A technique for slicing the rat cochlea around the onset of hearing

Abstract

The cochlea presents a considerable challenge to the study of sound transduction and auditory neurotransmission. This arises from the location of the sensory, supporting and secretory epithelia, and primary auditory neurons within a complex ossified spiral structure comprised of three separate fluid-filled chambers. We have developed a novel cochlear slice preparation, which provides access to the highly differentiated tissues while retaining structural integrity and cell viability. Our technique for slicing the cochlea and imaging tissue structure facilitates the study of peripheral auditory signaling in situ. The preparation was developed in the neonatal rat (postnatal days 4–14) and is based on the use of vibrating blade microtome slicing after perfusing the perilymphatic compartments with chilled Pluronic F127 NF, a block copolymer gel. This material is liquid when cold, and sets when warmed to room temperature, stabilizing the cochlear fluid-filled compartments and thereby supporting the cochlear partition during slicing. Slices (150–300 μm) of neonatal rat cochlea, imaged using infrared videomicroscopy, allow tight-seal voltage clamp recordings from a variety of cell types. Recordings obtained from primary auditory neurons, hair cells, supporting cells, and Reissner's membrane epithelial cells verify the viability of the tissues in the preparation. Data includes novel evidence for glutamatergic and purinergic co-transmission by primary auditory neurons. The preparation has considerable potential in a range of molecular physiological applications requiring cell-specific targeting with retention of cell connectivity.