Maturation of Heterogeneity in Afferent Synapse Ultrastructure in the Mouse Cochlea

Abstract

Auditory nerve fibers (ANFs) innervating the same inner hair cell (IHC) may have identical frequency tuning but different sound response properties. In cat and guinea pig, ANF response properties correlate with afferent synapse morphology and position on the IHC, suggesting a causal structure-function relationship. In mice, this relationship has not been fully characterized. Here we measured the emergence of synaptic morphological heterogeneities during maturation of the C57BL/6J mouse cochlea by comparing postnatal day 17 (p17, ∼3 days after hearing onset) with p34, when the mouse cochlea is mature. Using serial block face scanning electron microscopy and three-dimensional reconstruction we measured the size, shape, vesicle content, and position of 70 ribbon synapses from the mid-cochlea. Several features matured over late postnatal development. From p17 to p34, presynaptic densities (PDs) and post-synaptic densities (PSDs) became smaller on average (PDs: 0.75 to 0.33; PSDs: 0.58 to 0.31 μm²) and less round as their short axes shortened predominantly on the modiolar side, from 770 to 360 nm. Membrane-associated synaptic vesicles decreased in number from 53 to 30 per synapse from p17 to p34. Anatomical coupling, measured as PSD to ribbon distance, tightened predominantly on the pillar side. Ribbons became less spherical as long-axes lengthened only on the modiolar side of the IHC, from 372 to 541 nm. A decreasing gradient of synaptic ribbon size along the modiolar-pillar axis was detected only at p34 after aligning synapses of adjacent IHCs to a common reference frame (median volumes in nm³ × 10⁶: modiolar 4.87; pillar 2.38). The number of ribbon-associated synaptic vesicles scaled with ribbon size (range 67 to 346 per synapse at p34), thus acquiring a modiolar-pillar gradient at p34, but overall medians were similar at p17 (120) and p34 (127), like ribbon surface area (0.36 vs. 0.34 μm²). PD and PSD morphologies were tightly correlated to each other at individual synapses, more so at p34 than p17, but not to ribbon morphology. These observations suggest that PDs and PSDs mature according to different cues than ribbons, and that ribbon size may be more influenced by cues from the IHC than the surrounding tissue.

Keywords: FIB-SEM; developmental maturation; modiolar; pillar; postsynaptic density; presynaptic density; ribbon synapse ultrastructure; synaptic vesicles.

FIGURE 1

Block face imaging with Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). Schematic of the dual beam method for serial block face imaging of inner hair cell (IHC) ribbon synapses. The milling beam and the imaging beam are offset by 52°. A protective pad made of carbon and platinum (C, Pt) was built over the IHCs by plasma-enhanced chemical vapor deposition using the gallium (Ga) FIB. The FIB was then used to bulk-trench the tissue in front of the IHCs, exposing these cells to the SEM. Images were acquired of this face at a resolution of 7 nm/pixel using backscatter detection. After collecting an image of the block face, the milling beam removed 7 nm of the sample from the surface before collecting the next image. This process was repeated until the sample was milled through the IHCs to the tunnel of Corti.

FIGURE 2

Segmentation of ribbons and membrane densities to generate 3-dimensional models of cochlear ribbon synapses. (A) Each of the 5 rows of images illustrates one ribbon synapse from the p17 sample. The volume of tissue surrounding each synapse was cropped in 3 dimensions. Column 1 shows images from the center of the stack, oriented with the presynaptic IHC on top, the postsynaptic afferent bouton on bottom, and the ribbon synapse in the center. The faint yellow dashed line in column 1 of row 1, passing through the center of the ribbon and parallel to the synaptic cleft, indicates the position of an orthogonal plane or section shown in column 2. The area inside the faint yellow dashed box in column 2 is enlarged in column 3, where the segmentation of synaptic structures is demonstrated. Column 4 contains a 2-dimensional representation of the 3-dimensional model generated from the series of segmented images. (B) Same as (A), for the p34 sample. Red, presynaptic ribbon; green, pre-synaptic density; blue, post-synaptic density. Scale bars are 1 μm.

