Similar to the development of speech in human infants, song learning in juvenile songbirds starts with memorization of adult vocalizations. Young birds develop their own song as a replica of a memorized adult song model through trial and error learning through abundant vocal practice. The internal model of this adult song, termed the ‘template’ is therefore central for guiding the process of vocal learning. However, even the most fundamental aspects of the template, such as when, where and how it is encoded in the brain, remain poorly understood. This dissertation attempts to define a paradigm where the onset of vocal learning and template formation can be time-locked to a small window of exposure to the tutor song. Using this paradigm, I further characterize the molecular changes accompanying the initial phase of vocal learning. The work described here shows that, in the zebra finch, template encoding can be time locked to, on average, a two-hour period of juvenile life and based on just 75 seconds of cumulative tutor song exposure. Rapid changes in vocal output induced by this exposure can be used as markers for predicting the future success of song imitation. To investigate the brain regions involved in this initial phase of vocal learning, a new approach was developed to interrogate global patterns of activity induced gene expression. Statistical parametric mapping, a method used for analysis of functional activity, was adapted to analyze regional activation across 3D reconstructions of whole brain gene expression maps. Using this approach, regions engaged at different stages of vocal learning were identified. To track the molecular mechanisms underlying these activation patterns, changes in the transcriptome of specific brain nuclei were analyzed as the young males undergo rapid changes in the vocal output in response to training. Significant changes in gene expression patterns were detected with divergent gene expression patterns across individual brain regions. The behavioral and molecular tools developed here present an important advance for understanding how the template is instantiated in the songbird brain.

<p>The ability of young children to vocally imitate the speech of adults is critical for speech learning. Vocal imitation requires exposure to an external auditory model and the use of auditory feedback to adaptively modify vocal output to match the model. Despite the importance of vocal imitation to human communication and social behavior, it remains unclear how these two types of sensory experience, model exposure and feedback, act on sensorimotor networks controlling the learning and production of learned vocalizations. Using a combination of longitudinal in vivo imaging of neuronal structure and electrophysiological measurements of neuronal function, I addressed the questions of where, when, and how these two types of sensory experience act on sensorimotor neurons important to singing and song learning in zebra finches. The major finding of these experiments is that synaptic inputs onto neurons in HVC, a sensorimotor nucleus important to singing and song learning, are sensitive to tutor song experience and deafening. Thus, these findings for the first time link auditory experiences important to vocal imitation to synaptic reorganization in sensorimotor neurons important to behavior.</p> / Dissertation

Animal communication systems are complex, but in many species relatively little is known about how they develop. Insight into the development of adult repertoires, considering how factors such as genetics, environment and learning contribute to signal changes, provides a more comprehensive understanding of communication. This thesis documented vocal learning and development in the grey seal. In chapter 3, vocal repertoires of grey seal pups were recorded from birth through their first year, and were compared to calls of other seals across life stages, from pups to adults. By examining call parameters that were similar in animals of the same age, size, and sex, changes that may be attributed to physical development and morphology were identified. Past studies have indicated that seals may also have advanced vocal learning abilities, however the extent of these capabilities was unknown. In this thesis, vocal learning in juvenile grey seals was tested considering both how the seals learn to produce calls as well as gain information from perceived sounds. In chapter 4, grey seals were shown to be capable of vocal usage learning by producing specific calls in specific contexts. In chapter 5, grey seals displayed control over the structure of their calls as they could imitate novel sound models, demonstrating vocal production learning. Lastly, in chapter 6 grey seals gained information from perceived sound signals and used them to their benefit to locate food, demonstrating acoustic comprehension learning. Adult grey seals have complex call repertoires, and in this thesis I have shown how their calls develop with physiological growth and experience, as well as how they can gain information from perceived sound signals.

