Limiting Hearing Loss in Transgenic Mouse Models

Genotyping Cdh23 SNP responsible for hearing loss in C57BL/6 mice. A, Schematic of Cdh23 locus (Exon 9), where C57BL/6 mice exhibit a SNP. Primers are designed such that the SNP is offset from the center of the amplified region. B, Sequence of the region denoted in A from C57BL/6 (top) and B6.CAST-Cdh23^Ahl+/Kjn (bottom) mice. A BsrI restriction enzyme site is present in C57BL/6. C, An image of gel following PCR amplification and BsrI restriction enzyme digest of DNA region shown in A in mice carrying zero, one, or two ahl alleles.

Two-photon imaging of sound-evoked responses in the auditory cortex. A, Left, Schematic of widefield imaging in the auditory cortex of adult (P60) Ahl+ B6; Thy1-GCaMP6s mice with overlaid responses to 70 dB SPL tones. Right, Two-photon imaging of the primary auditory cortex (A1, 250 µm from pial surface) with overlaid responses to 70 dB SPL tones. B, Fluorescence traces from an individual neuron within the field of view depicted in A as a function of frequency (x-axis) and sound level (y-axis). Gray traces are individual trials; black traces are the trial averages. C, The plot of the proportion of sound-responsive neurons within each genotype. Light markers indicate individuals and dark markers are mean ± SEM; n = 8 mice for each genotype. Two-way ANOVA, genotype, F_(2,15) = 2.42; p = 0.12; sex, F_(1,15) = 1.95; p = 0.18; interaction, F_(2,15) = 0.00; p = 0.99. n.s., not significant. D, Average response across all sound-responsive neurons. Gray traces represent individual mice, and the black trace represents the group average. A total of 16 mice were analyzed (n = 8 per genotype). In ahl B6 mice, 3,247 total sound-responsive neurons were analyzed (mean ± SD, 406 ± 190 neurons per animal), while in Ahl+ B6 mice, 3,888 total sound-responsive neurons were analyzed (mean ± SD, 486 ± 258 neurons per animal). Two-way ANOVAs revealed no significant effects of genotype or sex on either the total number of detected neurons (genotype, F_(1,12) = 0.0006; p = 0.980; sex, F_(1,12) = 0.083; p = 0.778; interaction, F_(1,12) = 1.32; p = 0.273) or the number of sound-responsive neurons (genotype, F_(1,12) = 0.665; p = 0.431; sex, F_(1,12) = 0.379; p = 0.550; interaction, F_(1,12) = 0.528; p = 0.481). E, Schematic indicating measurements reported in F–H. The color black indicates the response of an individual neuron to different frequencies and sound levels, with lighter colors indicating larger responses. The bandwidth is defined as the width in octaves separating the highest and lowest responding frequencies; a bandwidth of 0 indicates the neuron was responsive to a single tone. Best frequency is defined as the frequency with the highest amplitude response, regardless of the sound level. Characteristic frequency is defined as the frequency eliciting the highest amplitude response at the lowest sound level. F–H, Plots of the proportion of neurons as a function of bandwidth at 70 dB, best frequency, and characteristic frequencies across genotypes. Markers are mean ± SEM; n = 8 mice for each genotype. Two-way ANOVAs with genotype and measurement, followed by post hoc t tests with Benjamini–Hochberg FDR correction. *p < 0.05; **p < 0.01; ***p < 0.001. Extended Data Figures 3-1 and 3-2 extend this analysis to include comparisons to Ahl+ B6.CBA; Thy1-GCaMP6s mice.

