Selective Impairment in Frequency Discrimination in a Mouse Model of Tinnitus

<div><p>Tinnitus is an auditory disorder, which affects millions of Americans, including active duty service members and veterans. It is manifested by a phantom sound that is commonly restricted to a specific frequency range. Because tinnitus is associated with hearing deficits, understanding how tinnitus affects hearing perception is important for guiding therapies to improve the quality of life in this vast group of patients. In a rodent model of tinnitus, prolonged exposure to a tone leads to a selective decrease in gap detection in specific frequency bands. However, whether and how hearing acuity is affected for sounds within and outside those frequency bands is not well understood. We induced tinnitus in mice by prolonged exposure to a loud mid-range tone, and behaviorally assayed whether mice exhibited a change in frequency discrimination acuity for tones embedded within the mid-frequency range and high-frequency range at 1, 4, and 8 weeks post-exposure. A subset of tone-exposed mice exhibited tinnitus-like symptoms, as demonstrated by selective deficits in gap detection, which were restricted to the high frequency range. These mice exhibited impaired frequency discrimination both for tones in the mid-frequency range and high-frequency range. The remaining tone exposed mice, which did not demonstrate behavioral evidence of tinnitus, showed temporary deficits in frequency discrimination for tones in the mid-frequency range, while control mice remained unimpaired. Our findings reveal that the high frequency-specific deficits in gap detection, indicative of tinnitus, are associated with impairments in frequency discrimination at the frequency of the presumed tinnitus.</p></div

Timeline of the experimental protocol and testing.

<p>(A) Habituation to the test environment and apparatus, baseline recording of auditory brainstem responses (ABRs), and behavioral testing for gap detection and frequency discrimination; (B) 60 min tone exposure to a 10kHz tone; (C) Post-exposure ABR recording, gap detection, and frequency discrimination testing at 1 week, 4 weeks, and 8 weeks.</p

Frequency discrimination threshold, in % frequency change (<i>Th</i>_<i>40</i>).

<p>Data from panels A, B, C: Frequency discrimination in mid-frequency range. D, E, F: Frequency discrimination in high-frequency range. A, D: Control group. B, E: Tinnitus(+) group. C, F: Tinnitus(-) group. Error bars: standard deviation taken from 1000 repeats generated using parametric bootstrap method.</p

Tone evoked ABR thresholds were measured in a subset of exposed mice at baseline, and at 1, 4, and 8 weeks post-exposure.

<p>(A) Tone exposure in Tinnitus(+) mice induced significant hearing loss at 28 kHz at all post-exposure timepoints, and at 16 kHz 1 week post-exposure. No hearing impairments developed for frequencies between 8 kHz and 22 kHz at 4 and 8 weeks post-exposure. (B) Tone exposure also induced hearing impairments in Tinnitus(-) mice for 28 kHz tone 1 and 4 weeks post-exposure, but not at 8 weeks post-exposure. (C-D) Frequency discrimination thresholds are shown for the subset of mice used for ABR recordings. Thresholds were defined as the frequency shift that caused 50% inhibition of the maximum ASR. (C) Tone exposure in Tinnitus(+) mice led to a decrease in frequency discrimination for the 12 kHz tone at 8 weeks post-exposure, and at 4 weeks post-exposure for the 22 kHz tone. (D) Frequency discrimination thresholds did not significantly change after tone exposure in Tinnitus(-) mice. Each data point represents population mean ± SEM. Open circles represent a significant difference from baseline and closed circles a non-significant difference from baseline (significance at p<0.05); <i>n</i> refers to the number of mice; *: p<0.05.</p

Gap detection of the pre-pulse induced inhibition of the acoustic startle response (ASR) for Control (N = 6), Tinnitus(+) (N = 14), and Tinnitus(-) (N = 8) mice at baseline, and 1, 4, and 8 weeks post-exposure.

