Age-related cochlear synaptopathy: an early-onset contributor to auditory functional decline

Abstract

Aging listeners experience greater difficulty understanding speech in adverse listening conditions and exhibit degraded temporal resolution, even when audiometric thresholds are normal. When threshold evidence for peripheral involvement is lacking, central and cognitive factors are often cited as underlying performance declines. However, previous work has uncovered widespread loss of cochlear afferent synapses and progressive cochlear nerve degeneration in noise-exposed ears with recovered thresholds and no hair cell loss (Kujawa and Liberman 2009). Here, we characterize age-related cochlear synaptic and neural degeneration in CBA/CaJ mice never exposed to high-level noise. Cochlear hair cell and neuronal function was assessed via distortion product otoacoustic emissions and auditory brainstem responses, respectively. Immunostained cochlear whole mounts and plastic-embedded sections were studied by confocal and conventional light microscopy to quantify hair cells, cochlear neurons, and synaptic structures, i.e., presynaptic ribbons and postsynaptic glutamate receptors. Cochlear synaptic loss progresses from youth (4 weeks) to old age (144 weeks) and is seen throughout the cochlea long before age-related changes in thresholds or hair cell counts. Cochlear nerve loss parallels the synaptic loss, after a delay of several months. Key functional clues to the synaptopathy are available in the neural response; these can be accessed noninvasively, enhancing the possibilities for translation to human clinical characterization.

Figure 1.

Aging mice show late-onset elevation of cochlear thresholds, preceded by selective decline of neural suprathreshold amplitudes. A, B, Mean thresholds (±SEM) for DPOAEs and ABR Wave 1, as measured at eight ages. The key in C applies to all panels. Actual ages were within ±5% of the target. When responses were not observed at the highest sound level, threshold was assigned a value 5 dB greater than the stimulus maximum; this was common only at 144 weeks, where threshold curves are displayed with dashed lines. Group sizes were 4 weeks (12), 16 weeks (20), 32 weeks (12), 64 weeks (16), 80 weeks (10), 96 weeks (20), 128 weeks (20), and 144 weeks (10). C, D, Mean amplitude versus level functions (±SEM) at 12 kHz for DPOAEs (C) and ABR Wave 1 (D) for the same animals shown in A and B (except for 144 weeks, where waveforms were too small and irregular to reliably parse). In all panels, symbols for 80 week animals are filled with red.

Figure 2.

ABR waveforms in aging mice suggest an auditory neuropathy. A, Representative ABR waveforms (12 kHz, 80 dB SPL) at 4 weeks (black) versus 80 weeks (red) show a large reduction in Wave 1 with minimal reduction in Wave 5; dotted lines mark Wave 1 peak and trough. B, Mean amplitude ratios for ABR Wave 5 to Wave 1, at 4 or 80 weeks (12 kHz at 80 dB SPL). C, D, Mean waveforms at different ages for the early (cochlear) components of the ABR (12 kHz). In C, responses are shown at equal SPL, i.e., 80 dB SPL. Wave 1 peak to trough is indicated for the 4 week response. In D, responses are shown for equal SL, i.e., 50 dB above ABR threshold. Data are shown for ages to 80 week, the oldest age at which 50 dB SL data are available. The key in C applies to all panels; group sizes are as given for Figure 1.

Figure 3.

Accelerating threshold deterioration beyond 80 weeks is mirrored by an accelerating loss of OHCs. A–C, Survival of OHCs, IHCs, and spiral ganglion cell (SGCs) at three cochlear locations and eight ages (key in B also apples to A and C). Data are means (±SEM) expressed as percentage of survival at 4 weeks. Mean 4 week values were as follows: OHCs (per row), 14.88, 15.57, and 14.64 cells per 100 μm of cochlear length; IHCs, 12.99, 13.00, and 12.91 cells per 100 μm; SGCs, 8.80, 10.60, and 10.40 cells per 540 μm² at 5.6, 11.3, and 32 kHz, respectively. D–F, Mean cell-survival data from panels A–C are replotted (left-hand axis) as a function of age at each of the three cochlear locations, where they are compared to mean threshold shifts (4 week means) from the same animals (right-hand axis). Shifts at 144 week are underestimated, since there was no response at the stimulus maximum in some cases. OHC counts are averages of all three rows. The key in D also applies to E and F. Group sizes are as given for Figure 1, except for SGC counts, which were obtained for a subset of ears: 4 weeks (6), 16 weeks (15), 32 weeks (12), 64 weeks (13), 80 weeks (8), 96 weeks (15), 128 weeks (9), and 144 weeks (8).

