SEM and measurements

The inner ear (cochlear and vestibular system) of mice aged P5 and P21 were harvested and immersed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.35, with 3 mM CaCl2 for 24 h or more. The tissues were postfixed in 1% OsO4 in the same buffer, dissected and double processed with thiocarbohydrazide followed by OsO4 (ref. ⁶¹) before dehydration through an alcohol series and critical point drying with CO2. After mounting on specimen support stubs, samples were sputter coated with platinum. Samples were examined with a JEOL 6700 F cold field emission scanning electron microscope operating at 3 or 5 kV. Measurements were made on the longest row of stereocilia closest to the kinocilium on 84 control stereocilia/16 HCs/2 mice and 75/24/2 (for P5 and P21), on Gpsm2 cKO 36/8/2 and 108/21/2 (For P5 and P21), and on Gnai3 cKO 95/18/2 and 60/14/2 (For P5 and P21) in at least four different cochleae. The quantification of the number of stereocilia were made on 23 control HCs/2 mice and 12/2 (for P5 and P21), on Gpsm2 cKO 16/2 and 23/2 (for P5 and P21) and on Gnai3 cKO 19/2 and 32/2 (for P5 and P21) in at least four different cochleae.

Images were collected from the basal or middle turn of the cochlea, defined as ∼20/30% and 50/60% of the total length of the organ of Corti from the base. At each location, the hair bundles were viewed both from behind the longest row of stereocilia to view the height of the hair bundle as well as approximately perpendicular to the apical surface of the HC or toward the inner aspect of the bundle to examine its overall morphology and composition. To estimate bundle height, measurements were made from images at calibrated instrument magnifications of × 20,000 or occasionally × 10,000. Although we might have underestimated the actual stereocilia length, all efforts were taken to minimize the effect of parallax. Measurements were taken from bundles viewed from the lateral side toward the medial side (from the stria toward the modiolus), so that the row of longest stereocilia were imaged from the ‘rear.’ Samples were tilted and rotated so that the row of longest stereocilia was approximately perpendicular to the direction of view. Stereo-imaging was used in a few cases to gain an indication of possible errors in length measurements from 2D images. From the anaglyphs generated by a pair of images separated by 8 degrees of tilt, height measurements were obtained using analySIS software. These revealed little difference in the height measurement from that obtained from the 2D image of a stereocilium at close to perpendicular to its long axis. Measurements were made using ImageJ software.