<p>Vocal learning is the ability modify vocal output based on auditory input and is the basis of human speech acquisition. It is shared by few distantly related bird and mammal orders, and is thus very likely to be an example of convergent evolution, having evolved independently in multiple lineages. This complex behavior is presumed to require networks of regulated genes to develop the necessary neural circuits for learning and maintaining vocalizations. Deciphering these networks has been limited by the lack of high throughput genomic tools in vocal learning avian species and the lack of a solid computational framework to understand the relationship between gene expression and behavior. This dissertation provides new insights into the evolution and mechanisms of vocal learning by taking a top-down, systems biology approach to understanding gene expression regulation across avian and mammalian species. First, I worked with colleagues to develop a zebra finch Agilent oligonucleotide microarray, including developing programs for more accurate annotation of oligonucleotides and genes. I then used these arrays and tools in multiple collaborative, but related projects, to measure transcriptome expression data in vocal learning and non-learning avian species, under a number of behavioral paradigms, with a focus on song production. To make sense of the avian microarray data, I compiled microarray data from other sources, including expression analyses across over 900 human brain regions generated by Allen Brain Institute. To compare these data sets, I developed and performed a variety of computational analyses including clustering, linear models, gene set enrichment analysis, motif discovery, and phylogenetic inference, providing a novel framework to study the gene regulatory networks associated with a complex behavior. Using the developed framework, we are able to better understand vocal learning at different levels: how the brain regions for vocal learning evolved and how those brain regions function during the production of learned vocalizations. At the evolutionary level, we identified genes with unique expression patterns in the brains of vocal learning birds and humans. Interesting candidates include genes related to formation of neural connections, in particular the SLIT/ROBO axon guidance pathway. This algorithm also allowed us to identify the analogous regions that are a part of vocal learning circuit across species, providing the first quantitative evidence relating the human vocal learning circuit to the avian vocal learning circuit. With the avian song system verified as a model for human speech at the molecular level, we conducted an experiment to better understand what is happening in those brain regions during singing by profiling gene expression in a time course as birds are producing song. Surprisingly, an overwhelming majority of the gene expression identified was strongly enriched in a particular region. We also found a tight coupling between the behavioral function of a particular region and the gene expression pattern. To gain insight into the mechanisms of this gene regulation, we conducted a genomic scan of transcription factor binding sites in zebra finch. Many transcription factor binding sites were enriched in the promoters of genes with a particular temporal patterns, several of which had already been hypothesized to play a role in the neural system. Using this data set of gene expression profiles and transcription factor binding sites along with separate experiments conducted in mouse, we were able uncover evidence that the transcription factor CARF plays a role in neuron homeostasis. These results have broadened our understanding of the molecular basis of vocal learning at multiple levels. Overall, this dissertation outlines a novel way of approaching the study of the relationship between genes and behavior.</p> / Dissertation

<p>The goal of my thesis is to understand the genetics of a complex behavioral trait, vocal learning, which serves as a critical substrate for human spoken language. With the available genomes of 23 mammals, I developed a novel approach based on molecular homoplasy to reveal Single Non-random Amino Acids Patterns (SNAAPs) that are associated with convergent traits, a task that proved intractable for standard approaches, e.g. dN/dS analyses. Of 73 genes I identified in mammalian vocal learners, ~25% function in neural connectivity, auditory or speech processing. Remarkably, these include a group of 6 genes from the ROBO1 axon guidance pathway. In birds, I found ROBO1 and its ligand SLIT1 show convergent differential expression in the motor output song nucleus of the three independent lineages of vocal learners but not in analogous brain areas of vocal non-learners, and ROBO1 is developmentally regulated during song learning critical periods in songbirds. In a different set of genes, I came across an unexpected discovery of the excess sharing of homoplastic substitutions in humans and domesticated species. I revealed biased nucleotide transitions (mostly favoring A/G mutation) for above amino acid substitutions and found that this rule was significantly relaxed during domestication for artificial selection. Overall, my thesis has resulted in a novel approach for studying convergent complex traits and provided critical insights into the evolution of vocal learning specifically, and complex traits generally.</p> / Dissertation

<p>Mice produce many ultrasonic vocalizations (USVs) in the 30 - 100 kHz range including pup isolation calls and adult male songs. These USVs are often used as behavioral readouts of internal states, to measure effects of social and pharmacological manipulations, and for behavioral phenotyping of mouse models for neuropsychiatric and neurodegenerative disorders; however, little is known about the biophysical and neurophysiological mechanisms of USV production in rodents. This lack of knowledge restricts the interpretation of data from vocalization-related experiments on mouse models of communication disorders and vocal medical conditions. Meanwhile, there has been increased interest in the social communication aspect of neural disorders such as autism, in addition to the common disorders involving motor control of the larynx: stroke, Parkinson's disease, laryngeal tremor, and spasmodic dysphonia. Therefore, it is timely and critical to begin assessing the neural substrate of vocal production in order to better understand the neuro-laryngeal deficits underlying communication problems.</p><p>Additionally, mouse models may generate new insight into the molecular basis of vocal learning. Traditionally, songbirds have been used as a model for speech learning in humans; however, the model is strongly limited by a lack of techniques for manipulating avian genetics. Accordingly, there has long been strong interest in finding a mammalian model for vocal learning studies. The characteristic features of accepted vocal learning species include programming of phonation by forebrain motor areas, a direct cortical projection to brainstem vocal motoneurons, and dependence on auditory feedback to develop and maintain vocalizations. Unfortunately, these features have not been observed in non-human primates or in birds that do not learn songs. Thus, in addition to elucidating vocal brain pathways it is also critical to determine the extent of any vocal learning capabilities present in the mouse USV system.</p><p>It is generally assumed that mice lack a forebrain system for vocal modification and that their USVs are innate; however, these basic assumptions have not been experimentally tested. I investigated the mouse song system to determine if male mouse song behavior and the supporting brain circuits resemble those of known vocal learning species. By visualizing activity-dependent immediate early gene expression as a marker of global activity patterns, I discovered that the song system includes motor cortex and striatal regions active during singing. Retrograde and anterograde tracing with pseudorabies virus and biodextran amines, respectively, revealed that the motor cortical region projects directly to the brainstem phonatory motor nucleus ambiguus. Chemical lesions in this region showed that it is not critical for producing innate templates of song syllables, but is required for producing more stereotyped acoustic features of syllables. To test for the basic components of adaptive learning I recorded the songs of mechanically and genetically deaf mice and found that male mice depend on auditory feedback to develop and maintain normal ultrasonic songs. Moreover, male mice that display natural strain specific song features may use auditory experience to copy the pitch of another strain when housed together and stimulated to compete sexually. I conclude that male mice have neuroanatomical and behavioral features thought to be unique to humans and song learning birds, suggesting that mice are capable of adaptive modification of the spectral features of their songs.</p> / Dissertation