<p>(A) Control mice exhibited no significant changes within each test frequency when comparing baseline and post-exposure ASR-inhibition. (B) Tinnitus(+) mice exhibited evidence of decreased performance on gap detection in high frequency bands after tone exposure. ASR-inhibition was significantly attenuated at 4 weeks post-exposure for the 2–32 kHz (BBN), 18–20 kHz, and 26–28 kHz bands, and at 8 weeks post-exposure for the 2–32 kHz and 26–28 kHz bands. (C) Tone exposure did not attenuate gap detection at high frequencies in Tinnitus(-) mice. Instead, Tinnitus(-) mice demonstrated gap detection deficits for BBN at 4 and 8 weeks post-exposure, and improved detection in the 6–8 kHz band at 8 weeks post-exposure. Each data point represents population mean ± SEM. *: p<0.05; **: p<.01.</p

Average pre-pulse inhibition (PPI) of the acoustic-startle response to pre-pulse frequency shifts.

<p>(A-C) PPI due to increasing frequency shifts from a 12 kHz background tone for Control (N = 6), Tinnitus(+) (N = 14), and Tinnitus(-) (N = 8) groups at baseline and at 1, 4, and 8 weeks post-exposure. (A) Frequency shift detection remained unchanged in Control mice. (B) Tone exposure in Tinnitus(+) mice led to impaired frequency shift detection at 4 weeks post-exposure, but not at 8 weeks post-exposure. (C) In Tinntius(-) mice, tone exposure also led to a significant decrease in frequency shift detection at 4 weeks post-exposure, but not at 8 weeks post-exposure. (D-F) PPI due to decreasing frequency shifts from a 22 kHz background tone at baseline and at 1, 4, and 8 weeks post-exposure. (D) Frequency shift detection did not change over time in Control mice. (E) Tinnitus(+) demonstrated sustained impairments in frequency shift detection at 4 weeks and 8 weeks post-exposure. (F) There was no significant difference in post-exposure frequency shift detection relative to baseline in Tinnitus(-) mice. Each data point represents population mean ± SEM. *: denotes frequency discrimination thresholds</p

Cortical inhibitory neurons bidirectionally regulate frequency discrimination acuity.

<p>A. Experimental design. On each trial, a sequence of three acoustic stimuli was presented: background tone (f₁, 15 kHz, 10–20 s), prepulse tone (f₂, 10.2–15 kHz, 80 ms), and startle broadband noise (SN, 20 ms). In light-On trials, the laser (1 s, blue bar) was activated overlapping with the prepulse. In light-Off trials, the laser did not overlap with the prepulse. B, D, F. Left. Diagram shows circuits targeted by photomodulation. Right. Representative examples of the ASR (pressure applied by the mouse on the load cell platform in responses to startle noise) in PV-ChR2 (B), PV-Arch (D), and CamKIIα-ChR2 (F) mice. Top. Mean ASR for 10 light-Off trials in one session. Bottom. Mean ASR for 10 light-On trials in the same session. Note that ASRs decrease as the frequency shift between 15 kHz background tone and prepulse tone (f₂) increases. C, E, G. Left. PPI as a function of frequency shift between the prepulse and the background tone on light-On (color) and light-Off (gray) trials. Vertical dashed lines: <i>Th</i>. Error bars: Mean ± SEM. Right. <i>Th</i> threshold for light-On and light-Off trials and for separate “no light” session, in which no photostimulation was presented. C. Photoactivation of PVs in PV-ChR2 group decreased <i>Th</i> (paired t-test with Bonferroni adjustment for comparison between performance on "light-on" trials to "no-light" session and "light-off" trials, <i>t</i>₁₄ = 3.2, <i>p</i> = 0.01; <i>t</i>₁₄ = 3.6, <i>p</i> = 0.006; <i>n</i> = 15 mice). E. Photosupression of PVs in PV-Arch group increased <i>Th</i> PV-Arch group (t₁₅ = 2.6, <i>p</i> = 0.034; <i>t</i>₁₅ = 3.2, <i>p</i> = 0.012; <i>n</i> = 16). G. Increasing activity level of excitatory neurons in CamKIIα-ChR2 mice did not affect behavioral <i>Th</i>. ns: paired <i>t</i> test, <i>n</i> = 6, <i>t</i>₅ = 0.78, <i>p</i> = 0.47; <i>t</i>₅ = 0.36, <i>p</i> = 0.73. Dots depict data for an individual subject. Bars depict mean value for each group. See data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002308#pbio.1002308.s001" target="_blank">S1 Data</a>.</p

Activating PVs increases tone-evoked responses, whereas suppressing PVs has the opposite effect.

