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 1

The different distributions of NMDARs and AMPARs at afferent dendritic terminals around IHCs in the normal mature cochlea. Postsynaptic AMPARs, NMDARs, and nerve fibers were identified by immunostaining for GluA2 (red), GluN1 (green), and neurofilament (NF) (grey), respectively. Nuclei were labeled with DAPI (blue). (a) The ISBs appeared as a dense meshwork of neuronal processes at the basal pole region of the IHCs. (b) AMPAR patches were mostly distributed among the ISBs around the basal poles of the IHCs. (c) Most of the NMDAR patches were distributed on the neural dendritic terminals, which were near the IHC nuclei region. (d) A diagram of the different locations of NMDARs and AMPARs at type I ANFs under physiological conditions. To count the number of synaptic elements, we drew one line along the top of the IHC nuclei and another line along the bottom of the ISBs. Then the field between these two lines was divided into two parts, the “IHC nuclei region” and the “IHC basal pole region.” The IHC nuclei region was defined as the half region proximal to the nuclei and the bundle poles of the IHCs, and the IHC basal pole region was defined as the half region proximal to the ISBs and the basal poles of the IHCs. (e) Enlarged view of merged images (a), (b), and (c). Most of the NMDAR patches were distributed on the neural dendritic terminals between the adjoining IHCs and closer to the IHC nuclei region, while most of the AMPAR patches were observed among the ISBs around the basal poles of the IHCs. In the IHC nuclei region, some NMDARs colocalized with GluA2-positive AMPAR puncta (the dashed white circles), which were smaller than AMPAR patches in the IHC basal pole region (the dashed yellow circles). (f, g) The 3D-reconstructed images showing spatial distributions of AMPARs and NMDARs in SGN fibers around the IHCs. Postsynaptic AMPARs, NMDARs, and nerve fibers were identified by immunostaining for GluA2 (red), GluN1 (magenta), and NF (green), respectively. Nuclei were labeled with DAPI (blue). IHC: inner hair cell; OHC: outer hair cell; ISBs: inner spiral bundles; ANFs: afferent nerve fibers. Scale bar = 10 μm.