Double labeling for β-tubulin and peripherin distinguishes type I and type II spiral ganglion neurons (SGNs) and their peripheral innervation during the first postnatal week of mouse development. Single plane (A,C,D,E,G,H) and maximum projections (B,F) of confocal image stacks show β-tubulin (green) and peripherin (red) immunofluorescence in cochleae from P1 (A-D) and P7 (E-H) mice. (A-D) At P1 peripheral fibers from type I SGNs (immunolabeled for β-tubulin only) form the inner spiral plexus (isp) beneath the inner hair cell (ihc). Type II SGN soma and peripheral fibers (β-tubulin and peripherin immunolabeled) are numerous in the spiral ganglion (sgn) and osseus spiral lamina (osl) of P1 cochleae (A,B). Their neurites are present beneath the IHCs (arrow in (C,D)) and beneath the outer hair cells (ohc) in the outer spiral bundles (osb). (E-H) At P7, the majority of neurons in the spiral ganglion are type I neurons (E,F) and their neurites innervate the IHCs (G). Peripherin and β-tubulin double-immunolabeled type II SGNs are now localized laterally in the spiral ganglion near the intra-ganglionic spiral bundle (igsb), their fibers form the outer spiral bundle and innervate the OHC only; no peripherin-immunolabeled fibers extend toward the IHC (H). Variation in the color (yellow to red) of double labeled (type II) neuron soma and their neurite processes arises from differences in the relative intensity of the β-tubulin (green fluorescence) in soma and neurites and also peripherin (red) fluorescence in merged confocal images. This is particularly evident at P7, where the type II soma appear red as there is a decline in β-tubulin labeling, whereas the outer spiral bundle region is yellow, due to approximate equivalence of the intensity of the red and green immunofluorescence in this region (see (E)). We attribute double labeling (type II SGNs) versus single (β-tubulin) labeling (type I SGNs) from analysis of individual channels. Panels (A,C-E,G,H) are from cryosectioned tissue; (B,F) are whole-mount preparations. Scale bars: 50 μm (A,B,E,F); 25 μm (C,D,G,H).