch songs. Auditory forebrain neurons in cross-fostered and isolated birds showed increases in firing rate and decreases in responsiveness and selectivity. In cross-fostered birds, decreases in responsiveness and selectivity relative to white noise were specific to conspecific song stimuli, which paralleled behavioral attentiveness to conspecific songs in those same birds. This study shows that auditory and social experience can already impact song 'type' processing in the brains of nestlings, and that brain changes at this age can portend the effects of natal experience in adults.

Seasonal patterns of neurogenesis in European starlings (Sturnus vulgaris) are region- and sex-specific.

Songbird vocal behavior, physiology, and brains-including neurogenesis-change between seasons. We examined seasonal differences in neurogenesis in three brain regions associated with vocal production and learning, HVC (letter-based proper name), robust nucleus of the arcopallium (RA), and Area X, and two brain regions associated with auditory perception, caudomedial nidopallium (NCM) and caudomedial mesopallium (CMM). To do this, we captured wild male and female European starlings (Sturnus vulgaris) in spring and fall, collected a blood sample, and minimized time from capture to tissue collection to limit suppressive effects of captivity on neurogenesis. We quantified neurogenesis using doublecortin (DCX) immunohistochemistry, counting new neurons of three DCX cell morphologies (multipolar, fusiform, and round). We found regional differences in types of morphologies expressed, and amount of neurogenesis across regions: NCM had more fusiform cells than all other regions, and RA had more round cells than other regions. Males had more neurogenesis in HVC in fall than in spring, but there was no seasonal difference in neurogenesis in HVC of females, perhaps reflecting sexually dimorphic vocal learning demands related to repertoire size and complexity. Plasma corticosterone was higher in spring than fall and was correlated with testis volume in males, but it was not correlated with another purported measure of stress, heterophil:lymphocyteratio (HLR), nor with neurogenesis. Our results suggest that the addition of new neurons to specific regions and circuits may serve different functions for males and females, particularly in the context of vocal production, learning, and perceptual demands across seasons.

Female calls promote song learning in male juvenile zebra finches

Social interactions promote vocal learning, but the impact of social feedback on this process and its neural circuitry is not well understood. We studied song imitation in juvenile male zebra finches raised either in the presence or absence of adult females. Juveniles learned songs more accurately with a female present, suggesting her presence improves imitation. When female calls correlated with practice, tutees’ songs better resembled the tutor’s, hinting toward the possibility that females provide practice-specific vocalizations. Intracellular recordings of HVC projection neurons revealed that a subset of these neurons in both juveniles and adults is sensitive to female calls during listening, suggesting a consistent neural mechanism for processing important vocalizations, regardless of age. However, call-related neural responses during singing were observed only in juveniles. These findings highlight how vocalizations, beyond those of the tutor, influence the neural circuits for vocal learning and production.

Vocal usage learning and vocal comprehension learning in harbor seals

BackgroundWhich mammals show vocal learning abilities, e.g., can learn new sounds, or learn to use sounds in new contexts? Vocal usage and comprehension learning are submodules of vocal learning. Specifically, vocal usage learning is the ability to learn to use a vocalization in a new context; vocal comprehension learning is the ability to comprehend a vocalization in a new context. Among mammals, harbor seals (Phoca vitulina) are good candidates to investigate vocal learning. Here, we test whether harbor seals are capable of vocal usage and comprehension learning.ResultsWe trained two harbor seals to (i) switch contexts from a visual to an auditory cue. In particular, the seals first produced two vocalization types in response to two hand signs; they then transitioned to producing these two vocalization types upon the presentation of two distinct sets of playbacks of their own vocalizations. We then (ii) exposed the seals to a combination of trained and novel vocalization stimuli. In a final experiment, (iii) we broadcasted only novel vocalizations of the two vocalization types to test whether seals could generalize from the trained set of stimuli to only novel items of a given vocal category. Both seals learned all tasks and took ≤ 16 sessions to succeed across all experiments. In particular, the seals showed contextual learning through switching the context from former visual to novel auditory cues, vocal matching and generalization. Finally, by responding to the played-back vocalizations with distinct vocalizations, the animals showed vocal comprehension learning.ConclusionsIt has been suggested that harbor seals are vocal learners; however, to date, these observations had not been confirmed in controlled experiments. Here, through three experiments, we could show that harbor seals are capable of both vocal usage and comprehension learning.