FIGURE 3

Ribbon synapse reconstructions from inner hair cells at p17 and p34. (A) 2-dimensional renderings of 3-dimensional reconstructions for p17 synapses. Each synapse is shown in, both, the en face or top-down view (upper) and a side view (lower). (B) Same as in (A), for p34. Red, presynaptic ribbon; green, pre-synaptic density; blue, post-synaptic density. Scale bars are 1 μm.

FIGURE 4

Morphological maturation of pre- and post-synaptic membrane densities and presynaptic ribbons. (A) Binned histogram of post-synaptic density surface areas (PSD SA) at p17 (filled gray bars) and p34 (hollow black bars). (B) Scatter plot of PSD SA vs. PSD short axis shows reduction in both metrics from p17 to p34 (SA, p = 6.5e^–4; short axis, p = 7.9e^–6; Wilcoxon). (C) PSD SA vs. PSD long axis shows that long axes are relatively unchanged between p17 and p34 (p = 0.97). (D) Pre-synaptic density (PD) SA vs. PSD SA for p17 and p34 (ρ = 0.90 and 0.87, respectively, Spearman’s Rho). (E) Long: short axis ratio of the PD vs. Long: Short axis ratio of the PSD shows positive correlation at both ages and an increase in both ratios from p17 to p34. (F) Binned histogram of synaptic ribbon volumes at p17 to p34, showing change in shape of distribution without significant difference of the mean (p = 0.07; Wilcoxon). (G) Scatter plot of ribbon volume vs. ribbon SA for p17 and p34. Neither changed significantly with maturation (p = 0.6). (H) PSD short axis vs. ribbon long axis. From p17 to p34, narrowing of the PSD short axis distribution and significant shortening (p = 7.9e^–6) was accompanied by an increase in the upper range of ribbon long axes, although the increase in central tendency was not significant (p = 0.07). (I) Ribbon volume vs. PSD SA shows no clear relationship at p17 or p34. (J) Ribbon Long: Short axis ratio vs. PSD Long: Short axis ratio shows a developmental shift toward less spherical ribbons and less round PSDs from p17 to p34, but the degree of elongation on one side of the synapse is not well predicted by the degree of elongation on the other side.

FIGURE 5

Inner hair cell segmentation and 3-dimensional reconstruction of ribbon synapse position in the organ of Corti. The diagram on the left indicates three orthogonal planes (A, B, and C) through the basolateral compartment of the IHC demonstrated in panels A, B, and C, respectively. The hair bundle is on the apical side (top) and the synapses are on the basal side (bottom). The cochlear spiral extends to the left and right. The modiolar-pillar axis is perpendicular to the page. (A) Original view–from the microscope perspective as the images were collected–for p17 (upper) and p34 (lower). On left, the central IHC (pink) is shown with segmented synapses overlaid on the raw data in the XY plane. On right, the IHC is colored in gray in a 2-dimensional rendering of the 3-dimensional reconstruction, oriented with the modiolar face in front. Red, presynaptic ribbon; green, pre-synaptic density (PD); blue, post-synaptic density (PSD). (B) The virtual XZ plane, showing the top-down view of the image in panel (A). The modiolar side is on bottom and the pillar side is on top. (C) The virtual ZY plane, showing the side view of the image in panel (A). Isotropic voxels enabled virtual sectioning in any orientation without distortion. Scale bars are approximately 3 μm.

