Molecular Tools to Study Regeneration of the Avian Cochlea and Utricle

The avian inner ear can regenerate sensory hair cells after damage and has served as a model for the study of hearing regeneration for more than 30 years. Here we present a detailed surgical protocol to induce rapid apoptosis of all hair cells in the chicken cochlea and utricle with a single, local infusion of the aminoglycoside sisomicin. S-phase entry of supporting cells engaged in proliferative regeneration peaks at 48 h and newly regenerated hair cells emerge as early as 4–5 days post-sisomicin. We provide reliable read-outs for hair cell loss, such as overt manifestations of vestibular deficiencies, and quick validation of regeneration using reliable markers that can be detected with commercial antibodies. Titrating down the dose of sisomicin reveals differential susceptibilities of hair cell subtypes: cochlea versus utricle, cochlear tall versus cochlear short hair cells, vestibular type I versus type II hair cells, and proximal versus distal location along the cochlea. We provide a method to quantitate cells within the sensory epithelium in 3D, leveraging vibratome sectioning and imaging methods that are presented in a companion chapter. Finally, we present the technique of cold-peeling the cochlear sensory epithelium for the purposes of RNA or protein extraction, and single-cell dissociation in preparation for RNA-seq.