(A–D) Maximum intensity projections of confocal z-sections of inner hair cells (IHCs) in apical cochlea turns of P22 KO and wildtype littermates, immunolabeled for CtBP2/RIBEYE (A), Homer (B), Bassoon (C) and Homer-Bassoon Overlay (D). Virtually no punctated extra-nuclear CtBP2 staining that is colocalized with other synaptic proteins can be seen in KO mice, whereas Bassoon and Homer appear generally unaffected. (E) 100 nm thick transmission electron microscopy (TEM) sections of pre- and postsynaptic densities in WT and KO mice at P28 confirm the absence of electron-dense ribbon structures in KO mice. (For E-–G) Black arrows point to ribbon structure, white arrows point to synaptic vesicles and white arrowheads point to the remaining density resembling the arciform density. (F) Single slice from a 10-nm thick tomographic section. (G) Reconstruction of complete synapses from data used to create E,F. Presynaptic density in cyan and postsynaptic density in green. Tethered synaptic vesicles modeled in yellow, electron-dense structure attributed to synaptic ribbons in magenta. (H) Confocal maximum intensity projection of IHCs immunolabeled for CtBP1 (green) and Homer (magenta). (I) Quantification of RIBEYE, Homer and Bassoon puncta in the organ of corti normalized to the number of IHCs in analyzed regions of interest, showing complete loss of synaptic RIBEYE staining in KO. The amount of Homer and Bassoon staining is unaffected (p>0.05). WT in blue, n = 20 mice, quantified puncta —RIBEYE = 3796, Bassoon = 10,215, Homer = 4340; KO in red, n = 19 mice, n puncta — RIBEYE = 33, Bassoon = 10,825, Homer = 4201. (J) Paired synaptic puncta per IHC is lost in the absence of RIBEYE staining (p≤0.001) between WT and KO. The number of synapses defined by colocated postsynaptic Homer and presynaptic Bassoon puncta (p=0.18) is unaffected in KO. (K) The number of extra-nuclear CtBP1 puncta is greatly reduced (p=0.003) in KO mice. (WT in blue, n = 9 mice, n puncta — CtBP1 = 19,929, Homer 4940; KO in red, n = 7 mice, n puncta — CtBP1 = 11,960, Homer = 5033). (L) Remaining CtBP1 puncta show significantly reduced (p≤0.001) pairing with postsynaptic Homer staining. Scale bars: A–D, H: 5 µm; E, F: 100 nm. Boxes represent standard deviations of the mean. Significance levels of two-tailed unpaired t-tests: n.s., not significant; *p≤0.05; **p≤0.01; ***p≤0.001.