FIGURE 6

Translation of synapse positions to a common IHC reference frame. (A) Lower: Schematic indicating the three orthogonal planes demonstrated in panels A, B, and C (as in Figure 5). X-axis is the cochlear spiral; Y-axis is apical-basal; Z-axis is modiolar-pillar. Upper: Image acquired in the XY plane (original view) from the p17 sample. Two IHCs are colored light blue and blue; two afferent fibers are colored yellow and green; one synaptic ribbon is colored orange. Green dashed line indicates plane of virtual section displayed in panel (B). (B) Upper: Virtual section indicated in panel A, showing top-down view in the ZX plane. Z-axis is the modiolar-pillar dimension. IHCs shifted to the modiolar side are light blue; IHCs shifted to the pillar side are darker blue; inner border cells are orange; inner pillar cells are violet. The terminal of the green fiber contacts the orange ribbon on the dark blue IHC. The terminal of the yellow fiber contacts a magenta ribbon on the light blue IHC. Black dashed line indicates plane of virtual section displayed in panel (C). Lower, left: Schematic of IHCs in the native positions from the top view. Right: Translated positions, after aligning IHCs in the modiolar-pillar dimension. (C) Virtual section indicated in panel B, showing side view in the ZY plane. Lower, left: Schematic of IHCs in the native positions from the side view. Right: Translated view, after aligning IHCs in the modiolar-pillar and apical-basal dimensions. Scale bars are approximately 5 μm.

FIGURE 7

Ribbon morphology by modiolar-pillar position. (A) Synapse position along the basal – apical (or habenular – cuticular) axis of the IHC versus synapse position along the modiolar – pillar (M-P) axis of the organ of Corti (native view, upper) or of the superimposed IHCs (translated view, lower) for p17 (left) and p34 (right). Marker diameter is proportional to ribbon volume; marker color identifies the ribbon with one of 3 IHCs in the imaging region. In the translated view, a schematic of an IHC (light blue) and its afferents (light yellow) is overlaid to indicate the modiolar and pillar halves of the IHC. (B) Ribbon surface area versus M-P position in the native view (upper) or the translated view (lower) for p17 (left) and p34 (right). Gold and purple markers correspond to modiolar and pillar groups, respectively. Ribbon SA showed a significant M-P gradient in the translated view at p34 (p = 4.4E-03; rho = –0.48). (C) Ribbon volume versus M-P position in the translated view at p17 (left) and p34 (right). Ribbon volume showed a significant M-P gradient at p34 (p34: p = 6.4E-11; rho = –0.51). (D) Ribbon Long axis: Short axis ratios for modiolar (gold) or pillar (purple) ribbons in the translated view for p17 and p34. Within each box, horizontal line denotes the median; box extends the interquartile range; vertical line denotes the 10-90 percentile range. Significant difference in ratios over development (p = 5.3E-06) came mainly from significant changes on the modiolar side (p = 5.2E-05). (E) Ribbon long axis (filled circles) and short axis (open circles) versus M-P position in the translated view for p17 (left) and p34 (right). The ribbon long axis showed a significant M-P gradient at p34 (p = 1.1E-03; rho = –0.54). *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 8

Post-synaptic density (PSD) morphology by modiolar-pillar position. (A) Synapse position along the basal – apical (or habenular – cuticular) axis of the IHC versus synapse position along the modiolar – pillar (M-P) axis of the organ of Corti (native view, upper) or of the superimposed IHCs (translated view, lower) for p17 (upper) and p34 (lower). Marker diameter is proportional to PSD surface area; marker color identifies the PSD with one of 3 IHCs in the imaging region. In the translated view, a schematic of an IHC (light blue) and its afferents (light yellow) is overlaid. (B) PSD surface area versus M-P position in the native view (upper) or the translated view (lower) for p17 (left) and p34 (right). Gold and purple markers correspond to modiolar and pillar groups, respectively. Significant gradients were observed at p17 in native (p = 7.6E-02; rho = 0.28) and translated views (p = 1.9E-02; rho = –0.36). (C) PSD (left) and PD (right) Long axis: Short axis ratios for modiolar (gold) or pillar (purple) ribbons in the translated view for p17 and p34. Significant difference in the PSD Long: Short axis ratio over development (p = 8.8E-09) came from changes on the modiolar side (p = 6.6E-09). Significant difference in the PD Long: Short axis ratio over development (p = 7.0E-07) came from changes on the modiolar side (p = 3.8E-09). (D) PSD long axis (filled circles) and short axis (open circles) versus M-P position in the translated view for p17 (left) and p34 (right). *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 9

