Age-related changes in the biophysical and morphological characteristics of mouse cochlear outer hair cells

Abstract

Key points: Age-related hearing loss (ARHL) is a very heterogeneous disease, resulting from cellular senescence, genetic predisposition and environmental factors (e.g. noise exposure). Currently, we know very little about age-related changes occurring in the auditory sensory cells, including those associated with the outer hair cells (OHCs). Using different mouse strains, we show that OHCs undergo several morphological and biophysical changes in the ageing cochlea. Ageing OHCs also exhibited the progressive loss of afferent and efferent synapses. We also provide evidence that the size of the mechanoelectrical transducer current is reduced in ageing OHCs, highlighting its possible contribution in cochlear ageing.

Abstract: Outer hair cells (OHCs) are electromotile sensory receptors that provide sound amplification within the mammalian cochlea. Although OHCs appear susceptible to ageing, the progression of the pathophysiological changes in these cells is still poorly understood. By using mouse strains with a different progression of hearing loss (C57BL/6J, C57BL/6NTac, C57BL/6NTac^Cdh23+ , C3H/HeJ), we have identified morphological, physiological and molecular changes in ageing OHCs (9-12 kHz cochlear region). We show that by 6 months of age, OHCs from all strains underwent a reduction in surface area, which was not a sign of degeneration. Although the ageing OHCs retained a normal basolateral membrane protein profile, they showed a reduction in the size of the K⁺ current and non-linear capacitance, a readout of prestin-dependent electromotility. Despite these changes, OHCs have a normal V_m and retain the ability to amplify sound, as distortion product otoacoustic emission thresholds were not affected in aged, good-hearing mice (C3H/HeJ, C57BL/6NTac^Cdh23+ ). The loss of afferent synapses was present in all strains at 15 months. The number of efferent synapses per OHCs, defined as postsynaptic SK2 puncta, was reduced in aged OHCs of all strains apart from C3H mice. Several of the identified changes occurred in aged OHCs from all mouse strains, thus representing a general trait in the pathophysiological progression of age-related hearing loss, possibly aimed at preserving functionality. We have also shown that the mechanoelectrical transduction (MET) current from OHCs of mice harbouring the Cdh23^ahl allele is reduced with age, highlighting the possibility that changes in the MET apparatus could play a role in cochlear ageing.

Keywords: OHCs; ageing; cochlea; electromotility; hearing loss; ion channels; mechanoelectrical transduction; mouse.

Figure 1. ABR thresholds evoked by frequency specific stimuli.

Aand B, Mean ABR thresholds for clicks (A) and frequency-specific pure tone stimulation from 3 kHz to 42 kHz (B) obtained from C3H, 6N, 6N-Repaired and 6J mice at 12-14 months of age. The arrows in B represent values above the upper threshold limit of our equipment (95 dB SPL). ABR thresholds were significantly elevated in 6J and 6N mice compared to both C3H and 6N-Repaired mice. For clicks: one-way ANOVA from single data points was: P < 0.0001. For frequency-specific stimulations, two-way ANOVA was P < 0.0001. For post-test comparisons, see Results.). *indicates statistical significance. Number of animals tested (males and females) is indicated by “n” and shown next to the different mouse strains. Values are mean ± S.D.

Figure 2. DPOAE thresholds in aged male and female mice

A-E, DPOAE thresholds measured in both males and females from 13-14 months old C3H (A, 15 mice), 6N-Repaired (B, 21 mice), 6N (C, 15 mice) and 6J (D, 14 mice) mice. Panel E shows the direct comparison of age-related changes in the median DPOAE threshold values for the four mouse strains. Because of the presence of “not-found” values (i.e. above the upper threshold limit of our system, 80dB) in all mouse strains investigated (see Methods), in this and the following figures DPOAE threshold values are shown as median (black line and circles), first (red lines) and third (blue lines) quartiles. Single values are reported as open circles. The number of mice with “found” and “not-found” values at each frequency is shown below (black) and above (grey) the median, respectively.

Figure 3. Age-related changes in DPOAE thresholds in male mice

A-D, DPOAE thresholds from males of C3H (A), 6N-Repaired (B) and 6N (C) at different ages for frequencies between 6 and 24 kHz. The number of mice with “found” and “not-found” values at each frequency is shown below and above the median, respectively. D-F, Comparison of age-related changes in the median DPOAE threshold values (from panels A-C) for the different mouse strains. Number of mice used for each strain/age is also shown.

