Nicotinic acetylcholine receptors in a songbird brain.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission and cell signaling, which contribute to learning, memory, and the execution of motor skills. Birdsong is a complex learned motor skill in songbirds. Although the existence of 15 nAChR subunits has been predicted in the avian genome, their expression patterns and potential contributions to song learning and production have not been comprehensively investigated. Here, we cloned all the 15 nAChR subunits (ChrnA1-10, B2-4, D, and G) from the zebra finch brain and investigated the mRNA expression patterns in the neural pathways responsible for the learning and production of birdsong during a critical period of song learning. Although there were no detectable hybridization signals for ChrnA1, A6, A9, and A10, the other 11 nAChR subunits were uniquely expressed in one or more major subdivisions in the song nuclei of the songbird brain. Of these 11 subunits, ChrnA3-5, A7, and B2 were differentially regulated in the song nuclei compared with the surrounding anatomically related regions. ChrnA5 was upregulated during the critical period of song learning in the lateral magnocellular nucleus of the anterior nidopallium. Furthermore, single-cell RNA sequencing revealed ChrnA7 and B2 to be the major subunits expressed in neurons of the vocal motor nuclei HVC and robust nucleus of the arcopallium, indicating the potential existence of ChrnA7-homomeric and ChrnB2-heteromeric nAChRs in limited cell populations. These results suggest that relatively limited types of nAChR subunits provide functional contributions to song learning and production in songbirds.

Seasonal regulation of singing-driven gene expression associated with song plasticity in the canary, an open-ended vocal learner.

Songbirds are one of the few animal taxa that possess vocal learning abilities. Different species of songbirds exhibit species-specific learning programs during song acquisition. Songbirds with open-ended vocal learning capacity, such as the canary, modify their songs during adulthood. Nevertheless, the neural molecular mechanisms underlying open-ended vocal learning are not fully understood. We investigated the singing-driven expression of neural activity-dependent genes (Arc, Egr1, c-fos, Nr4a1, Sik1, Dusp6, and Gadd45ß) in the canary to examine a potential relationship between the gene expression level and the degree of seasonal vocal plasticity at different ages. The expression of these genes was differently regulated throughout the critical period of vocal learning in the zebra finch, a closed-ended song learner. In the canary, the neural activity-dependent genes were induced by singing in the song nuclei throughout the year. However, in the vocal motor nucleus, the robust nucleus of the arcopallium (RA), all genes were regulated with a higher induction rate by singing in the fall than in the spring. The singing-driven expression of these genes showed a similar induction rate in the fall between the first year juvenile and the second year adult canaries, suggesting a seasonal, not age-dependent, regulation of the neural activity-dependent genes. By measuring seasonal vocal plasticity and singing-driven gene expression, we found that in RA, the induction intensity of the neural activity-dependent genes was correlated with the state of vocal plasticity. These results demonstrate a correlation between vocal plasticity and the singing-driven expression of neural activity-dependent genes in RA through song development, regardless of whether a songbird species possesses an open- or closed-ended vocal learning capacity.