Figure 1.

α9 nAChR is required for target recognition and navigation of efferent fibers during olivocochlear synaptogenesis. Mid-modiolar paraffin cross sections of cochlea from α9 null and wild-type littermate control mice were double stained with Pep19 (red; hair cell marker) and synaptophysin (green, vesiculated efferent fiber marker) at P3, P5, P7, P10, P13, and P18 postnatal stages. (A) P3 wild-type cochleae possessed synaptophysin-positive efferent terminals adjacent to inner hair cells (IHC), but synaptophysin immunoreactive terminals were not detected in the outer hair cell (OHC) region. (B) P3 α9 null mice already possessed small numbers of synaptophysin-positive efferent terminals below the first and second rows of OHCs (arrows). (C) No significant change in innervation to IHCs occurred at P5 in the wild-type mice. (D) Synaptophysin staining intensity increased at the IHCs of α9 null mice. A greater number of efferent fibers migrating across the tunnel of Corti towards the OHCs and an increase in number and intensity of synaptophysin-positive efferent terminals were also observed in the OHC region in the α9 nulls (arrow). (E) At P7 in wild-type mice, an increase in intensity of synaptophysin staining was observed at the IHCs, and the first clear indication of vesiculated synaptic endings (arrow) occurred under OHCs. (F) Similarly, there was an increase in synaptic terminal innervation to the IHCs of the α9 null mice at P7. At the OHCs, synaptophysin labeled efferent fibers (arrowhead) overshot the synaptic (basal) pole of the first row hair cell (asterisk) and enveloped the modiolar side lateral wall of the OHC. Large immunopositive profiles of accumulated efferent crossing fibers (arrow) were also located near the base of the first row OHC. (G) At P10, the robust innervation to the wild-type IHCs decreased, and innervation to OHCs took on a mature profile under all three rows. (H) In the α9 null mice, large numbers of distinct synaptophysin labeled terminals were present directly over supranuclear regions of the OHCs (arrows). Synaptophysin labeling at the IHCs remained greater than that observed in wild-type mice. (I) At P13, the efferent innervation in wild-type mice remained stable and adult-like. (J) However, in α9 null mice, synaptophysin labeled efferent fibers created a lattice-like network surrounding the OHC soma of all three rows in α9 null mice (arrowhead) while also hypertrophying at the base of the cells (arrows). (K) At P18, the innervation to the wild-type hair cells remained unchanged and adult-like. (L) In the α9 nulls, direct efferent fiber contacts with the IHCs decreased significantly, and fibers were present in a position below the IHCs. In the OHC region, the lattice-like innervation to these cells also disappeared, leaving only the hypertrophied basal terminal (arrow). All scale bars represent 10 μm.