N-Methyl-D-Aspartate Receptors Involvement in the Gentamicin-Induced Hearing Loss and Pathological Changes of Ribbon Synapse in the Mouse Cochlear Inner Hair Cells

Figure 4

The number of cochlear ribbon synapses decreased and their locations changed from the basal pole region toward the nuclei region of the IHCs after gentamicin treatment. The IHCs were outlined by myosin 7a fluorescence in the cytoplasm (grey), and the nuclei were stained with DAPI (blue). The afferent synapses on the IHCs were labeled by immunostaining presynaptic ribbons with anti-CtBP2 (green) and the postsynaptic AMPARs with anti-GluA2 (red). (a) Ribbons and AMPARs were almost perfectly paired at the basal poles of the IHCs under physiological conditions. (b) After gentamicin treatment for 4 days, the ribbons and AMPARs moved towards the bundle poles of the IHCs. (c) After gentamicin treatment for 7 days, ribbons and AMPARs migrated towards the bundle poles of the IHCs, and some of them reached to or across the IHC nuclei region. The dashed white circles indicate some variegated ribbons and/or AMPARs at the IHC basal pole region. (d) The 3D-reconstructed image. The dashed yellow circles show orphan AMPARs near the bundle poles of the IHCs after 4 days of gentamicin treatment. (e) The numbers of CtBP2-positive ribbons. (f) The numbers of GluA2-positive AMPARs. (g) The numbers of CtBP2 and GluA2 double-positive ribbon synaptic pairs. GM 4 d, 7 d: gentamicin treatment for 4 days or 7 days; IHC: inner hair cell. Scale bar = 10 μm. .