A molecular neuroethological approach for identifying and characterizing a melody of behaviorally regulated genes

Songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior. However, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. To overcome this limitation, here we generated a resource of full-length cDNAs for gene expression analyses and gene manipulation in songbirds. We constructed 21 regular, normalized, and subtracted full-length cDNA libraries from brains of songbirds in 57 behavioral and developmental conditions in an attempt to clone as much of the brain transcriptome as possible. From these libraries ~14,000 transcripts were isolated, representing an estimated 4,738 genes, or ~40% of the genes expressed in the brain. With these cDNAs, we created a novel draft transcriptome database and large scale songbird brain cDNA microarrays. We used the arrays to reveal a set of 33 genes regulated in brain vocal nuclei by singing behavior. These genes clustered into 4 anatomical and 6 temporal brain expression patterns. Their functions spanned broad range of cellular and molecular categories, including signal transduction, trafficking, structural, and synaptically released molecules. With the full-length cDNAs, we over-expressed proteins of representative singing-regulated genes in vocal nuclei in the absence of singing, using a lentiviral vector system. This resource now allows investigators to comprehensively study molecular neuroethological mechanisms of behavior. Keywords: songbird, zebra finch, transcriptome, learned vocalization, immediate early genes, lentivirus For the HVC there are 3 replicates of a dual Cy-dye experiement; samples GSM103451 and GSM103453 are dye swaped technical replicates. Sample GSM103740 is a biological replicate. For the RA there are 4 replicates of a dual Cy-dye experiement; samples GSM103741 and GSM103742 are dye swaped technical replicates. Samples GSM103743 and GSM103744 are biological replicates. For the Area X there are 4 replicates of a dual Cy-dye experiement; samples GSM103748 and GSM103750 are technical replicates. Samples GSM103749 and GSM103751 are biological replicates. For the lateral MAN there are 3 replicates of a dual Cy-dye experiement; samples GSM103745 and GSM103746 are technical replicates. Sample GSM103747 is a biological replicate. HVC, RA, Area X or MAN cDNA from singing and quiet zebra finches was labeled with either Cy5 or Cy3 and hybridized to the array.