Maturation of pre- and post-synaptic morphological relationships by modiolar-pillar position. (A) PSD SA vs. PD SA (left) or Ribbon SA (right). The correlations between PSD SA and PD SA were significant at p17 (p = 3.7E-03; rho = 0.9) and p34 (p = 3.3E-03; rho = 0.94). In all panels: open circles are p17, filled circles are p34; gold circles are modiolar, purple circles are pillar. (B) PSD L:S axis ratio vs. PD L:S axis ratio (left) or Ribbon L:S axis ratio (right). (C) PSD SA (left) or PD SA (right) vs. Ribbon volume. (D) PSD short axis (left) or PD short axis (right) vs. Ribbon long axis.

FIGURE 10

Comparing synapse morphology with models of synapse geometry. (A–D) Left: Observations of diversity in PSD shape in the raw data. Each row is one synapse shown from different perspectives. Right: Reconstructions in side views and top views. (E–F) Geometric models used for comparison with PSD shape, shown from the side (E) and top views (F). Yellow lines show measurements of shortest distance from points on the PSD to the ribbon. Scale bars are approximately 1 μm.

FIGURE 11

Post-synaptic density (PSD) shape measurements and comparison with geometric models. (A,B) Proximity of the ribbon and the synaptic cleft was quantified with shortest-distance measurements from points on the PSD to the synaptic ribbon, expressed as cumulative probability density functions (Cum. PDF). Synapses were grouped as modiolar (top) or pillar (bottom), according to the translated view, for p17 (A) and p34 (B). The red lines in panels (A,B) denote synapses assigned as pillar in the native view; the blue lines denote synapses assigned as modiolar in the native view. (C) Box plots of the median distance (top) and 15% percentile distance (bottom) from PSD to Ribbon at p17 and p34. Gold is modiolar; purple is pillar. Significant shortening of median distances between p17 and p34 (p = 2.8E-03) came mainly from the pillar side (2.6E-03) which was also significant for the 15th percentile distance (4.7E-02). (D,E) Average cumulative probability density functions of each group in the native (D) and translated (E) views. Top graphs are on a log scale and bottom graphs are on a linear scale. (F) Cumulative probability density functions of the four model synapses from Figure 10 (solid lines). Dashed lines are data from synapses depicted in Figure 10: Black dashed line is 10A; Green dashed line is 10B; Blue dashed line is 10C; Red dashed line is 10D. *p < 0.05, ***p < 0.001.

FIGURE 12

Vesicle content vs. synapse size by M-P position at p17 and p34. (A) Box plots of ribbon associated vesicles. (B,C) p17 (left) and p34 (right) ribbon-associated synaptic vesicles per synapse vs. Ribbon SA (B) or PSD SA (C) for modiolar (gold) or pillar synapses (purple). The correlations between #Vesicles/synapse and Ribbon SA were significant (p17 Pillar, p = 3.0E-04; Modiolar, p = 3.1E-02; p34 Pillar, p = 1.7E-09; Modiolar, p = 3.4E-04). (D) Box plots of membrane-associated vesicles. (E,F) p17 (left) and p34 (right) membrane-associated synaptic vesicles per synapse vs. ribbon SA (E) and PD SA (F). (G,H) Reconstructions of synapses in the top (G) and side views (H). Ribbon is red; PD is green; PSD is blue; Ribbon-associated vesicles are yellow; Membrane-associated vesicles are magenta. Scale bars are approximately 1 μm. **p < 0.01, ****p < 0.0001.