Vocal functional flexibility in a nonprimate vocal learning species

Abstract Can nonhuman animals use the same acoustic signal to transmit different illocutions on different occasions? This communicative capacity is known as vocal functional flexibility and occurs, for example, in speech, when a sentence serves different illocutionary forces or functions on different occasions based on changes to visual and intonational cues. Although common in human speech, there is a lack of clear evidence for this ability in other species. Here, we examined a likely candidate, the Brown-headed Cowbird (Molothrus ater), which is a vocal-learning songbird species that develops a repertoire of structurally distinct song types. Most of this species’ songs are directed towards conspecific males and females less than a meter away, making it unusually easy to determine the apparent target of songs, unlike the broadcast songs done by most songbirds. Songs directed to other males have clear aggressive/threatening intent, while those to females involve courtship/sexual intent. Extensive prior work shows that male cowbirds perform the visual display that accompanies singing differently in these two social settings and also modulate the intonation of song types differently. Because of these display and tonal modulations, constancy of song type usage across male- vs female-directed singing would provide evidence of vocal functional flexibility. Herein, we examined 4,828 songs in three captive flocks containing twenty-four males and thirty females during the breeding season. Males did not use their song types randomly and had strongly favored songs and less commonly used ones. Importantly, favored song types and less commonly used ones were the same whether directing courtship song to a female, aggressive song to another male or singing nonsocially with no receiver nearby. Results were consistent within and across the three flocks, providing strong evidence of vocal functional flexibility. These findings indicate that some species may evolve the ability to modulate and exaggerate visual display components and prosody more than vocal presentation per se because a learned phonological system in this and possibly other species is constrained by its vital role as an indicator trait.

Benefits of Learning Vocal Music with a Systematic Strategy

Abstract: There are many possible approaches to learning a song, some of which may involve multi-tasking across musical elements, despite research showing that multi-tasking is ineffective. This study compared singer-chosen song learning strategies with a systematic approach. The results showed that the systematic approach resulted in statistically significant improvement in accuracy across words, rhythms, and notes. Further, singers who demonstrated vocal quality changes with their personal approach, showed no evidence of vocal deterioration with the systematic approach. These findings suggest that a systematic approach to vocal music learning enhances both accuracy and efficiency, critical components of vocal performance.

Vocal labeling of others by nonhuman primates.

Humans, dolphins, and elephants are the only known species that vocally label their conspecifics. It remains unclear whether nonhuman primates share this ability. We recorded spontaneous "phee-call" dialogues between pairs of marmoset monkeys. We discovered that marmosets use these calls to vocally label their conspecifics. Moreover, they respond more consistently and correctly to calls that are specifically directed at them. Analysis of calls from multiple monkeys revealed that family members use similar calls and acoustic features to label others and perform vocal learning. These findings shed light on the complexities of social vocalizations among nonhuman primates and suggest that marmoset vocalizations may provide a model for understanding aspects of human language, thereby offering new insights into the evolution of social communication.

