Figure 7.

CRFR1^−/− mice possess normal numbers of afferent synapses but abnormal synapse distribution. A, Presynaptic ribbons and postsynaptic terminals were localized on the inner hair cell by double immunolabeling for CTBP2 (green), a maker for synaptic ribbons in hair cells, and GluR4 (red). While a coincident overlay of ribbons and GluR immunopositive puncta was present in both CRFR1^+/+ and CRFR1^−/− mice, GluR4 puncta appeared larger in CRFR1^−/− mice. Additionally, GluR4 puncta were more often apposed to two CTBP2 (synaptic ribbon) puncta in the CRFR1^−/− mice (arrows) compared with CRFR1^+/+ mice. The distance between the nucleus and the edge of the synaptic zone (d) was shorter in CRFR1^−/− mice compared with wild-type mice, generating a tighter clustering of GluR4/CTBP2 ensembles compared with wild-type mice. Scale bar, 5 μm. B, Three-dimensional reconstruction of the basal region of IHCs following double labeling for GluR4 (red) and CTBP2 (green). Large aggregations of GluR4 puncta (arrows) were present in CRFR1^−/− mice that were only occasionally observed in wild-type mice. Space-filling reconstructions allowed better visualization of doubled ribbon structures (arrowheads) in CRFR1^−/− mice. These were often discerned from simple large ribbons by contours representing the upper and lower limits of the individual ribbons in the aggregate. By contrast, very few double ribbons were observed in wild-type mice (e.g., A, arrow). Few GluR4/CTBP2 profiles were observed on the pillar side of the IHC nuclei in CRFR1^−/− mice. C, Western blot of cochlear samples revealed a significant increase in GluR4 expression in CRFR1^−/− mice compared with CRFR1^+/+ mice (**p = 0.0074, error bars are SEM). D, Measurement of GluR4 puncta in the x–y-plane revealed a bimodal distribution of GluR4 puncta size in both CRFR1^+/+ (blue) and CRFR1^−/− (red) mice. CRFR1^−/− mice possessed more puncta 0.6 μm² and larger (upward shift of curve) and a statistically significant increase in mean puncta size (p = 0.0007). E, Three-dimensional analysis revealed an abnormal synaptic ribbon distribution in the CRFR1^−/− mice (right), with more ribbons clustered toward the modiolar side of the hair cell in CRFR1^−/− mice and fewer falling on the pillar side compared with wild type (right). WT, Wild-type; KO, knock-out.