FIG. 4.

Visualizing IHC synapses in confocal z-stacks from immunostained cochlear whole mounts, acquired in the x-y plane (A) and re-projected into y-z (B) plane. A Pre- and post-synaptic elements in the IHC area are counted in cochlear whole mounts quadruple-immunostained for CtBP2 (red), GluA2 (green), Na-K ATPase (blue), and myosin VIIa (white). White arrows indicate several synapses, i.e., closely juxtaposed red and green puncta; red-filled arrows indicate several terminal swellings of cochlear nerve fibers. B The size gradients in pre- and post-synaptic elements are quantified by defining a new set of axes in the y-z plane designed to (1) minimize the habenular-cuticular spread of synaptic distances from new modiolar-pillar axis while (2) keeping the new origin as close to the IHC midline as possible. This maximum projection in the y-z plane is from the same z-stack shown in the acquisition (x-y) plane in panel A.