Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using two-photon microscopy

Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing, loss are commonly available. However, the mouse cochlea is substantially smaller than other animal models routinely used to study cochlear physiology. This makes study of their hair cells difficult. We develop a novel methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea. Fresh cochleae are harvested, left intact within their otic capsule bone, and fixed in a recording chamber. The bone overlying the cochlear epithelium is opened and Reissner's membrane is incised. A fluorescent calcium indicator is applied to the preparation. A custom-built upright two-photon microscope was used to image the preparation using 3-D scanning. We are able to image about one third of a cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we find that outer hair cells demonstrate increased fluorescence compared with surrounding supporting cells. This methodology is then used to visualize hair cell calcium changes during mechanotransduction over a region of the epithelium. Because the epithelium is left within the cochlea, dissection trauma is minimized and artifactual changes in hair cell physiology are expected to be reduced.