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Songbirds communicate through learned vocalizations, using a forebrain circuit with convergent similarity to vocal-control circuitry in humans. This circuit is incomplete in female zebra finches, hence only males sing. We show that the UTS2B gene, encoding Urotensin-Related Peptide (URP), is uniquely expressed in a key pre-motor vocal nucleus (HVC), and specifically marks the neurons that form a male-specific projection that encodes timing features of learned song. UTS2B-expressing cells appear early in males, prior to projection formation, but are not observed in the female nucleus. We find no expression evidence for canonical receptors within the vocal circuit, suggesting either signalling to other brain regions via diffusion or transduction through other receptor systems. Urotensins have not previously been implicated in vocal control, but we find an annotation in Allen Human Brain Atlas of increased UTS2B expression within portions of human inferior frontal cortex implicated in human speech and singing. Thus UTS2B (URP) is a novel neural marker that may have conserved functions for vocal communication.

Songbirds are important models for the study of social behaviour and communication. To complement the recent genome sequencing of the domesticated zebra finch, we sequenced the brain transcriptome of a closely related songbird species, the violet-eared waxbill (Uraeginthus granatina). Both the zebra finch and violet-eared waxbill are members of the family Estrildidae, but differ markedly in their social behaviour. Using Roche 454 RNA sequencing, we generated an assembly and annotation of 11 084 waxbill orthologues of 17 475 zebra finch genes (64%), with an average transcript length of 1555 bp. We also identified 5985 single nucleotide polymorphisms (SNPs) of potential utility for future population genomic studies. Comparing the two species, we found evidence for rapid protein evolution (ω) and low polymorphism of the avian Z sex chromosome, consistent with prior studies of more divergent avian species. An intriguing outlier was putative chromosome 4A, which showed a high density of SNPs and low evolutionary rate relative to other chromosomes. Genome-wide ω was identical in zebra finch and violet-eared waxbill lineages, suggesting a similar demographic history with efficient purifying natural selection. Further comparisons of these and other estrildid finches may provide insights into the evolutionary neurogenomics of social behaviour.

Songbirds have unique value as a model for memory and learning. In their natural social life, they communicate through vocalizations that they must learn to produce and recognize. Song communication elicits abrupt changes in gene expression in regions of the forebrain responsible for song perception and production--what is the functional significance of this genomic response? For 20 years, the focus of research was on just a few genes [primarily ZENK, now known as egr1 (early gene response 1)]. Recently, however, DNA microarrays have been developed and applied to songbird behavioral research, and in 2010 the initial draft assembly of the zebra finch genome was published. Together, these new data reveal that the genomic involvement in song processing is far more complex than anticipated. The concepts of neurogenomic computation and biological embedding are introduced as frameworks for future research.

Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical "constitutive plasticity" (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as "ribosome" (expressed more highly in juvenile brain) and "dopamine metabolic process" (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience.

Songbirds share some essential traits but are extraordinarily diverse, allowing comparative analyses aimed at identifying specific genotype-phenotype associations. This diversity encompasses traits like vocal communication and complex social behaviors that are of great interest to humans, but that are not well represented in other accessible research organisms. Many songbirds are readily observable in nature and thus afford unique insight into the links between environment and organism. The distinctive organization of the songbird brain will facilitate analysis of genomic links to brain and behavior. Access to the zebra finch genome sequence will, therefore, prompt new questions and provide the ability to answer those questions.