Pronounced early differentiation underlies zebra finch gonadal germ cell development

The diversity of germ cell developmental strategies has been well documented across many vertebrate clades. However, much of our understanding of avian primordial germ cell (PGC) specification and differentiation has derived from only one species, the chicken (Gallus gallus). Of the three major classes of birds, chickens belong to Galloanserae, representing less than 4% of species, while nearly 95% of extant bird species belong to Neoaves. This represents a significant gap in our knowledge of germ cell development across avian species, hampering efforts to adapt genome editing and reproductive technologies developed in chicken to other birds. We therefore applied single-cell RNA sequencing to investigate inter-species differences in germ cell development between chicken and zebra finch (Taeniopygia castanotis), a Neoaves songbird species and a common model of vocal learning. Analysis of early embryonic male and female gonads revealed the presence of two distinct early germ cell types in zebra finch and only one in chicken. Both germ cell types expressed zebra finch Germline Restricted Chromosome (GRC) genes, present only in songbirds among birds. One of the zebra finch germ cell types expressed the canonical PGC markers, as did chicken, but with expression differences in several signaling pathways and biological processes. The second zebra finch germ cell cluster was marked by proliferation and fate determination markers, indicating beginning of differentiation. Notably, these two zebra finch germ cell populations were present in both male and female zebra finch gonads as early as HH25. Using additional chicken developmental stages, similar germ cell heterogeneity was identified in the more developed gonads of females, but not males. Overall, our study demonstrates a substantial heterochrony in zebra finch germ cell development compared to chicken, indicating a richer diversity of avian germ cell developmental strategies than previously known.

AVN: A Deep Learning Approach for the Analysis of Birdsong.

Deep learning tools for behavior analysis have enabled important new insights and discoveries in neuroscience. Yet, they often compromise interpretability and generalizability for performance, making it difficult to quantitively compare phenotypes across datasets and research groups. We developed a novel deep learning-based behavior analysis pipeline, Avian Vocalization Network (AVN), for the learned vocalizations of the most extensively studied vocal learning model species - the zebra finch. AVN annotates songs with high accuracy across multiple animal colonies without the need for any additional training data and generates a comprehensive set of interpretable features to describe the syntax, timing, and acoustic properties of song. We use this feature set to compare song phenotypes across multiple research groups and experiments, and to predict a bird's stage in song development. Additionally, we have developed a novel method to measure song imitation that requires no additional training data for new comparisons or recording environments, and outperforms existing similarity scoring methods in its sensitivity and agreement with expert human judgements of song similarity. These tools are available through the open-source AVN python package and graphical application, which makes them accessible to researchers without any prior coding experience. Altogether, this behavior analysis toolkit stands to facilitate and accelerate the study of vocal behavior by enabling a standardized mapping of phenotypes and learning outcomes, thus helping scientists better link behavior to the underlying neural processes.

USING JEROME BRUNER'S COGNITIVE THEORY IN VOCAL TEACHING AT UNIVERSITY

This paper examines the teaching of vocal music according to a number of provisions of Jerome Bruner's cognitive theory. The impact of this theory on the development of students as creative individuals, their understanding of the learning process, and the link between thinking and music learning is explored. By utilising Bruner's cognitive theory, teachers can create an engaging learning environment and encourage students to actively participate in the vocal learning process. This will help students' creativity to develop harmoniously.

Note similarities affect syntactic stability in zebra finches.

The acquisition of an acoustic template is a fundamental component of vocal imitation learning, which is used to refine innate vocalizations and develop a species-specific song. In the absence of a model, birds fail to develop species typical songs. In zebra finches (Taeniopygia guttata), tutored birds produce songs with a stereotyped sequence of distinct acoustic elements, or notes, which form the song motif. Songs of untutored individuals feature atypical acoustic and temporal structure. Here we studied songs and associated respiratory patterns of tutored and untutored male zebra finches to investigate whether similar acoustic notes influence the sequence of song elements. A subgroup of animals developed songs with multiple acoustically similar notes that are produced with alike respiratory motor gestures. These birds also showed increased syntactic variability in their adult motif. Sequence variability tended to occur near song elements which showed high similarity in acoustic structure and underlying respiratory motor gestures. The duration and depth of the inspirations preceding the syllables where syntactic variation occurred did not allow prediction of the following sequence of notes, suggesting that the varying duration and air requirement of the following expiratory pulse is not predictively encoded in the motor program. This study provides a novel method for calculation of motor/acoustic similarity, and the results of this study suggest that the note is a fundamental acoustic unit in the organization of the motif and could play a role in the neural code for song syntax.

