Hyperexcitability of inferior colliculus and acoustic startle reflex with age-related hearing loss

Abstract

Chronic tinnitus and hyperacusis often develop with age-related hearing loss presumably due to aberrant neural activity in the central auditory system (CAS) induced by cochlear pathologies. However, the full spectrum of physiological changes that occur in the CAS as a result age-related hearing loss are still poorly understood. To address this issue, neurophysiological measures were obtained from the cochlea and the inferior colliculus (IC) of 2, 6 and 12 month old C57BL/6J mice, a mouse model for early age-related hearing loss. Thresholds of the compound action potentials (CAP) in 6 and 12 month old mice were significantly higher than in 2 month old mice. The sound driven and spontaneous firing rates of IC neurons, recorded with 16 channel electrodes, revealed mean IC thresholds of 22.8 ± 6.5 dB (n = 167) at 2 months, 37.9 ± 6.2 dB (n = 132) at 6 months and 47.1 ± 15.3 dB (n = 151) at 12 months of age consistent with the rise in CAP thresholds. The characteristic frequencies (CF) of IC neurons ranged from 3 to 32 kHz in 2 month old mice; the upper CF ranged decreased to 26 kHz and 16 kHz in 6 and 12 month old mice respectively. The percentage of IC neurons with CFs between 8 and 12 kHz increased from 36.5% in 2 month old mice, to 48.8% and 76.2% in 6 and 12 month old mice, respectively, suggesting a downshift of IC CFs due to the high-frequency hearing loss. The average spontaneous firing rate (SFRs) of all recorded neurons in 2 month old mice was 3.2 ± 2.5 Hz (n = 167). For 6 and 12 month old mice, the SFRs of low CF neurons (<8 kHz) was maintained at 3-6 spikes/s; whereas SFRs of IC neurons with CFs > 8 kHz increased to 13.0 ± 15.4 (n = 68) Hz at 6 months of age and then declined to 4.8 ± 7.4 (n = 110) spikes/s at 12 months of age. In addition, sound-evoked activity at suprathreshold levels at 6 months of age was much higher than at 2 and 12 months of age. To evaluate the behavioral consequences of sound evoked hyperactivity in the IC, the amplitude of the acoustic startle reflex was measured at 4, 8 and 16 kHz using narrow band noise bursts. Acoustic startle reflex amplitudes in 6 and 12 month old mice (n = 4) were significantly larger than 2 month old mice (n = 4) at 4 and 8 kHz, but not 16 kHz. The enhanced reflex amplitudes suggest that high-intensity, low-frequency sounds are perceived as louder than normal in 6 and 12 month old mice compared to 2 month olds. The increased spontaneous activity, particularly at 6 months, may be related to tinnitus whereas the increase in sound-evoked activity and startle reflex amplitudes may be related to hyperacusis.

Keywords: Age-related hearing loss; Hyperacusis; Hyperexcitability; Inferior colliculus; Tinnitus.

Figure 1.

The thresholds of the compound action potentials (CAP) recorded from C57BL/6J mice showed a significant increase with age. The mean averaged CAP thresholds in the 6 month old group (G-6M, n=6) and 12 month old group (G-12M, n=6) were significantly higher than the 2 month old group (G-2M, n=6) (One-way ANOVA, df = 2, P<0.001. “↑” indicates there was no response at the maximum tested intensity at 80 dB SPL).

Figure 2.

Distribution of the minimal thresholds (MT) and the characteristic frequencies (CF) of the inferior colliculus (IC) neurons recorded in 2, 6, and 12-month-old mice. (A) In the young mice (G-2M), the CFs was evenly distributed from 3 to 32 kHz with MTs from 10–45 dB SPL (n = 167). (B) In the 6 month old mice, animals displayed fewer high frequency neurons at above 16 kHz and the averaged MT increased about 20 dB compared to the 2 month old mice. (C) In the 12 month old mice (G-12M), all of the IC neurons showed a CFs less than 12 kHz and the preponderance of neurons concentrated in around the 8.5 kHz.

Figure 3.

Changes of the distribution of the characteristic frequencies (CF) and minimal threshold changes with age. (A) The percentage of recorded neurons with different characteristic frequencies. There was a clear increment of the percentage of the 8–12 kHz neurons in the G-12M group (76.16%) and a reduction of the high frequency neurons at above 12 kHz in the G-6M and G-12M groups. (B) The averaged minimal threshold of IC neurons at different age groups. The mean minimal threshold increased with age (One-way ANOVA, df =2, *** P<0.0001, ** P<0.001).

Figure 4.

Tuning curves recorded from inferior colliculus neurons at 2, 6 and 12 month old mouse groups. (A-B) Examples of a low frequency neuron and a high frequency neuron in a 2-month old mouse. The tuning curve showed a steep slope on the frequency above the characteristic frequencies (CF) and a broad tuning on below the CF. (C-D) Examples of tuning curve recorded from a 6-month old mouse, which developed broad tunings on both side of the CF. (E-F) Examples of tuning curves recorded from a 12-month old mouse, which showed significant elevated thresholds and broad tunings compared the 2-month age group.

Figure 5.

The Q10 and Q30 values of the tuning curve of the inferior colliculus neurons recorded at 2, 6 and 12 month old groups. There was a significant decrease of Q10 and Q30 at low frequencies IC neurons (<8 kHz) in the 12 month old mice compared to the 2 and 6 month old mice (One-way ANOVA, F_Q10=16.46, df = 2, *** P<0.0001; F_Q30=11.62, df = 2, *** P<0.0001, ** P<0.001). There was no significant difference of Q10 and Q30 in the IC neurons at other frequencies.