Background: Songbirds hold great promise for biomedical, environmental and evolutionary research. A complete draft sequence of the zebra finch genome is imminent, yet a need remains for application of genomic resources within a research community traditionally focused on ethology and neurobiological methods. In response, we developed a core set of genomic tools and a novel collaborative strategy to probe gene expression in diverse songbird species and natural contexts., Results: We end-sequenced cDNAs from zebra finch brain and incorporated additional sequences from community sources into a database of 86,784 high quality reads. These assembled into 31,658 non-redundant contigs and singletons, which we annotated via BLAST search of chicken and human databases. The results are publicly available in the ESTIMA:Songbird database. We produced a spotted cDNA microarray with 20,160 addresses representing 17,214 non-redundant products of an estimated 11,500-15,000 genes, validating it by analysis of immediate-early gene (zenk) gene activation following song exposure and by demonstrating effective cross hybridization to genomic DNAs of other songbird species in the Passerida Parvorder. Our assembly was also used in the design of the "Lund-zfa" Affymetrix array representing approximately 22,000 non-redundant sequences. When the two arrays were hybridized to cDNAs from the same set of male and female zebra finch brain samples, both arrays detected a common set of regulated transcripts with a Pearson correlation coefficient of 0.895. To stimulate use of these resources by the songbird research community and to maintain consistent technical standards, we devised a "Community Collaboration" mechanism whereby individual birdsong researchers develop experiments and provide tissues, but a single individual in the community is responsible for all RNA extractions, labelling and microarray hybridizations., Conclusion: Immediately, these results set the foundation for a coordinated set of 25 planned experiments by 16 research groups probing fundamental links between genome, brain, evolution and behavior in songbirds. Energetic application of genomic resources to research using songbirds should help illuminate how complex neural and behavioral traits emerge and evolve.

Background: Vocal learning is a rare and complex behavioral trait that serves as a basis for the acquisition of human spoken language. In songbirds, vocal learning and production depend on a set of specialized brain nuclei known as the song system., Methodology/principal Findings: Using high-throughput functional genomics we have identified approximately 200 novel molecular markers of adult zebra finch HVC, a key node of the song system. These markers clearly differentiate HVC from the general pallial region to which HVC belongs, and thus represent molecular specializations of this song nucleus. Bioinformatics analysis reveals that several major neuronal cell functions and specific biochemical pathways are the targets of transcriptional regulation in HVC, including: 1) cell-cell and cell-substrate interactions (e.g., cadherin/catenin-mediated adherens junctions, collagen-mediated focal adhesions, and semaphorin-neuropilin/plexin axon guidance pathways); 2) cell excitability (e.g., potassium channel subfamilies, cholinergic and serotonergic receptors, neuropeptides and neuropeptide receptors); 3) signal transduction (e.g., calcium regulatory proteins, regulators of G-protein-related signaling); 4) cell proliferation/death, migration and differentiation (e.g., TGF-beta/BMP and p53 pathways); and 5) regulation of gene expression (candidate retinoid and steroid targets, modulators of chromatin/nucleolar organization). The overall direction of regulation suggest that processes related to cell stability are enhanced, whereas proliferation, growth and plasticity are largely suppressed in adult HVC, consistent with the observation that song in this songbird species is mostly stable in adulthood., Conclusions/significance: Our study represents one of the most comprehensive molecular genetic characterizations of a brain nucleus involved in a complex learned behavior in a vertebrate. The data indicate numerous targets for pharmacological and genetic manipulations of the song system, and provide novel insights into mechanisms that might play a role in the regulation of song behavior and/or vocal learning.

Zebra finches show a multifaceted response to playback of tape-recorded birdsong. A novel song induces an overt listening behavior, accompanied by a distinct electrophysiological signature and a wave of gene expression in the auditory telencephalon. With repetition of the same song these responses all habituate; habituation can persist for a day or more and is specific for the repeated song. This habituation is a form of selective memory. Here we describe four experiments to probe the nature of the information stored in that memory, using habituation of the genomic response as a physiological assay for stimulus recognition. Adult male zebra finches (awake and unrestrained) were exposed to repetitions of a test song to habituate the genomic response. The same song was then presented in one of four different contexts: (1) from a speaker on the opposite side of the cage; (2) at a reduced sound pressure level; (3) paired with constant illumination of colored lights next to the speaker; (4) paired with colored lights that were turned on and off in synchrony with each song bout. In all cases except the third, a "familiar" (habituated) song re-induced a new wave of gene expression in the auditory telencephalon when presented in the new context. These results reveal that memory for a specific song, as indicated by the initial gene habituation, incorporates more than just an acoustic description of the song. We suggest that habituation in the auditory telencephalon is controlled in part by an extrinsic system that allows detection of synchronous activity in different sensory or representational modalities.

