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.
Keywords: Zebra finch, Area X, miR-9, Lentivirus, Song learning, Song performance
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. In recent years, several laboratories began using viral vectors to manipulate gene expression in the zebra finch brain and to study the functional consequences. These efforts are best illustrated by studies of the FoxP2 gene, which encodes the forkhead box p2 transcription factor. The FoxP2 protein controls the expression of hundreds of downstream genes that have important roles in nervous system development. Mutations in the human FoxP2 gene cause speech and language impairments (Lai et al., 2001). In songbirds, knockdown or overexpression of the FoxP2 gene in Area X of zebra finches, a basal ganglia nucleus critical for vocal learning, profoundly impairs song behavior (Haesler et al., 2007; Murugan et al., 2013; Heston and White, 2015). These studies significantly extended the usage of the zebra finch model to study gene functions in neural circuit development, vocal communication behavior, as well as in speech and language-related neural developmental disorders. 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). With minor modifications, these methods can be tailored to study other miRNAs or genes in vocal learning and performance in songbirds.
Materials and Reagents
Equipment
Software
Procedure
Experiments involving lentivirus and animals should be approved by the Institutional Animal Care and Use Committee and the Institutional Biosafety Committee and follow institutional or national regulations. When working with bacteria or virus, all glassware, pipet tips, tubes, and solutions should be autoclaved when applicable before use. All surgery tools should be autoclaved before use, and surgical procedures should be performed under aseptic conditions.
Notes
Recipes
Acknowledgments
This work was funded by the National Science Foundation grant 1258015 (XCL) and the National Institute of Health grant R01MH105519 (XCL). The funders had no roles in study design, data collection and interpretation, or the decision to submit the work for publication. We thank Drs. M. Sheng and D. Edbauer for generously providing the lentiviral vector and Dr. H. Xia for the lentiviral packaging plasmids. We thank many members of the birdsong community for their constructive inputs throughout the course of this work.
Competing interests
The authors declare no competing financial interests.
Ethics
Experiments involving animals are approved by the Institutional Animal Care and use committee (IACUC) protocol (#3187) of the LSU School of Medicine.
References
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