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Background: Production of contextually appropriate social behaviors involves integrated activity across many brain regions. Many songbird species produce complex vocalizations called 'songs' that serve to attract potential mates, defend territories, and/or maintain flock cohesion. There are a series of discrete interconnect brain regions that are essential for the successful production of song. The probability and intensity of singing behavior is influenced by the reproductive state. The objectives of this study were to examine the broad changes in gene expression in brain regions that control song production with a brain region that governs the reproductive state., Results: We show using microarray cDNA analysis that two discrete brain systems that are both involved in governing singing behavior show markedly different gene expression profiles. We found that cortical and basal ganglia-like brain regions that control the socio-motor production of song in birds exhibit a categorical switch in gene expression that was dependent on their reproductive state. This pattern is in stark contrast to the pattern of expression observed in a hypothalamic brain region that governs the neuroendocrine control of reproduction. Subsequent gene ontology analysis revealed marked variation in the functional categories of active genes dependent on reproductive state and anatomical localization. HVC, one cortical-like structure, displayed significant gene expression changes associated with microtubule and neurofilament cytoskeleton organization, MAP kinase activity, and steroid hormone receptor complex activity. The transitions observed in the preoptic area, a nucleus that governs the motivation to engage in singing, exhibited variation in functional categories that included thyroid hormone receptor activity, epigenetic and angiogenetic processes., Conclusions: These findings highlight the importance of considering the temporal patterns of gene expression across several brain regions when engaging in social behaviors.

A male zebra finch begins to learn to sing by memorizing a tutor's song during a sensitive period in juvenile development. Tutor song memorization requires molecular signaling within the auditory forebrain. Using microarray and in situ hybridizations, we tested whether the auditory forebrain at an age just before tutoring expresses a different set of genes compared with later life after song learning has ceased. Microarray analysis revealed differences in expression of thousands of genes in the male auditory forebrain at posthatch day 20 (P20) compared with adulthood. Furthermore, song playbacks had essentially no impact on gene expression in P20 auditory forebrain, but altered expression of hundreds of genes in adults. Most genes that were song-responsive in adults were expressed at constitutively high levels at P20. Using in situ hybridization with a representative sample of 44 probes, we confirmed these effects and found that birds at P20 and P45 were similar in their gene expression patterns. Additionally, eight of the probes showed male-female differences in expression. We conclude that the developing auditory forebrain is in a very different molecular state from the adult, despite its relatively mature gross morphology and electrophysiological responsiveness to song stimuli. Developmental gene expression changes may contribute to fine-tuning of cellular and molecular properties necessary for song learning.

Background: Recent evidence suggests that some sex differences in brain and behavior might result from direct genetic effects, and not solely the result of the organizational effects of steroid hormones. The present study examined the potential role for sex-biased gene expression during development of sexually dimorphic singing behavior and associated song nuclei in juvenile zebra finches., Results: A microarray screen revealed more than 2400 putative genes (with a false discovery rate less than 0.05) exhibiting sex differences in the telencephalon of developing zebra finches. Increased expression in males was confirmed in 12 of 20 by qPCR using cDNA from the whole telencephalon; all of these appeared to be located on the Z sex chromosome. Six of the genes also showed increased expression in one or more of the song control nuclei of males at post-hatching day 25. Although the function of half of the genes is presently unknown, we have identified three as: 17-beta-hydroxysteroid dehydrogenase type IV, methylcrotonyl-CoA carboxylase, and sorting nexin 2., Conclusion: The data suggest potential influences of these genes in song learning and/or masculinization of song system morphology, both of which are occurring at this developmental stage.

Studies over roughly the last decade have emphasized the importance of gene expression in the development of structure and function of the songbird forebrain. However, few tools have been available to efficiently identify the critical factors. To that end, we have produced a normalized cDNA library from juvenile zebra finch telencephalon, and have spotted inserts from 2400 randomly selected cDNA clones on microarrays (1664 unique sequences). We have also added several previously cloned cDNAs of interest, including three representing genes encoded on sex chromosomes. Hybridizations comparing Cy3- and Cy5-labeled cDNA from the telencephalon of day 25 male and female zebra finches confirmed sexually dimorphic expression of the Z- and W-linked genes, demonstrating the utility of these microarrays for detecting differential expression and providing information about the relative expression of these genes in the brains of juveniles of this age.

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]

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]

Highlights: [•] Genome and transcriptome data are rapidly becoming available in avian species. [•] Genomics can transform research by allowing study of large sets of genes at once instead one gene at a time. [•] Environmental and behavioral signals trigger massive shifts in gene expression in the brain. [•] Comparative approaches may help uncover the genetic roots of behavioral variation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log2 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity. [ABSTRACT FROM AUTHOR]

