Quantification of synaptic puncta

The quantification procedures were based on an established method⁷ with modifications to accommodate guinea pig tissues. The cochlear distance from the apex was measured using ImageJ plugin (http://www.masseyeandear.org/research/otolaryngology/investigators/laboratories/eaton-peabody-laboratories/epl-histology-resources/imagej-plugin-for-cochlear-frequency-mapping-in-whole-mounts) and this distance was converted into frequency based on the calculation by Greenwood.⁴⁰ Confocal z-stacks of the 8, 16, and 32 kHz regions from each cochlea were taken with a 60× (1× digital zoom) oil immersion lens. For synaptic punctum counts, the z-stacks (0.25 µm step size) were set to include the entire length of inner hair cells so that all the synaptic puncta could be imaged. In these stacked images, we counted all puncta labeled for presynaptic marker (RIBEYE and CtBP2), using tpsDig software (Version 2.12; F. James Rohlf, Ecology & Evolution, SUNY at Stony Brook, NY). Counts in each z-stack were divided by the number of inner hair cell nuclei, which could be visualized by staining of CtBP2 antibody. Each image usually contained 20–27 inner hair cells. For Statistical analysis of punctum density, MANOVA was used to test for differences in punctum counts among the three groups (NS, Adv.Ntf3 and AAV.Ntf3), analyzing all tested frequency areas simultaneously. Subsequently, to test which groups were different, we performed pairwise comparisons of groups using MANOVA, then for those pairs with statistical significance, univariate ANOVA was performed to determine which frequency areas differed.