Figure 9. Presynaptic and postsynaptic marker counts are normal in KNa1 DKO in 1-3-, but not 6-9-mo old mice. Synapses between the spiral ganglion neurons (SGNs) and inner hair cells (IHCs) were quantified at four tonotopic locations (4, 8, 16, and 32 kHz) in the organ of Corti isolated from 1-9-mo old WT and DKO mice. (A–D) There were no obvious differences between WT and DKO mice in the organization of afferent synapses, identified as paired CtBP2 (red) and PSD95-(green) immunopuncta. Images presented as Z-projections were made using stacks of confocal micrographs from the 4, 8, 16, and 32 kHz region as indicated. (E–H) Quantification of the average number of synapses per IHC showed no statistically significant differences between WT (black) and DKO (grey) animals at 1 month of age. However, with increasing age from 3- to 9-mo, statistical differences among the synapse counts appeared and gradually encompassed more tonotopic regions. Statistical differences were observed first at the high-frequency segment of the cochlea. Values (mean ± SEM) are illustrated (p < 0.05, 0.01, 0.001 = *, **, ***). Scale bar = 3 μm.