Association of intracellular and synaptic organization in cochlear inner hair cells revealed by 3D electron microscopy

Abstract

The ways in which cell architecture is modelled to meet cell function is a poorly understood facet of cell biology. To address this question, we have studied the cytoarchitecture of a cell with highly specialised organisation, the cochlear inner hair cell (IHC), using multiple hierarchies of three-dimensional (3D) electron microscopy analyses. We show that synaptic terminal distribution on the IHC surface correlates with cell shape, and the distribution of a highly organised network of membranes and mitochondria encompassing the infranuclear region of the cell. This network is juxtaposed to a population of small vesicles, which represents a potential new source of neurotransmitter vesicles for replenishment of the synapses. Structural linkages between organelles that underlie this organisation were identified by high-resolution imaging. Taken together, these results describe a cell-encompassing network of membranes and mitochondria present in IHCs that support efficient coding and transmission of auditory signals. Such techniques also have the potential for clarifying functionally specialised cytoarchitecture of other cell types.

Keywords: 3D electron microscopy; Inner hair cell; Intracellular membranes; Synapse.

Fig. 1.

SBF-SEM analysis of IHCs from the middle cochlear coil of C57/Bl6 mice. (A) Diagram of the organ of Corti. Direction of modiolus and pillar cells are shown. (B) Whole-cell sections from five hair cells. Concentration of internal membranes and mitochondria on one side of the cell is shown (white arrowheads). Images from cell 9 show the internal lumen of the membranes. (C) SEM micrograph showing IHCs viewed from the modiolar side of the cell. (D) Reconstruction of five IHCs viewed from the same position. Innervation of three of the cells is also reconstructed. Cells are non-uniformly orientated and either the rounded (purple and green) or flattened (yellow, blue and red) side can face the modiolus. (E) Flatness of cells measured by contact of the cell membrane with a perpendicular line. Lines were placed on either side of each cell (n=11). Mean±s.e.m. measurements of three regions showed that one side of the cell was consistently flatter than the other. Scale bars: 2 µm (B, cells 1, 2, 4, 5 and 8); 1 µm (B, cell 9); 10 µm (C); 5 µm (D).

Fig. 2.

Distribution of afferent terminals around IHCs. (A) Distribution of afferent terminals (dark blue) on five cells. Terminals were either manually segmented (cell 2) or represented by a sphere at the approximate centre of the bouton. Comparison between manual segmentation and sphere representation is shown for cell 2. (B) Number of afferent terminals present on each cell studied. (C) Mean±s.e.m. distribution of afferent terminals in the pillar and modiolar, and rounded and flattened hemispheres of n=8 cells. **P<0.05. Scale bars, 2 µm. See also supplementary material Table S1.

Fig. 3.

Distribution of membranes and mitochondria revealed by stereology analysis of IHCs. (A,B) Stereology grid superimposed on an image of cell 2, showing the longitudinal (A) and radial (B) section. (C,D) Stereology models of whole cells (cell 2 and cell 8). (E,F) 3-µm sections around the nucleus of the same cells. The flattened side of the cell is indicated by ‘/’ and rounded side by ‘)’; P and M indicate pillar and modiolar orientation, respectively. The concentration of membranes and mitochondria (pink and yellow) to the flattened side of the cell is visible. (G) Mean±s.e.m. distribution of membranes and mitochondria between flattened and rounded hemispheres of the IHC in 3-µm sections and whole-cell reconstructions (n=9 and n=11, respectively). (H) Orientation of the IHC on the x-, y- and z-axes, and orientation of division along the anterior-posterior axis (An and Po) and the rounded-flattened axis (R and F). The sides of this axis can be pillar or modiolar (P/M) depending of the orientation of the cell in the organ of Corti. Scale bars: 2 µm. See also supplementary material Table S1.

Fig. 4.

