Dissection of the Mouse Utricle
Adult mice (usually 5–8 weeks of age) are euthanized by overdose of Nembutal (Ovation Pharmaceuticals, Inc. Deerfield, IL). Heads are removed using medium scissors, and the external auditory canal is snipped beneath the skin on either side using small scissors. The skin of the head is then pulled forward from back to front, and the head is bisected (also from back to front) using small scissors. The brain is removed from each half using the tip of the small scissors. The VIIIth nerve root and posterior semicircular canal can then be visualized near the back of the skull. The skull is broken (or cut) to obtain the auditory bulla and a small bit of surrounding bone. Once this gross dissection is complete, the fine dissection is completed in a tissue culture hood equipped with a stereomicroscope.
For the fine dissection, dissecting tools are cleaned and immersed in 70% ethanol for 15 minutes prior to being placed at a convenient location in the tissue culture hood. The necessary tools are as follows:
- 2 pairs #55 forceps (such as Fine Science Tools #11255-20)
- 1 pair #5 forceps
- 1 pair #3 forceps
- 1 scalpel with #11 blade
- 1 fine probe (such as half of a broken #55 forceps)
The fine dissection is carried out using sterile technique. Each ear is placed in a 35mm sterile Petri dish and covered with dissecting medium consisting of a 2:1 v/v mixture of Basal Medium Eagle and Earle’s Balanced Salt Solution (BME/EBSS). BME consists of Basal Medium Eagle (Sigma, St. Louis, MO #B-9638) supplemented with 25mM sodium bicarbonate (Sigma #S-6297) and 25mM glucose (Sigma #G5767). Earle’s Balanced Salt Solution is obtained from Sigma (#E2888).
The first step in the fine dissection is to locate the external auditory canal (EAC) on one side of the preparation and the VIIIth nerve root on the other. The auditory bulla is broken open by inserting one fork of both the #3 forceps and the #5 forceps all the way into the EAC until the tips reach the bone at the apical end (). The forceps are then pulled apart to break the bulla, exposing the bony cochlea. After the bulla is removed, the bony cochlear spiral is visible along with the ossicles and the oval and round windows. On the opposite (medial) surface of the preparation, the VIIIth nerve root and posterior semicircular canal are visible ().
With the preparation held such that the cochlear spiral and ossicles are on the bottom of the dish and the nerve root is on top, the apex of the cochlea is removed using the scalpel. The scalpel blade is used to cut all the way through the apex of the cochlea between the VIIIth nerve root and the apex (). The preparation is then rotated until the remaining cochlear spiral is pointing up and the preparation is resting on the semicircular canals. The posterior semicircular canal is used as a handle to hold the preparation steady by placing one fork of the #3 forceps in the canal. The remaining cochlear spiral (soft tissue) is then removed at the modiolus using #5 forceps.
After the cochlea (except for the basal hook region) is removed, the remainder of the osseous spiral lamina is removed using the fine probe (). The tip of the fine probe is placed beneath the remainder of the osseous spiral lamina, and the bone is carefully lifted up. Just beneath this bony shelf lies the saccule. When the saccule is removed, the utricle (surrounded by bone) is visible directly across from the stapes footplate (). The utricle is carefully removed using #55 forceps.
Culture Conditions and Ototoxic Drug Exposures
Utricles are cultured free-floating (2–8 utricles per well) in sterile 24-well tissue culture plates. Culture medium consists of dissecting medium (BME and EBSS, 2:1 v/v) supplemented with 5% fetal bovine serum (FBS). For ototoxic drug experiments, neomycin sulfate solution (Sigma) is added to culture medium at final concentrations ranging from 0.1mM to 5.0mM. Cisplatin is supplied as a 1 mg/ml stock solution (Bedford Laboratories, Bedford, OH) and diluted into culture medium at final concentrations ranging from 1.0 μg/ml to 10.0 μg/ml. No neomycin or cisplatin is added to control cultures. Utricles are incubated at 37°C in a 5% CO2/95% air environment.
At the end of the culture period, utricles are fixed overnight at 4°C in 4% paraformaldehyde. Utricles are then washed in phosphate buffered saline (PBS). Otoconia are removed by holding the edge of the utricle with a pair of #55 forceps and directing a stream of PBS across the utricle from a syringe with a 23-gauge needle. This technique can also be used to remove otoconia from fresh utricles prior to culturing them.
Labeling Hair Cells
For hair cell counts, fixed utricles can be prepared for whole mount double-label fluorescent immunohistochemistry using a monoclonal antibody against calmodulin (Sigma C-3545) and a polyclonal antibody against calbindin (Chemicon AB1778) (Cunningham et al., 2002
; Cunningham et al., 2004
). The double label immunohistochemistry protocol allows for separate counts of the two types of hair cells in the mouse utricle: anti-calmodulin labels all hair cells in the utricle, and anti-calbindin labels only the hair cells in the striolar region. Striolar hair cells are more sensitive than non-striolar hair cells to aminoglycoside-induced death (Cunningham et al., 2002
After staining, whole utricles are mounted on glass slides in Fluoromount-G (Southern Biotechnology, Birmingham, AL) and coverslipped.