Figure 4. Biophysical properties of OHCs in one-year old males and females

A-D, Potassium currents recorded from apical coil OHCs of 12-13 months old females of C3H (A), 6N (B), 6N-Repaired (C) and 6J (D) using 10 mV depolarizing voltage steps from – 124 mV to the various test potentials shown by some of the traces. The activation of the adult-type current I _K,n (indicated by the arrows) was present in all OHCs. E, Average currentvoltage (I-V) curves obtained from 12-13 months old OHCs from females and males of the above four mouse strains. F, Average size of the peak K⁺ current measured at the membrane potential of 0 mV, as previously described (Marcotti & Kros, 1999), in OHCs from males and females of the different mouse strains. G and H, Average OHC membrane capacitance (C_m: G) and resting membrane potential (V_m: H) in the different mouse strains. In panels F-H, individual cell values are also plotted as open symbols. Note that 6N-Repaired was abbreviated as 6N-Rep. Number of OHCs investigated is shown above the data points. Number of mice used: 12 C3H, 15 6N, 12 6N-Repaired and 9 6J. Values are mean ± S.D.

Figure 5. Age-related changes in OHC biophysical characteristics in males and females

A and B, Example of K⁺ currents recorded from apical coil OHCs of male 6N-Repaired (A) and 6N (B) mice at 1 month (left panels), 12 months (middle panels) and 16-17 months (right panels) of age. Currents were recorded using the voltage protocol described in Fig. 4. C, Peak K⁺ current (I _K) measured at the step potential of 0 mV from all four mouse strains at different ages. D, Membrane capacitance (C_m) of OHCs as function of age in the four mouse strains. E, Normalized peak IK (panel C) to the correspondent C _m (panel D) for each OHC tested. F, Resting membrane potential (V_m) as a function of age in all above mouse strains. In panels C-F, single cell values are also shown behind the average data. Number of OHCs investigated is shown above the average data points. Number of mice used: C3H (1 month: 6; 6 months: 6; 12-13 months: 12), 6N (1 month: 5; 6 months: 8; 12-13 months: 15; 15-17 months: 4), 6N-Repaired (1 month: 7; 6 months: 5; 12-13 months: 12; 15-17 months: 4) and 6J (1 month: 19; 6 months: 7; 12-13 months: 9; 15-17 months: 2). For statistical analysis see Results. Values are mean ± S.D.

Figure 6. Prestin expression and age-related changes in the number of OHCs

A-D, Maximum intensity projections of confocal z-stacks taken from the apical cochlear region of 1 month and aged 6N (A and B) and 6N-Repaired (C and D) mice. OHCs were stained with antibodies against prestin (green). Scale bars: 10 μm. E-H, Number of OHCs present in a 140 μm region from the apical coil of 1, 6 and 14-16 months old mice from the four mouse strains used in the previous electrophysiological experiments. OHC counting from individual cochleae (open symbols) are plotted behind the average data and were collected from both males and females. Number of mice investigated is shown above or below the data points. Values are mean ± S.D. For statistical analysis, see Results.

Figure 7. Aged OHCs exhibit electromotility

A and B, Examples of voltage-dependent non-linear capacitance (C _N-L) recorded in apical-coil OHCs from 1 and aged 6N (A) and 6J (B) mice by applying a voltage ramp from –154 mV to +96 mV over 2s. Note that the cell membrane capacitance (Cm) was added to the measured C _N-L. C _N-L was absent in the non-electromotile IHCs, which was used as a control for our experiments. C and D, Average C _N-L was reduced in OHCs from both mouse strains with age (C), but not after normalisation to the OHC membrane capacitance C _m (D). C _N-L was calculated as the difference between the peak of the recording near -40 mV and the lowest value at positive membrane potentials. For statistical analysis, see Results. Number of cells investigated is shown above the data. Individual measurements are plotted behind the average data points and were collected from both males and females. Values are mean ± S.D. E,F, Quantitative real time PCR (qPCR) from 6N and 6N-Repaired mice at 1 and 15 months of age. Genes investigated: Slc26a5 (E, prestin) and Ocm (F, oncomodulin). Number of mice tested is shown above the data. Values are mean ± S.D. G and H, Toluidine blue stained semithin plastic sections from the 8 kHz and 20 kHz region of the cochlea of 17 months old 6N (G) and 6N-Repaired mice (H) mice. The tectorial membrane (TM) is attached to the spiral limbus (arrows) and laying on top of the OHCs (arrowheads).