Figure 4.

Confocal imaging of presynaptic ribbons and postsynaptic glutamate receptors shows age-related cochlear neuropathy. A, Schematic cross-section showing three of the ∼20 auditory nerve fibers (ANFs) making synaptic contact with an IHC. Presynaptic ribbons and postsynaptic receptor patches are also schematized. The x–y–z axis shows the viewing angle for the confocal x–y projections shown in B and C. B, C, Maximum projections of confocal z-stacks obtained in the 32 kHz region from a 4 week (B) and a 144 week (C) ear. These epithelial whole mounts were triple stained for presynaptic ribbons (CtBP2; red), postsynaptic glutamate receptors (GluA2; green), and hair cells (myosin VIIA; blue). The anti-CtBP2 also faintly stains IHC nuclei. One juxtaposed ribbon–receptor pair is indicated by the red and green arrow (B); one orphan ribbon is indicated by the red arrow (C). The cuticular plate of an IHC is indicated by the white and blue arrow (B). D, High-power views of selected synaptic complexes illustrating the juxtaposition between presynaptic and postsynaptic elements and the occasional orphan ribbons (red arrows) seen in both young and old ears. Scale bars: (in C) B, C, 5 μm; D, 1 μm.

Figure 5.

Synaptic counts in IHCs, but not OHCs, decrease steadily throughout the cochlea from 4 to 144 weeks. A, B, Counts of presynaptic ribbons in IHCs (A) and OHCs (B) at three cochlear locations versus age. Means (±SEMs) are normalized to 4 week values: 2.45, 2.52, and 2.19 ribbons per OHC and 9.25, 17.91, and 17.11 ribbons per IHC at 5.6, 11.3, and 32 kHz, respectively. C, IHC ribbon counts from A were averaged across cochlear frequency, replotted versus life span, and compared to frequency-averaged spiral ganglion cell (SGC) counts from the same animals. D, IHC ribbon counts from A are plotted versus mean Wave 1 amplitudes (at 80 dB SPL) from the same animals (except the 144 week group, where ABR waveforms were so abnormal that wave classification was unreliable). Red-circled points are those from the 80 week group. Best-fit lines are shown for each stimulus/cochlear frequency. Group sizes are as given for Figures 1 and 3.

Figure 6.

Age-related declines in threshold and ganglion cell survival in mouse and man. A, Age-related threshold shifts versus percentage mean life span at two cochlear regions in humans versus two common strains of laboratory mice. Human audiometric thresholds are values for males from Pearson et al. (1995). ABR thresholds for CBA/CaJ are from the present study; data for C57BL/6 are from Hequembourg and Liberman (2001). To normalize for difference in mean life spans, we used 2.1 years for male CBA/CaJ and C57BL/6 mice (http://research.jax.org/faculty/harrison/ger1vi_LifeStudy1.html) and 76 years for human males (Kochanek et al. 2012). To normalize for difference in frequency range, we matched cochlear location, choosing loci 35 and 70% of the distance from the base, which, according to the relevant cochlear frequency maps (Schuknecht, 1994; Müller et al., 2005), correspond to 32 and 12 kHz in mice versus 4 and 0.75 kHz in humans. B, Spiral ganglion cell counts for CBA/CaJ are from the present study, averaged across all cochlear regions and displayed as the best-fit straight line. Group sizes are as given for Figure 3. Best-fit lines for C57BL/6 and human are computed from data in previous studies (Hequembourg and Liberman, 2001; Makary et al., 2011) and are also collapsed across all cochlear regions. For both panels, when data points represent a range of ages, the midpoint was used.