Gunaratne et al. BMC Genomics 2011, 12:277 http://www.biomedcentral.com/1471-2164/12/277 The net rate of neuronal addition and loss in the adult songbird has been shown to depend on social and envir- onmental influences [48-51]. Perhaps tgu-miR-124 is a regulatory link between experience and neurogenesis - further study of this fascinating possibility is clearly warranted. Although miRNAs can have diverse functions, they often act by altering the concentrations of specific mRNAs they target via complementary base pairing. We used the TargetScan algorithm [52] to predict binding sites of tgu-miR-2954-3p in chicken genes, and then we confirmed the presence of the same conserved target sequence in the zebra finch genome assembly. We found eight targets that met these criteria and were also regulated by song in the Dong et al. microarray data [14]. These eight genes have a provocative coherence in their function, as they are all implicated in control of cell proliferation and neuronal differentiation. Six oper- ate by affecting gene expression and chromatin remodel- ing as we briefly review here. ELAVL2 is a member of a protein family that binds AU-rich regions in the 3’UTR of genes such as c-fos and promotes the shift from cell proliferation into cellular differentiation [53-57]. TLK2 is a kinase tightly associated with DNA replication dur- ing cell division [58]. At least one of its targets, the his- tone chaperone Asf1, controls chromatin assembly, thus TLK2 activity can regulate transcription and elongation [59-61]. BTG1 is also regulated during the cell cycle [62]. It acts as a cofactor for Hoxb9, a transcription fac- tor that controls cell proliferation and differentiation, and BTG1 reduces rates of cell proliferation [62-64]. CHD2 can potentially affect transcription of many genes by remodeling chromatin [65,66]; disruption of CHD2 has profound consequences for development and is implicated in many human diseases [67-69]. HMGB1 is another DNA binding protein that facilitates transcrip- tion by altering chromatin structure to ease promoter binding [70-73]. Some of the genes regulated by HMGB1 may play a role in cell proliferation and migra- tion [74,75]. Neuronal migration and neurite outgrowth are affected by CRKL, a transcriptional activator that is a component of the reelin pathway [76-79]. Unlike the other six genes, NEGR1 and LINGO2 do not seem to alter transcription but they do have established roles in neuronal differentiation. NEGR1 affects cell-cell adhe- sion to modulate neurite outgrowth and synapse forma- tion [80-82]. LINGO2 is one member of a family of transmembrane proteins that are involved in neural and axonal regeneration [83,84]. The function of LINGO2 is untested, but expression of a related protein, LINGO1, is attenuated in cortical areas deprived of sensory input and is a partner in a signaling pathway that correlates with neuronal activity during a learning paradigm [85,86]. Page 9 of 14 Conclusions In conclusion, these data reveal a network of miRNAs in the zebra finch’s auditory forebrain, responsive to the experience of hearing another bird sing. The network includes well-characterized conserved miRNA known to have roles in neuronal differentiation (miR-124), and novel miRNAs that can target genes that control neuro- nal differentiation (tgu-miR-2954-3p). Our data suggest this miRNA network may influence the fundamental shift we have observed in the transcriptional and metabolic state of the auditory forebrain during the process of song-specific habituation [14,87]. Further study of song responses in the zebra finch may reveal general insights into the neurogenomic mechanisms that underlie learn- ing, memory and the ongoing adaptation to experience. Methods Song stimulation and brain dissections Zebra finches were obtained from aviaries maintained at the University of Illinois. All procedures involving ani- mals were conducted with the approval of the University of Illinois Institutional Animal Care and Use Commit- tee. The birds were raised in a standard breeding aviary and were tutored under normal social conditions (i.e., by their parents or other adult birds in the breeding col- ony). All birds used in this study were adults (older than 90 days after hatching). The song playback procedures and brain dissections were performed exactly as in pre- vious microarray analyses, using the same equipment [14,88]. Briefly, each bird was put individually into a sound isolation chamber for 18 hours on the first day, and on the second day those in the song group heard 30 minutes of a song not heard previously ("novel song”). Matched controls collected in parallel heard no song playback ("silence”). Birds were sacrificed in song- silence pairs, so that 5 minutes before the end of the song playback to one bird, a bird in the silence group was sacrificed and its auditory forebrain was dissected and frozen in dry ice. Then the auditory forebrain of the song-stimulated bird was dissected and frozen in dry ice. The auditory forebrain dissection (also referred to as auditory lobule) is described in [89] and collects NCM (caudomedial nidopallium), CMM (caudomedial mesopallium) and the enclosed Field L subregions. At the end of the song stimulation procedure, all auditory forebrains were transferred and stored at -80C until RNA isolation. For the comparison of responses after overnight isolation to song versus SEN (Additional File 2, Figure S3), we used two matched stimuli derived from bird “C7” as previously described [34]. RNA Samples For Illumina analyses: Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) from three