Self-efficacy is theorized to represent our ability, capability, or capacity to accomplish particular tasks. One’s belief in that ability (self-efficacy belief) has been identified as the greatest predictor of successful performance and is influenced by four primary sources (enactive mastery experience, vicarious experience, verbal/social persuasion, physiological and affective states), in addition to personal and contextual influences. However, the development of accurate self-perceptions may be particularly challenging for the collegiate vocalist. In the development of singing technique—where self-assessment is complicated by the corporal nature of the vocal instrument—Bandura’s (1997) sources of self-efficacy provide a framework whereby assessment of ability and capability may become more tangible. The aim of the present study, therefore, was to investigate how collegiate vocal students’ beliefs in their vocal performance abilities may be influenced by the four self-efficacy sources and personal/contextual factors. I distributed the Vocal Performance Self-Efficacy Survey (adapted from Zelenak, 2011) to 46 voice majors at a private university in the western United States. Nine interview participants, who represented diversity of performance beliefs, were subsequently selected from the survey participant pool. Interview participants completed an initial interview based on a priori themes (four sources of self-efficacy); and a follow-up interview, which explored contextual factors (i.e., student/teacher relationship, environment, cognitive self-regulation, practice habits, and gender). In addition, participants documented three experiences—in a voice lesson, practice session, and performance—that fostered or hindered their performance belief. Vocal students in this study described how they progressed in self-belief by moving from a reliance on external assessments of ability to a reliance on self-appraisal as they (a) developed their technique through practice, studio learning, and performance (enactive mastery experience); (b) watched coping and master models (vicarious experience); (c) received feedback (verbal/social persuasion); (d) knew and felt physically when they were singing freely (physiological and affective states); and (e) learned to exercise agency (cognitive self-regulation). A particularly important finding from this study was the common and consistent reliance singers placed on physiological and affective states. Eight of nine interview participants responded that, of all the self-efficacy sources, physiological and affective states most affected their performance belief. Interview data indicate the importance of nurturing vocal students’ performance beliefs through utilizing the four sources of self-efficacy, fostering qualities of persistence and resilience, facilitating cognitive self-regulation, working toward productive student/teacher relationships, and creating safe learning and performance environments.

Aspects of the behavioral ecology of bird song learning are examined in three parts. First, an approach from image analysis is extended to allow rapid, quantitative description of animal sounds. In this approach, sounds are summarized as sets of time-frequency-amplitude landmarks. Second, the role of dawn song in tree swallow (Tachycineta bicolor) breeding biology is examined. Song syllable sharing among tree swallows was found to be high among birds nesting at the same site, but sharing was lower between birds nesting at different sites. When birds nested at different sites, the distance between those sites was not related to the amount of difference between the birds' syllable repertoire compositions. All tree swallow song repertoires did not remain constant during the breeding season; some individuals added new syllable types, others modified existing types. Singing performance was correlated with reproductive success in tree swallows: males that sang more precise repetitions of their syllable types attracted more extra-pair mates. Furthermore, pairwise comparisons between the social and genetic fathers of extra-pair young found that the genetic fathers averaged higher syllable consistency than the cuckolded males. Third, a comparative study of the phylogenetic distribution of vocal mimicry examined the evolutionary history of song learning in oscine passerines. Vocal mimicry, defined as the habitual incorporation of heterospecific sounds into song displays, was found in twenty-eight separate clades of oscines. These clades were found in every major oscine superfamily, but made up a higher proportion of daughter groups within the most ancient superfamilies of oscines. The most plesiomorphic lineages of oscines were found to contain many highly-skilled mimics. These observations support the hypothesis that the course of song learning in oscines has run repeatedly from permissive learning rules that permit mimicry to restrictive learning rules that limit mimicry.

While much work has been done on regional vocal variation in birds, relatively few studies have found evidence of similar variation in mammalian vocalizations. This study quantifies individual, colonial, and regional level variation in T. brasiliensis songs in the southeastern United States. Brazilian free-tailed bats (Tadarida brasiliensis) are among a handful of mammals that produce complex, hierarchically structured vocalizations. Their songs are composed of multiple syllables that are combined into three phrases that vary in number and order across renditions. Tadarida brasiliensis songs showed considerable amount of variation, and differed significantly between locations in terms of syllable structure and song syntax. Some of the variation observed was not correlated to geographical distance, and is unlikely to be explained by genetic divergence or differences in habitat use. These results indicate the existence of vocal dialects and a possible role of vocal production learning in dialect formation in this species.

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