The sound of tape-recorded birdsong triggers a set of behavioral and physiological responses in zebra finches, including transcriptional activation of the zenk gene in the auditory forebrain. Song repetition leads to the stimulus-specific habituation of these responses. To gain insight into the mechanisms that couple auditory experience to gene regulation, we monitored the phosphorylation of the zebra finch extracellular signal-regulated kinase (ERK) protein by immunoblotting. Initial presentations of novel song (but not tones or noise) resulted in a rapid increase in ERK phosphorylation, followed by a return to basal levels within 5 min. This response was localized to the auditory forebrain where the zenk gene is activated. Sustained repetition of one song caused a selective habituation of the ERK response: a different song triggered another cycle of ERK phosphorylation without altering the habituated response to the first. To test directly for a role of ERK in experience-dependent zenk gene regulation, we infused an inhibitor of mitogen-activated and extracellular-regulated protein kinase kinase (MEK-1; the enzyme responsible for ERK activation) unilaterally into one auditory lobule just before song stimulation. The song-induced increase in zenk mRNA was blocked on the side of the injection, but not on the contralateral (uninfused) side. These results show that ERK phosphorylation is necessary for the initiation of the zenk gene response to novel song and identify ERK as a plausible site of signal integration underlying the selective habituation of genomic responses to a repeated song.

The biology of songbirds poses fundamental questions about the interplay between gene, brain, and behavior. New tools of genomic analysis will be invaluable in pursuing answers to these questions. This review begins with a summary of the broad properties of the songbird genome and how songbird brain gene expression has been measured in past studies. Four key problems in songbird biology are then considered from a genomics perspective: What role does differential gene expression play in the development, maintenance, and functional organization of the song control circuit? Does gene regulation set boundaries on the process of juvenile song learning? What is the purpose of song-induced gene activity in the adult brain? How does the genome underlie the profound sexual differentiation of the song control circuit? Finally, the range of genomic technologies currently or soon to be available to songbird researchers is briefly reviewed. These technologies include online databases of expressed genes ("expressed sequence tags" or ESTs); a complete library of the zebra finch genome maintained as a bacterial artificial chromosome (BAC) library; DNA microarrays for simultaneous measurement of many genes in a single experiment; and techniques for gene manipulation in the organism. Collectively, these questions and techniques define the field of songbird neurogenomics.

Zebra finches respond to certain auditory stimuli with the activation of the immediate early gene zenk. It has been shown that the amount of sound-mediated zenk gene expression varies in the zebra finch caudomedial neostriatum (NCM), apparently correlated with stimulus type (conspecific>heterospecific>noise>tones) and familiarity. Here we tested the impact of two additional factors-song-specific acoustical properties and testing conditions-on the specificity of the sound-mediated zenk response, as assessed by in situ hybridization. A variant of a normal conspecific song was first produced by randomizing the spectral content while retaining the amplitude envelope ('song-enveloped noise'). This stimulus and related controls were presented to birds which were either free in cages or restrained in a stereotaxic instrument, after isolation either overnight or for only 1 h prior to testing. We confirmed prior results that unrestrained birds show a greater zenk response to normal conspecific song than to other acoustic stimuli. However, under restraint, birds showed little or no selectivity for conspecific song compared to matched stimuli lacking a song organization. Thus the specificity of the zenk response to song is not determined simply by the acoustic structure and familiarity of the stimulus. We conclude that the intrinsic selectivity of sensory responses measured in the CNS may be influenced by factors associated with attention, arousal or vigilance, and may be significantly altered by experimental conditions that involve physical restraint.