<p>A, C, E. Scaled time course of the firing rate of the neurons in response to a tone (outlined by black dashed lines) on light-On (color) and light-Off (gray) trials. Time of laser onset and offset is outlines by vertical color dashed lines. Mean ± SEM. A. PV-ChR2 mice. C. PV-Arch mice. E. CamKIIα-ChR2 mice. Inset diagram shows circuits targeted by photomodulation. B, D, F. Left. Scaled responses to tones on light-On trials plotted against responses on light-Off trials for putative excitatory neurons. Response magnitude is defined as a difference in mean scaled FR_base (0–50 ms before tone onset) and mean response to tone (FR_tone, 0–50 ms after tone onset). Right. Mean ± SEM. responses to tones from the left panel. See data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002308#pbio.1002308.s001" target="_blank">S1 Data</a>. B. PV-ChR2 mice: Tone-evoked responses on light-On trials (blue) were significantly higher than on light-Off trials (gray). Paired <i>t</i> test, <i>n</i> = 550, <i>t</i>₅₄₉ = 5.81, <i>p</i> = 1.1e-8. Data are combined for three laser powers used to activate PV interneurons (0.2, 0.5, and 10 mW/mm²). D. PV-Arch mice: Tone-evoked responses on light-On trials (green) were significantly lower than on light-Off trials (gray). Paired <i>t</i> test, <i>n</i> = 127, <i>t</i>₁₂₆ = 6.70, <i>p</i> = 6.3e-10. F. CamKIIα-ChR2 mice: Tone-evoked responses were not significantly affected by light. Paired <i>t</i> test, <i>n</i> = 130, <i>t</i>₁₂₉ = 1.19, <i>p</i> = 0.22. G. Change in the magnitude of scaled response to tones is correlated with change in behavioral <i>Th</i> due to manipulation of PVs activity. Each dot represents data averaged for single units from each subject at one light intensity (only subjects with >5 identified single units were included). Blue: PV-ChR2 group (<i>n</i> = 28); Green: PV-Arch group (<i>n</i> = 5). Magenta: CamkIIα-ChR2 group (<i>n</i> = 6, not included in regression analysis). <i>p</i> = 0.01.</p

Mutually coupled excitatory–inhibitory neuronal model accounts for differential effects of PV and excitatory neuronal modulation on tone-evoked response magnitude.

<p>A. Diagram of model of inhibitory and excitatory mutually coupled neuronal populations. Closed circles: excitatory inputs; open circles: inhibitory inputs; -: depressing synapse. Blue boxes: excitatory pathway. Red boxes: inhibitory pathway. B. Tone-evoked responses of model neuronal excitatory population under different optogenetic manipulations. Tone is from 200 to 250 ms. Left: ChR2 in inhibitory neurons. Center: Arch in inhibitory neurons. Right: ChR2 in excitatory neurons. Black trace: Light-off condition; Color trace: Light-on condition. See matlab code in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002308#pbio.1002308.s016" target="_blank">S1 Model</a>. C. Mean magnitude of tone-evoked responses under different stimulation conditions.</p

Modulating PV activity does not affect frequency tuning, but bidirectionally affects frequency selectivity.