Background: Male song sparrows (Melospiza melodia) are territorial year-round; however, neuroendocrine responses to simulated territorial intrusion (STI) differ between breeding (spring) and non-breeding seasons (autumn). In spring, exposure to STI leads to increases in luteinizing hormone and testosterone, but not in autumn. These observations suggest that there are fundamental differences in the mechanisms driving neuroendocrine responses to STI between seasons. Microarrays, spotted with EST cDNA clones of zebra finch, were used to explore gene expression profiles in the hypothalamus after territorial aggression in two different seasons. Methodology/Principal Findings: Free-living territorial male song sparrows were exposed to either conspecific or heterospecific (control) males in an STI in spring and autumn. Behavioral data were recorded, whole hypothalami were collected, and microarray hybridizations were performed. Quantitative PCR was performed for validation. Our results show 262 cDNAs were differentially expressed between spring and autumn in the control birds. There were 173 cDNAs significantly affected by STI in autumn; however, only 67 were significantly affected by STI in spring. There were 88 cDNAs that showed significant interactions in both season and STI. Conclusions/Significance: Results suggest that STI drives differential genomic responses in the hypothalamus in the spring vs. autumn. The number of cDNAs differentially expressed in relation to season was greater than in relation to social interactions, suggesting major underlying seasonal effects in the hypothalamus which may determine the differential response upon social interaction. Functional pathway analyses implicated genes that regulate thyroid hormone action and neuroplasticity as targets of this neuroendocrine regulation. [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]

The microarray technology has revolutionized biological research in the last decade. By monitoring the expression of many genes simultaneously, microarrays can elucidate gene function, as well as scan entire genomes for candidate genes encoding complex traits. However, because of high costs of sequencing and design, microarrays have largely been restricted to a few model species. Cross-species microarray (CSM) analyses, where microarrays are used for other species than the one they were designed for, have had varied success. We have conducted a CSM analysis by hybridizing genomic DNA from the common whitethroat ( Sylvia communis) on a newly developed Affymetrix array designed for the zebra finch ( Taeniopygia guttata), the Lund-zf array. The results indicate a very high potential for the zebra finch array to act as a CSM utility in other passerine birds. When hybridizing zebra finch genomic DNA, 98% of the gene representatives had higher signal intensities than the background cut-off, and for the common whitethroat, we found the equivalent proportion to be as high as 96%. This was surprising given the fact that finches and warblers diverged 25–50 million years ago, but may be explained by a relatively low sequence divergence between passerines (89–93%). Passerine birds are widely used in studies of ecology and evolution, and a zebra finch array that can be used for many species may have a large impact on future research directions. [ABSTRACT FROM AUTHOR]

Background: In animals with heteromorphic sex chromosomes, dosage compensation of sex-chromosome genes is thought to be critical for species survival. Diverse molecular mechanisms have evolved to effectively balance the expressed dose of X-linked genes between XX and XY animals, and to balance expression of X and autosomal genes. Dosage compensation is not understood in birds, in which females (ZW) and males (ZZ) differ in the number of Z chromosomes. Results: Using microarray analysis, we compared the male:female ratio of expression of sets of Z-linked and autosomal genes in two bird species, zebra finch and chicken, and in two mammalian species, mouse and human. Male:female ratios of expression were significantly higher for Z genes than for autosomal genes in several finch and chicken tissues. In contrast, in mouse and human the male : female ratio of expression of X-linked genes is quite similar to that of autosomal genes, indicating effective dosage compensation even in humans, in which a significant percentage of genes escape X-inactivation. Conclusions: Birds represent an unprecedented case in which genes on one sex chromosome are expressed on average at constitutively higher levels in one sex compared with the other. Sex-chromosome dosage compensation is surprisingly ineffective in birds, suggesting that some genomes can do without effective sex-specific sex-chromosome dosage compensation mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Abstract: The derivation of stably cultured cell lines has been critical to the advance of molecular biology. We profiled gene expression in the first two generally available cell lines derived from the zebra finch. Using Illumina RNA-seq, we generated ~93 million reads and mapped the majority to the recently assembled zebra finch genome. Expression of most Ensembl-annotated genes was detected, but over half of the mapped reads aligned outside annotated genes. The male-derived G266 line expressed Z-linked genes at a higher level than did the female-derived ZFTMA line, indicating persistence in culture of the distinctive lack of avian sex chromosome dosage compensation. Although these cell lines were not derived from neural tissue, many neurobiologically relevant genes were expressed, although typically at lower levels than in a reference sample from auditory forebrain. These cell lines recapitulate fundamental songbird biology and will be useful for future studies of songbird gene regulation and function. [Copyright &y& Elsevier]

We compared global patterns of gene expression between two bird species, the chicken and zebra finch, with regard to sex bias of autosomal versus Z chromosome genes, dosage compensation, and evolution of sex bias. Both species appear to lack a Z chromosome-wide mechanism of dosage compensation, because both have a similar pattern of significantly higher expression of Z genes in males relative to females. Unlike the chicken Z chromosome, which has female-specific expression of the noncoding RNA MHM (male hypermethylated) and acetylation of histone 4 lysine 16 (H4K16) near MHM, the zebra finch Z chromosome appears to lack the MHM sequence and acetylation of H4K16. The zebra finch also does not show the reduced male-to-female (M:F) ratio of gene expression near MHM similar to that found in the chicken. Although the M:F ratios of Z chromosome gene expression are similar across tissues and ages within each species, they differ between the two species. Z genes showing the greatest species difference in M:F ratio were concentrated near the MHM region of the chicken Z chromosome. This study shows that the zebra finch differs from the chicken because it lacks a specialized region of greater dosage compensation along the Z chromosome, and shows other differences in sex bias. These patterns suggest that different avian taxa may have evolved specific compensatory mechanisms. [ABSTRACT FROM AUTHOR]