Impact of experience-dependent and -independent factors on gene expression in songbird brain.

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.

Discrete molecular states in the brain accompany changing responses to a vocal signal.

New experiences can trigger changes in gene expression in the brain. To understand this phenomenon better, we studied zebra finches hearing playbacks of birdsong. Earlier research had shown that initial playbacks of a novel song transiently increase the ZENK (ZIF-268, EGR1, NGFIA, KROX-24) mRNA in the auditory forebrain, but the response selectively habituates after repetition of the stimulus. Here, using DNA microarray analysis, we show that novel song exposure induces rapid changes in thousands of RNAs, with even more RNAs decreasing than increasing. Habituation training leads to the emergence of a different gene expression profile a day later, accompanied by loss of essentially all of the rapid "novel" molecular responses. The novel molecular profile is characterized by increases in genes involved in transcription and RNA processing and decreases in ion channels and putative noncoding RNAs. The "habituated" profile is dominated by changes in genes for mitochondrial proteins. A parallel proteomic analysis [2-dimensional difference gel electrophoresis (2D-DIGE) and sequencing by mass spectrometry] also detected changes in mitochondrial proteins, and direct enzyme assay demonstrated changes in both complexes I and IV in the habituated state. Thus a natural experience, in this case hearing the sound of birdsong, can lead to major shifts in energetics and macromolecular metabolism in higher centers in the brain.

Birdsong "transcriptomics": neurochemical specializations of the oscine song system.

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.