Reorganization of the auditory cortex toward lower frequencies in ahl B6 mice. A, The plot of neuron location with characteristic frequencies indicated with color. B, Schematic of the analytic method to quantify the cortical area devoted to processing a given characteristic frequency. A Gaussian filter (σ = 60 µm) was used to create a weighted sum of each neuron's characteristic frequency response for each unit of area. The frequency with the highest value was assigned to that unit of area and proportion of area examined. C, Exemplar characteristic frequency maps and assigned areas for the indicated genotypes. D, The plot of the fractional area as a function of characteristic frequency range and genotype. Light lines are individual animals; dark lines are mean ± SEM; n = 8 mice for each genotype. Two-way ANOVA (characteristic frequency, F_(1,36) = 72.98; p < 0.001; genotype, F_(2,36) = 0.08; p = 0.92; interaction, F_(2,36) = 34.6; p < 0.001), followed by post hoc t tests with Benjamini–Hochberg FDR correction. n.s., not significant; **p < 0.01; ***p < 0.001. Extended Data Figure 3-1 extends this analysis to include comparisons to Ahl+ B6.CBA; Thy1-GCaMP6s mice.

The linear classifier of network response performs worse in ahl B6 mice. A, Schematic of a pipeline for prediction of frequency and the sound level based on population-level response using LDA. B, C, Left, Low-dimensional representation (t-SNE) of neuronal population response from an individual animal with each marker representing a single trial. Right, Confusion matrix of classifier performance from a single animal. D, The plot of overall classifier performance as a function of frequency. Markers are mean ± SEM; n = 8 mice for each genotype. Three-way ANOVA (genotype, F_(2,360) = 76.7; p < 0.001; frequency, F_(4,360) = 15.2; p < 0.001; sound level, F_(3,360) = 47.8; p < 0.001; interaction, F_(50,360) = 2.89; p < 0.001), all attenuation levels included for each frequency in post hoc t tests with Benjamini–Hochberg FDR correction. *p < 0.01; **p < 0.01; ***p < 0.001. E, The plot of classifier performance as a function of frequency (x-axis) and the sound level (plots arranged from highest sound level to lowest sound level). Markers are mean ± SEM; n = 8 mice for each genotype. The same three-way ANOVA as reported in D, post hoc t tests with Benjamini–Hochberg FDR correction. *p < 0.01; **p < 0.01; ***p < 0.001. Extended Data Figure 3-1 extends this analysis to include comparisons to Ahl+ B6.CBA; Thy1-GCaMP6s mice.

Low-threshold responses are intact in 6-month-old Ahl+ B6 mice. A, Left, The plot of fluorescence changes in 2.5-month-old ahl B6 mice over the imaging field to varying frequency (x-axis) and sound levels (y-axis). Right, The plot of fluorescence during sound presentation across animals; gray traces are individual mice; black traces are the average; n = 9 mice. B, The plot of fluorescence changes in 6-month-old ahl B6 mice over the imaging field to varying frequency (x-axis) and sound levels (y-axis). Right, The plot of fluorescence during sound presentation across animals; gray traces are individual mice; black is the average; n = 9 mice. Note the complete disappearance of responses to 32 and 4–16 kHz at low sound levels. C, The plot of average threshold as a function of frequency and time point. Dashed lines indicate measurements at 2.5 months; solid lines indicate measurements at 6 months; n = 9 mice. Markers at 110 dB SPL indicate no response was observed at 90 dB SPL. Three-way ANOVA (frequency, F_(4,70) = 10.6; p < 0.001; timepoint, F_(1,70) = 97.8; p < 0.001; sex, F_(1,70) = 0.29; p = 0.59; interaction, F_(13,70) = 1.51; p = 0.14) with post hoc paired t tests with Benjamini–Hochberg FDR correction. *p < 0.01; **p < 0.01; ***p < 0.001. D, Similar to A, but for Ahl+ B6 mice; n = 8 mice. E, Similar to B, but for Ahl+ B6 mice. n = 8 mice. F, Similar to C, but for Ahl+ B6 mice. n = 8 mice. Three-way ANOVA (frequency, F_(4,60) = 2.26; p = 0.07; timepoint, F_(1,60) = 7.83; p = 0.007; sex, F_(1,60) = 0.29; p = 0.009; interaction, F_(13,60) = 0.71; p = 0.74) with post hoc paired t tests with Benjamini–Hochberg FDR correction. n.s., not significant.