Reconstruction of mitochondria in IHCs. (A) Mitochondrial distribution in IHCs, reconstructed using manual segmentation or representative spheres. Top row of images, the view from the nucleus to the middle of the infranuclear region is shown, overlaid on the radial section image of the cell at the centre of the infranuclear portion. Bottom row of images, mitochondrial distribution compared to the positions of the afferent terminal population of each cell (dark blue). (B) IHC mitochondria (yellow) reconstructed by manual segmentation. Orientation of the cell relative to the pillar cell (P) and modiolus (M) is shown on the image. (C) Reconstructed mitochondria shown in conjunction with reconstructed intracellular membranes (pink). (D) Mitochondria alone, and the mitochondria and intracellular membrane population together viewed looking towards the basal pole of the cell from the position of the nucleus. Scale bars: 2 µm. See also supplementary material Table S1.

Fig. 5.

Reconstruction and classification of IHC intracellular membranes. (A) Histogram of internal membrane surface areas reconstructed from Cell 1. Sizes of very large membrane sheets are shown above their respective bars. (B) Histogram of intracellular membrane surface area, excluding very large membrane sheets. (C) Membranes are shown divided into the clusters shown in the histogram. Orientation of the cell to the pillar cell (P) and modiolus (M) is shown on images. Red spheres represent positions of ribbon synapses. Insets: membranes aligned perpendicular to the z-axis, showing their sheet characteristics. Only a portion of sheet c (red) is shown. >, a Type 2 membrane sheet close to a ribbon. (D) Type 3 (blue) membranes and sheet c (red) are shown together. (E) Type 2 and Type 3 membranes are shown together. (F) Top section of intracellular membrane shown from the position of the nucleus, and with a radial section image. (G) View through the complete length of the cell from the same position. Scale bars: 2 µm. See also supplementary material Table S2 and Movie 1.

Fig. 6.

Complete model of membranes, mitochondria and synapse distribution in cell 1. (A) Ribbon synapses (red spheres) are marked with asterisks for clarity. (B) Reconstructions of synaptic terminals are shown (blue). Scale bars: 2 µm. See supplementary material Movie 1.

Fig. 7.

Tomographic reconstruction of linkages between RER and mitochondria. (A–C) Slices from tomographic reconstructions of conventionally fixed mouse tissue showing linkages of three different mitochondria (Mt) to RER (R) (white arrows). (D) 3D reconstruction of mitochondria (blue) and membrane (green) showing the depth and arrangement of mitochondria–membrane links (purple). (E–G) Membrane–mitochondria links observed in HPF tissue from a guinea pig. Links (white arrows) are shown between mitochondria (M) and membranes (black arrows). Images E–G show links in an IHC. Scale bars: 50 nm. See supplementary material Movie 2.

Fig. 8.

Tomographic reconstruction of vesicle–membrane and vesicle–vesicle links on ER and at the ribbon synapse. (A–D) Linkages (white arrowheads) are shown between membrane vesicles (black arrowheads) and three different areas of the RER (R). Images show an average of five tomographic slices. Insets in C and D show reconstruction of these links. (E) Reconstruction of a section of RER (green with ribosomes in red) with linkages to mitochondria (blue) and vesicles (yellow) showing the linkages surrounding the membrane. (F,G) Tomographic reconstructions of vesicles in HPF guinea pig tissue. Images show an average of five tomographic slices. (F) Vesicle membrane linkages (G) HPF prepared vesicle showing apparent internal vesicle structure (black arrow). The inset shows the vesicle internal structure outlined in red. (H) Tomographic reconstruction of a mouse ribbon synapse; images show an average of nine consecutive tomographic slices. The 3D model shows vesicles (green) with vesicle–vesicle links (red) and vesicles without linkages (purple) around the synaptic ribbon (blue). The synapse is viewed from four positions: through the pre-synaptic membrane (grey), from the IHC cytoplasm, and from the top and bottom of the synaptic ribbon. Scale bars: 50 nm (A–D, H); 10 nm (inset in C), 20 nm (inset in D), 100 nm (E), 25 nm (F), 25 nm (G, inset in G). See supplementary material Movie 2.