Figure 8. Ribbon synapse number is reduced in OHCs from aged mice

A and B, Maximum intensity projections of confocal z-stacks taken from the apical cochlear region of 6N (A) and 6N-Repaired (B) mice at 1, 6 and 15 months using antibodies against CtBP2 (ribbon synaptic marker: white). Myosin 7a (Myo7a) was used as a hair cell marker (blue). Scale bar 10 μm. C-F, Number of CtBP2 puncta as a function of age in OHCs from C3H (C), 6N (D), 6N-Repaired (E) and 6J (F) mice. Data are plotted as mean values (± S.D.) and the individual OHC counts are shown by the open symbols. Numbers above the data represent the number of mice and (OHCs) used for each time point.

Figure 9. Efferent synapses are present in aged OHCs

A and B, Maximum intensity projections of confocal z-stacks taken from the apical cochlear region of 6N (A) and 6N-Repaired (B) mice at 1 and 15 months of age using antibodies against SK2 (green) and ChAT (red). Myosin 7a (Myo7a) was used as a hair cell marker. Arrowheads indicate OHCs with only efferent terminals; and arrows indicate OHCs with only anti-SK2 signals. Scale bars: 10 μm. C, Number of SK2 puncta per OHCs present in a 140 μm apical cochlea region as a function of age in C3H, 6N, 6N-Repaired and 6J mice. D, Percentage of juxtaposed SK2 and ChAT puncta as a function of age and strain. Data are plotted as mean values (± S.D.) and individual OHC counts are indicated by the open symbols. Numbers below or above the data in panel C represent the number of mice and (OHCs) used for each time point, which also apply to panels D.

Figure 10. Changes in the mechanoelectrical transducer apparatus in ageing OHCs

A, Scanning electron microscopy (SEM) showing the gross morphology of the OHC stereociliary bundle from one-year old 6N mice in the 8 kHz cochlear region. B, Quantitative real time PCR (qPCR) from organ of Corti RNA, from 6N, 6J and 6N-Repaired mice at 1 and 15 months of age. Genes investigated: Cdh23 (cadherin 23) and Pcdh15 (protocadherin 15). Number of mice tested is shown above the columns. Data are shown as mean ± S.D. C and D, SEM showing the gross morphology of the OHC stereociliary bundle from aged Tecta/b^-/- mice. E and F, Example of K⁺ currents recorded from apical-coil OHCs of 1 month (E) and 11 months (F) Tecta/b^-/- mice. Currents were recorded using the voltage protocol described in Fig. 4. G, Peak K⁺ current (I _K) measured from Tecta/b^-/- mouse OHCs at the step potential of 0 mV, from the holding of –84 mV, at 1 and 11 months of age. H and I, Saturating MET currents recorded from OHCs of 1 month (H) and 11 months (I) Tecta/b^-/- mice by applying sinusoidal force stimuli of 50 Hz to the hair bundles from a holding potential of –84 mV. The driver voltage (V Piezo) signal of ±40 V to the fluid jet is shown above the traces (positive halfcycles of the V Piezo o are inhibitory). The extracellular Ca²⁺concentration was 1.3 mM. The arrows indicate the complete closure of the transducer currents elicited during inhibitory bundle displacements (note that the difference in current between this level and the dashed lines represents the size of the resting MET current). Dashed lines indicate the holding current, which is the current at the holding membrane potential. J and K, Maximal size (J) and resting open probability (K) of the MET current recorded at the two different ages from OHCs of Tecta/b^-/- mice. The number of OHCs tested is shown above the data points.

Figure 11. Schematic representation of the biophysical and morphological profile of young adult and aged OHCs

A and B, Schematic representation of the basolateral membrane protein profile and innervation pattern of OHCs from young adult and aged 6N, 6J, C3H and 6N-Repaired mice. Note the reduction in size of OHCs that leads to the reduction in numbers of all the indicated membrane proteins in OHCs. The afferent (blue) and efferent (red) fibres are present at both ages, but become reduced in number in aged OHCs.