Fig. 5.

The location of synaptic elements after ouabain treatment suggests dynamic rearrangement of synaptic architecture in the IHC area. In all images, IHC cytoplasm is stained with anti-myosin VIIA (blue). A, C The double stain for presynaptic ribbons (anti-CtBP2—red) and postsynaptic glutamate receptors (green) shows that, in the normal ear (A), synapses are clustered near the cell’s basal pole (red/green arrows) and orphan ribbons are rare. After ouabain (B), orphan ribbons (red arrows) appear throughout the sub- and perinuclear cytoplasm. Both images are from the 32 kHz region and are maximum projections from four adjacent IHCs, acquired as z-stacks with a focal plane parallel to the basilar membrane and then re-projected to mimic radial sections; the ouabain ear was 1 week posttreatment. B, D The double stain for synaptic ribbons (anti-CtBP2—red) and ANF terminals (Na⁺/K⁺ ATPase—green) shows that, in the normal ear (B), terminals and associated presynaptic ribbons are confined to the subnuclear region. In the ouabain-treated ear, ectopic terminals climb near the cuticular plate (green arrows in D), where they are not juxtaposed to ribbons. Numerous orphan ribbons are visible: some are abnormally large (red arrow). Control image is from the 11 kHz region; ouabain image is from the 8 kHz region, 1 week posttreatment. Images were acquired as z-stacks with the x–y focal plane parallel to the basilar membrane and then re-projected in the x–z plane. Scale bar in B also applies to D; scale bar in B also applies to A. Approximate positions of nuclei are shown by dashed circles in all panels.