Figure 6.

The spontaneous firing rates (SFRs) of the inferior colliculus neurons increased with age. (A) The SFRs changed with IC neurons with different characteristic frequencies in 2, 6 and 12 month old mice. In G-2M (blue circle), the SFRs were lower than 10 spikes/s for all the neurons with CFs at 3 to 32 kHz. In contrast, the SFRs in G-6M (red dot) increased to 60 spikes/s for CFs near 12 kHz, which were much higher than the 2-month-old mice. In G-12M, the SFRs (black triangle) decreased compared to the 6-month-old mice, but still higher than the 2-month-old mice. (B) The SFRs of below 8 kHz CF neurons were 3.53 ± 1.95 (n = 53), 6.17 ± 4.07 (n = 34) and 4.13 ± 4.66 (n = 41) spikes/s for the 2, 6 and 12 month groups, respectively (One-way ANOVA test, F = 5.797, P<0.01. Bonferroni post-test, *** P <0.001 between G-6M and G-2M, * P <0.05 between G-6M and G-12M). Between 8 – 12 kHz, the SFRs further increased to 13.01 ± 15.45 (n = 68) spikes/s in 6 month old mice, but did not change in the 2 month old mice and 12 month old mice (3.16 ± 2.52 spikes/s, n =65, and 4.80 ± 7.37 spikes/s, n= 110, respectively) (One-way ANOVA test, F = 21.08, P<0.0001. Bonferroni post-test, *** P <0.0001 between G-6M and G-2M, G-6M and G-12M). For high frequency neurons at above 12 kHz, the SFRs in the 6 month old mice were much higher than the 2 month old mice (Student’s test, *** P<0.0001).

Figure 7.

The firing-rate-level function of the inferior colliculus (IC) neurons elicited by noise-bursts (50 ms, 0 to 100 dB SPL) recorded from 2, 6, and 12-month-old mice. For the 2 month old group (blue circle), the averaged firing rates maintained at a very low level to sound stimuli between 0 and 20 dB SPL, increased almost linearly from 30 to 60 dB SPL and saturated at above 60 dB and reached to 194 ± 15 Hz at 100 dB SPL. For the 6 month group (red square), the firing rates was low at 0–40 dB SPL and showed a rapid increase at above 50 dB SPL and reached to 334 ± 12 Hz at 100 dB SPL (n = 132). The mean discharge rate of the G-12M group) was low at 60–90 dB SPL (black triangle, n=151) and was slightly higher than the 2 month old group at 100 dB SPL. The averaged firing rates were significant different at 70, 80, 90 and 100 dB SPL (grey shade area) between the three groups (two-way ANOVA, F= 225.8, df=11, 780, P<0.0001). The rate-level functions were fitted with logarithm curves (dashed lines). The slope of the logarithm function was 0.04, 0.067 and 0.086, and the EC50 (the sound intensity that induced halfway response between the baseline to the maximum response) was 44, 60 and 95 dB SPL for the G-2M, G-6M and G-12M groups, respectively.

Figure 8.

The peristimulus time histograms (PSTHs) recorded from the inferior colliculus (IC) neurons elicited by tone bursts (4.8, 8.7, 15.7 and 23.3 kHz at 80 dB SPL) of 2, 6, and 12 month old mice. Compared to the 2 month old group (blue), the PSTHs in the 6 month old group (red) were much higher at 8.7 kHz and 10.6 kHz (One-way ANOVA, df = 2, P<0.001). In the 12-month-old group, the PSTHs showed significant reduction at high frequencies (10.6 to 23.3 kHz) compared to the 2-month-old group. However, at low frequencies (4.8 to 8.7 kHz), the 12-month old group showed no difference or even slightly higher peaks compared to the 2-month-old group. These results suggest there may be a frequency sensitivity shift from high to low with age.

Figure 9.

Examples of the peristimulus time histograms (PSTHs) recorded from 2, 6, and 12-month-old mice. PSTHs were elicited by tone bursts at different intensities (10 to 90 dB SPL) at 4.8 kHz (A), 8.7 kHz (B) and 15.7 kHz (C). (A) At 4.8 kHz, the PSTHs showed a significant reduction at low intensities (<50 dB SPL) and no obvious decrease at supra-threshold levels (>60 dB SPL) in the 6 month group (red, n = 132) and the 12 month group (black, n = 151) compared to 2 month group (blue, n=167). (B) At 8.7 kHz, the PSTHs showed a significant increase at high intensities (> 50 dB SPL) in the 6-month group (red) compared to the 2-month group (blue). The 12-month group showed a significant reduction at all tested intensities. (C) At 15.7 kHz, the peak amplitude of the PSTHs in the 6-month group showed a significant increase at high intensities (> 70 dB SPL) compared to the 2-month group (blue). In the 12-month group, there was no obvious response below 80 dB SPL.

Figure 10.

Response of acoustic startle reflex (ASR) of 2, 6 and 12 month old mice elicited by 4, 8, 16 kHz sound stimuli. (A) A raw startle response recorded from a mouse. (B) At 4 kHz, the mean amplitude of ASR in the 6-month-old mice was significantly higher than for 2 and 12 month old mice. (C) At 8 kHz, The ASR in the 6-month-old mice was also significant higher than the 2-month-old mice, but not to the 12-month-old mice. (D) At 16 kHz, the amplitude of ASR in the 6 and 12-month group was significantly lower than the 2-month-old mice.