Humans need auditory experience to produce typical volitional nonverbal vocalizations

Human nonverbal vocalizations such as screams and cries often reflect their evolved functions. Although the universality of these putatively primordial vocal signals and their phylogenetic roots in animal calls suggest a strong reflexive foundation, many of the emotional vocalizations that we humans produce are under our voluntary control. This suggests that, like speech, volitional vocalizations may require auditory input to develop typically. Here, we acoustically analyzed hundreds of volitional vocalizations produced by profoundly deaf adults and typically-hearing controls. We show that deaf adults produce unconventional and homogenous vocalizations of aggression and pain that are unusually high-pitched, unarticulated, and with extremely few harsh-sounding nonlinear phenomena compared to controls. In contrast, fear vocalizations of deaf adults are relatively acoustically typical. In four lab experiments involving a range of perception tasks with 444 participants, listeners were less accurate in identifying the intended emotions of vocalizations produced by deaf vocalizers than by controls, perceived their vocalizations as less authentic, and reliably detected deafness. Vocalizations of congenitally deaf adults with zero auditory experience were most atypical, suggesting additive effects of auditory deprivation. Vocal learning in humans may thus be required not only for speech, but also to acquire the full repertoire of volitional non-linguistic vocalizations.

Maternal in‐nest call structure reduces habituation risk in songbird embryos

Repetition of the same vocal stimulus during vocal learning may result in habituation. Therefore, selection may favor vocal tutors that produce vocal stimuli with characteristics that reduce the risk of habituation. Superb fairywren Malurus cyaneus mothers produce a two‐element (A and signature B) call to embryos, but embryos only produce one element type (B) after hatching. Why do mothers use the A element to embryos? We broadcast calls with one (B) or two (AB) element types and measured embryo response. Embryos habituated to calls with one element type and remained responsive to calls with both. We conclude that signal characteristics in tutors may guide learning to retain pupil attention during learning.

Persistent vocal learning in an aging open-ended learner reflected in neural FoxP2 expression

BackgroundMost vocal learning species exhibit an early critical period during which their vocal control neural circuitry facilitates the acquisition of new vocalizations. Some taxa, most notably humans and parrots, retain some degree of neurobehavioral plasticity throughout adulthood, but both the extent of this plasticity and the neurogenetic mechanisms underlying it remain unclear. Differential expression of the transcription factor FoxP2 in both songbird and parrot vocal control nuclei has been identified previously as a key pattern facilitating vocal learning. We hypothesize that the resilience of vocal learning to cognitive decline in open-ended learners will be reflected in an absence of age-related changes in neural FoxP2 expression. We tested this hypothesis in the budgerigar (Melopsittacus undulatus), a small gregarious parrot in which adults converge on shared call types in response to shifts in group membership. We formed novel flocks of 4 previously unfamiliar males belonging to the same age class, either “young adult” (6 mo − 1 year) or “older adult” (≥ 3 year), and then collected audio-recordings over a 20-day learning period to assess vocal learning ability. Following behavioral recording, immunohistochemistry was performed on collected neural tissue to measure FoxP2 protein expression in a parrot vocal learning center, the magnocellular nucleus of the medial striatum (MMSt), and its adjacent striatum.ResultsAlthough older adults show lower vocal diversity (i.e. repertoire size) and higher absolute levels of FoxP2 in the MMSt than young adults, we find similarly persistent downregulation of FoxP2 and equivalent vocal plasticity and vocal convergence in the two age cohorts. No relationship between individual variation in vocal learning measures and FoxP2 expression was detected.ConclusionsWe find neural evidence to support persistent vocal learning in the budgerigar, suggesting resilience to aging in the open-ended learning program of this species. The lack of a significant relationship between FoxP2 expression and individual variability in vocal learning performance suggests that other neurogenetic mechanisms could also regulate this complex behavior.

