Dynamic cortical auditory-motor neuronal projections regulate developmental song learning in zebra finches

Similar to how human infants learn to speak, juvenile zebra finches learn to sing by memorizing and then matching their vocalizations to their tutor’s song (TS）during the developmental critical period. We previously identified a neuronal substrate of tutor song memories in the avian analog of secondary auditory cortex, the caudomedial nidopallium (NCM) (Yanagihara and Yazaki-Sugiyama, 2016). Recently, we found a transient projection into the song premotor area, HVC, from the NCM neurons which were specifically activated by hearing TS and by using activity-dependent viral expression of EGFP (AAV9-cFos-TetON-EGFP). Ablation of those neurons with another AAV (AAV9-cFos-TetON-CaCaspase) prevented juveniles within the song critical period, but not the ones after the critical period, from song learning, suggesting a potential role of HVC-projecting NCM neurons in song learning (Louder et al., in submission). Here, we investigated the developmental timeline of NCM-HVC projection and disconnection. We found significantly lower densities of projections within HVC in juveniles which were in the end of sensorimotor learning (80- and 90-days post hatch (DPH)) than those in sensorimotor learning (70 DPH). We also found that the timing of decrease in NCM-HVC projections coincided with song crystallization. We further examined the axonal dynamics of NCM projections within HVC during song learning period with longitudinal structural two-photon in vivo imaging.Taken together, our results suggest that transient auditory-motor neuronal projections timely regulate memory-guided song learning during development.