|
Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing loss
are commonly available for research. However, the mouse cochlea is substantially smaller than other animal models
routinely used to study cochlear physiology. This makes the study of their hair cells difficult. We developed a novel
methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea.
Fresh cochleae were harvested, left intact within their otic capsule bone, and glued upright in a recording chamber. The
bone overlying the region of the cochlear epithelium to be studied was opened and Reissner's membrane was incised. A
fluorescent indicator was applied to the preparation to image intracellular calcium. A custom-built upright two-photon
microscope was used to image the preparation using three dimensional scanning. We were able to image about 1/3 of a
cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we found that
outer hair cells demonstrated increased fluorescence compared with surrounding supporting cells. Thus, this
methodology can be used to visualize hair cell calcium changes and 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 reduced.
|
