Abstract
Here we provide a detailed step-by-step protocol for using lentivirus to manipulate miRNA expression in Area X of juvenile zebra finches and for analyzing the consequences on song learning and song performance. This protocol has four parts: 1) making the lentiviral construct to overexpress miRNA miR-9; 2) packaging the lentiviral vector; 3) stereotaxic injection of the lentivirus into Area X of juvenile zebra finches; 4) analysis of song learning and song performance in juvenile and adult zebra finches. These methods complement the methods employed in recent works that showed changing FoxP2 gene expression in Area X with lentivirus or adeno-associated virus leads to impairments in song behavior.
Highlights
[Background] The zebra finch, with its well-characterized song behavior and the underlying neural circuitry, provides a unique animal model to study neural mechanisms underlying vocal communication and related sensory-motor learning
We recently reported that overexpression of miRNA miR-9 in Area X of juvenile zebra finches impairs song learning and performance (Shi et al, 2018)
Hoping others might benefit from this study, here we provide step-by-step protocols for lentivirus cloning and production, stereotaxic injection of the virus into Area X of juveniles, and analysis of the impact of miR-9 overexpression on song learning and performance using the software Sound Analysis Pro (Tchernichovski et al, 2000)
Summary
[Background] The zebra finch, with its well-characterized song behavior and the underlying neural circuitry, provides a unique animal model to study neural mechanisms underlying vocal communication and related sensory-motor learning. Hoping others might benefit from this study, here we provide step-by-step protocols for lentivirus cloning and production, stereotaxic injection of the virus into Area X of juveniles, and analysis of the impact of miR-9 overexpression on song learning and performance using the software Sound Analysis Pro (Tchernichovski et al, 2000). 10 cm cell culture plates (Corning, catalog number: 430167) 3. 24-well cell culture plates (Corning, Costar®, catalog number: 3524) 4.
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