Fig. 2. Both calretinin-rich and -poor nerve fibers juxtapose synaptic ribbons. (A) A 3D reconstructed image of an organ of Corti of a P19 rat that is tripleimmunolabeled with anti-myosin VIIa (blue), anti-C-terminal binding protein 2 (CtBP2; red), and anti-NKA (green). A 3D image of a single IHC and its associated nerve fibers (marked area) was extracted and processed for further analysis in Aa~Bc4. (Aa) ZY projection view. (B) XY projection view. (Ba1~Bc4) XY projection views from partial Z stacks, as described in diagram. (Ba2~Ba3) XY projection views of the marked area in Ba1. (Bb2~Bb3) XY projection views of the marked area in Bb1. (Bc2~Bc3) XY projection views of the marked area in Bc1. The trajectories of 7 exemplary NKA-positive nerve fibers were traced and displayed in curved cylinders according to their courses after passing the habenula perforata. Magenta: fibers with a modiolar-side course and terminating on the modiolar side of the IHCs. Cyan: fibers with a pillar-side course and terminating on the modiolar side of the IHCs. Yellow: fibers with a pillar-side course and terminating on the pillar side of the IHCs. (C) A 3D reconstructed image of an organ of Corti of a 11-week-old rat that is triple-immunolabeled with anti-calretinin (green), anti-CtBP2 (cyan), and anti-NKA (red). A 3D image of a single IHC and its associated nerve fibers (marked area) was extracted and processed for further analysis in Ca~Cc3. (Ca~Ca2) ZY projection view. (Ca3) XY projection view. (Cb1~Cb2) XY projection views of the marked area in Cb. (Cc1~Cc2) XY projection views of the marked area in Cc. Magenta: calretinin-poor fibers terminating on the modiolar side of the IHCs. Yellow: calretinin-rich fibers terminating on the pillar side of the IHCs. Regardless of their courses, all traced fibers juxtaposed CtBP2 puncta (insets). The pillar (P) and modiolar side (M) of IHC is marked with dotted lines (Aa and Ca).
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