Tutor auditory memory for guiding sensorimotor learning in birdsong.

Memory-guided motor shaping is necessary for sensorimotor learning. Vocal learning, such as speech development in human babies and song learning in bird juveniles, begins with the formation of an auditory template by hearing adult voices followed by vocally matching to the memorized template using auditory feedback. In zebra finches, the widely used songbird model system, only males develop individually unique stereotyped songs. The production of normal songs relies on auditory experience of tutor's songs (commonly their father's songs) during a critical period in development that consists of orchestrated auditory and sensorimotor phases. "Auditory templates" of tutor songs are thought to form in the brain to guide later vocal learning, while formation of "motor templates" of own song has been suggested to be necessary for the maintenance of stereotyped adult songs. Where these templates are formed in the brain and how they interact with other brain areas to guide song learning, presumably with template-matching error correction, remains to be clarified. Here, we review and discuss studies on auditory and motor templates in the avian brain. We suggest that distinct auditory and motor template systems exist that switch their functions during development.

CHIRP-Seq: FoxP2 transcriptional targets in zebra finch brain include numerous speech and language-related genes.

Vocal learning is a rare, convergent trait that is fundamental to both human speech and birdsong. The Forkhead Box P2 (FoxP2) transcription factor appears necessary for both types of learned signals, as human mutations in FoxP2 result in speech deficits, and disrupting its expression in zebra finches impairs male-specific song learning. In juvenile and adult male finches, striatal FoxP2 mRNA and protein decline acutely within song-dedicated neurons during singing, indicating that its transcriptional targets are also behaviorally regulated. The identities of these targets in songbirds, and whether they differ across sex, development and/or behavioral conditions, are largely unknown. Here we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to identify genomic sites bound by FoxP2 in male and female, juvenile and adult, and singing and non-singing birds. Our results suggest robust FoxP2 binding concentrated in putative promoter regions of genes. The number of genes likely to be bound by FoxP2 varied across conditions, suggesting specialized roles of the candidate targets related to sex, age, and behavioral state. We validated these binding targets both bioinformatically, with comparisons to previous studies and biochemically, with immunohistochemistry using an antibody for a putative target gene. Gene ontology analyses revealed enrichment for human speech- and language-related functions in males only, consistent with the sexual dimorphism of song learning in this species. Fewer such targets were found in juveniles relative to adults, suggesting an expansion of this regulatory network with maturation. The fewest speech-related targets were found in the singing condition, consistent with the well-documented singing-driven down-regulation of FoxP2 in the songbird striatum. Overall, these data provide an initial catalog of the regulatory landscape of FoxP2 in an avian vocal learner, offering dozens of target genes for future study and providing insight into the molecular underpinnings of vocal learning.

Expansion of learning capacity elicited by interspecific hybridization.

Learned behavior, a fundamental adaptive trait in fluctuating environments, is shaped by species-specific constraints. This phenomenon is evident in songbirds, which acquire their species-specific songs through vocal learning. To explore the neurogenetic mechanisms underlying species-specific song learning, we generated F1 hybrid songbirds by crossing Taeniopygia guttata with Aidemosyne modesta. These F1 hybrids demonstrate expanded learning capacities, adeptly mimicking songs from both parental species and other heterospecific songs more extensively than their parental counterparts. Despite the conserved size of brain regions and neuron numbers in the neural circuits for song learning and production, single-cell transcriptomics reveals distinctive transcriptional characteristics in the F1 hybrids, especially in vocal-motor projection neurons. These neurons exhibit enrichment for nonadditively expressed genes, particularly those related to ion channel activity and cell adhesion, which are associated with the degree of song learning among F1 individuals. Our findings provide insights into the emergence of altered learning capabilities through hybridization, linked to cell type-specific transcriptional changes.