<p>A, B, C. Frequency response function (top) and tuning curve (bottom) of a putative excitatory neuron in the absence of photostimulation (light-Off trials) and during photostimulation of AC(light-On trials). Inset diagram shows circuits targeted by photomodulation. A. PV-ChR2: light activates PVs. B. PV-Arch: light suppresses PVs. C. CamKIIα-ChR2: light activates excitatory neurons. D, E, F. Scatter plot (top) shows distribution of the BF for putative excitatory neurons in light-On and light-Off trials. Histogram (bottom) shows index of change in the BF due to photostimulation. See data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002308#pbio.1002308.s001" target="_blank">S1 Data</a>. D. PV-ChR2 group: Photoactivation of PVs had no significant effect on the BF of the frequency response function. One-sample <i>t</i> test. <i>n</i> = 233, mean ΔBF = −0.01, t₂₃₂ = 0.94, <i>p</i> = 0.35. E. PV-Arch group: Photosuppression of PVs did not significantly affect the BF of the frequency response function. One-sample <i>t</i> test. <i>n</i> = 83, mean ΔBF = −0.04, t₈₂ = 1.98, <i>p</i> = 0.051. F. CamKIIα-ChR2 group: Direct photoactivation of excitatory neurons did not significantly affect the BF of the frequency response function. One-sample <i>t</i> test. <i>n</i> = 82, mean ΔBF = −0.004, t₈₁ = 0.22, <i>p</i> = 0.82. G, H, I. Scatter plot (top) shows distribution of the tuning width for putative excitatory neurons in light-On and light-Off trials. Histogram (bottom) shows index of change in the tuning width due to photostimulation. See data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002308#pbio.1002308.s001" target="_blank">S1 Data</a>. G. PV-ChR2 group: Photoactivation of PVs significantly decreased the tuning width of the frequency response function. One-sample <i>t</i> test, mean ΔBW = −0.10, <i>t</i>₂₃₂ = 5.17, <i>p</i> = 5.2e-7. H. PV-Arch group. Photosuppression of PVs significantly increased the tuning width of the frequency response function. One-sample <i>t</i> test mean ΔBW = 0.13, <i>t</i>₈₂ = 4.31, <i>p</i> = 4.5e-5. I. CamKIIα-ChR2 group. Direct photoactivation of excitatory neurons significantly increased the tuning width of the frequency response function. One-sample <i>t</i> test mean ΔBW = 0.09, <i>t</i>₈₁ = 3.77, <i>p</i> = 4.5e-5. J, K, L. Scatter plot (top) shows distribution of sparseness for putative excitatory neurons in light-On and light-Off trials. Histogram (bottom) shows index of change in sparseness due to photostimulation of PVs. J. PV-ChR2 group: Photoactivation of PVs led to an increase in sparseness of the frequency response function. One-sample <i>t</i> test, mean ΔSparseness = −0.09, <i>t</i>₆₃₁ = 11.0, <i>p</i> = 6.6e-26. K. PV-Arch group. Photosuppression of PVs led to a decrease in sparseness. One-sample <i>t</i> test mean ΔSparseness = −0.04, <i>t</i>₁₅₈ = 2.96, <i>p</i> = 0.04. L. CamKIIα-ChR2 group. Direct photoactivation of excitatory neurons led to a decrease in sparseness. One-sample <i>t</i> test, mean ΔSparseness = −0.09, <i>t</i>₁₅₁ = 6.01, <i>p</i> = 1.3e-8. M–O. Change in sparseness due to photostimulation was negatively correlated with the change in tuning width in all tested groups: PV-ChR2 (M, <i>p</i> = 7.4e-61), PV-Arch (N, <i>p</i> = 2.7e-21), CamKIIα-ChR2 (O, <i>p</i> = 6.3e-19). P. Change in sparseness did not significantly correlate with behavioral <i>Th</i> due to manipulation of PVs activity. Each dot represents data averaged for single units from each subject at one light intensity (only subjects with >5 identified single units were included). Blue: PV-ChR2 group (<i>n</i> = 28); Green: PV-Arch group (<i>n</i> = 5). Magenta: CamKIIα-ChR2 group (<i>n</i> = 6, not included in regression analysis). <i>p</i> = 0.21. For PV-ChR2 mice, data are combined over three laser powers used to activate PV interneurons (0.2, 0.5, and 10 mW/mm²). J, K, L Data for putative excitatory neurons that showed increased FR in response to tones (“auditory” neurons). PV-ChR2: <i>n</i> = 632; PV-Arch: <i>n</i> = 159; CamKIIα-ChR2: <i>n</i> = 152. D–I, M–O. Data for “auditory” neurons fitted to Gaussian function at <i>R</i>^<i>2</i> > 0.4. PV-ChR2: <i>n</i> = 233; PV-Arch: <i>n</i> = 83, CamKIIα-ChR2: <i>n</i> = 82.</p