For immunostaining and quantification of synaptic degeneration, cochleae were perfused with 4% paraformaldehyde and 0.25% glutaraldehyde, post-fixed for 1–2 hrs, decalcified in EDTA, microdissected into 6 pieces and immunostained with antibodies to 1) C-terminal binding protein 2 (mouse anti-CtBP2 from BD Biosciences used at 1:200), and either 2) heavy neurofilaments (chicken anti-NF-H from Chemicon used at 1:1000) or 3) parvalbumin (goat anti-parvalbumin from Swant at 1:5000) and appropriate secondary antibodies coupled to Alexafluors in the red and green channels. A nuclear dye, TOPRO-3 was added to aid in hair cell counting, and in some cases, phalloidin (coupled to Alexafluor 568) was added to image stereocilia bundles. Immunostaining with post-synaptic markers such as glutamate receptors (rabbit anti-GluR2/3 from Chemicon) or proteins associated with the post-synaptic density (mouse anti-PSD-95 from Chemicon) did not survive the decalcification process required to reliably dissect entire cochleas from base to apex. Cochlear lengths were obtained for each case, and a cochlear frequency map computed to precisely localize IHCs from the 5.6, 8.0, 11.3, 22.6, 32, 45.2 and 64 kHz regions in each case. Confocal z-stacks of these 7 regions from each ear were obtained using a high-resolution (1.4 N.A.) oil-immersion objective and 2X digital zoom on a Leica TCS SP2. Care was taken to span the entire synaptic pole of the hair cells in the z-dimension, with a z-step-size of 0.25 mm, from the subjacent inner spiral bundle to the apical most ribbon or nerve terminal in the supranuclear region. Image stacks were ported to image-processing software (Amira®: Visage Imaging), where synaptic ribbons were counted and divided by the total number of IHC nuclei in the microscopic field (including fractional estimates, when necessary, at the apical and basal ends of the image stack). To avoid underestimating ribbon counts due to superposition in the image stacks, 3-D renderings were produced, using the “isosurface” feature in Amira®, and rotated to disambiguate the xy projection images.