Our understanding of bird song, a model system for animal communication and the neurobiology of learning, depends critically on making reliable, validated comparisons between the complex multidimensional syllables that are used in songs. However, most assessments of song similarity are based on human inspection of spectrograms, or computational methods developed from human intuitions. Using a novel automated operant conditioning system, we collected a large corpus of zebra finches' (Taeniopygia guttata) decisions about song syllable similarity. We use this dataset to compare and externally validate similarity algorithms in widely-used publicly available software (Raven, Sound Analysis Pro, Luscinia). Although these methods all perform better than chance, they do not closely emulate the avian assessments. We then introduce a novel deep learning method that can produce perceptual similarity judgements trained on such avian decisions. We find that this new method outperforms the established methods in accuracy and more closely approaches the avian assessments. Inconsistent (hence ambiguous) decisions are a common occurrence in animal behavioural data; we show that a modification of the deep learning training that accommodates these leads to the strongest performance. We argue this approach is the best way to validate methods to compare song similarity, that our dataset can be used to validate novel methods, and that the general approach can easily be extended to other species. Author summary: How do birds hear the differences between their songs? This fascinating question carries implications, since the study of bird song, a model system for the neurobiology of learning and animal communication, depends critically on our ability to assess the similarity of songs. Traditionally, researchers compare sounds by human assessment, or use computational methods based on human intuitions about similarity. However, neither approach is connected to birds' own perception of sound similarity. Here, using a novel automated operant conditioning system, we recorded many thousands of acoustic judgments of similarity from zebra finches, and used this perceptual decision data for the first time to train a deep learning system. The trained system outperforms other computational methods for the task of making the same judgments as birds. This algorithm to compare song similarity, together with the potential of extending the general approach to other species, places the study of bird song on a firmer footing. [ABSTRACT FROM AUTHOR]

Prolonged social isolation has negative effects on brain and behavior in humans and other social organisms, but neural mechanisms leading to these effects are not understood. Here we tested the hypothesis that even brief periods of social isolation can alter gene expression and DNA methylation in higher cognitive centers of the brain, focusing on the auditory/associative forebrain of the highly social zebra finch. Using RNA sequencing, we first identified genes that individually increase or decrease expression after isolation and observed general repression of gene sets annotated for neurotrophin pathways and axonal guidance functions. We then pursued 4 genes of large effect size: EGR1 and BDNF (decreased by isolation) and FKBP5 and UTS2B (increased). By in situ hybridization, each gene responded in different cell subsets, arguing against a single cellular mechanism. To test whether effects were specific to the social component of the isolation experience, we compared gene expression in birds isolated either alone or with a single familiar partner. Partner inclusion ameliorated the effect of solo isolation on EGR1 and BDNF, but not on FKBP5 and UTS2B nor on circulating corticosterone. By bisulfite sequencing analysis of auditory forebrain DNA, isolation caused changes in methylation of a subset of differentially expressed genes, including BDNF. Thus, social isolation has rapid consequences on gene activity in a higher integrative center of the brain, triggering epigenetic mechanisms that may influence processing of ongoing experience. [ABSTRACT FROM AUTHOR]

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken—the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour. [ABSTRACT FROM AUTHOR]

Abstract: Songbirds respond to initial playback of a recorded conspecific song in numerous ways, from changes in gene expression in the brain to changes in overt physical activity. When the same song is presented repeatedly, responses have been observed to habituate at multiple levels: molecular, cellular and organismal. Core criteria of habituation have been established at each level, although in no case have all the formal parameters been rigorously measured. At the level of overt behavior, classical field studies showed that territorial birds respond to the song of a potential challenger with a variety of behaviors, and many (but not all) of these behaviors decline with repeated stimulus presentation. More recent laboratory studies have defined analogous responses to song presentation in the zebra finch (Taeniopygia guttata), the dominant species in current molecular and neurobiological research and one that does not use song for territorial defense. Studies in the zebra finch have also demonstrated activation followed by habituation of responses measured at both electrophysiological and molecular (gene expression and signal transduction) levels. In all cases, habituation is specific for a very particular stimulus – an individual song presented in a particular context. There are strong correlations between habituation measurements made at these different levels, but some dissociations have also been observed, implying that molecular, electrophysiological and behavioral habituations are not equivalent manifestations of a single core process. [Copyright &y& Elsevier]