The experimental procedures on animals were approved by the Institutional Animal Care and Use Committee (IACUC) at Wayne State University. The Nefl^CreER/CreER (stock no. 008363), iDTR (stock no. 007900), and the reporter Ai9 (stock no. 007909) mouse strains were obtained from Jackson Laboratories (Bar Harbor, ME, United States) (Buch et al., 2005; Rotolo et al., 2008; Madisen et al., 2010). They were maintained and bred following the guidelines of the local Division of Laboratory Animal Resources. Nefl^CreER/CreER mice were crossed with iDTR or Ai9 mice, followed by genotyping. Heterozygous Nefl^CreER/+;iDTR^+/– and Nefl^CreER/+;Ai9^+/– mice were used in this study. The homozygous animals were used for breeding and maintenance of the strains.

A tail snip-based genotyping was performed to determine the genotypes of the mice (Fang et al., 2012). Two millimeters of the tail was snipped and placed in alkaline lysis buffer (25 mM NaOH and 0.2 mM ethylenediaminetetraacetic acid (EDTA; E5134, Sigma, St. Louis, MO, United States) for the hotshot procedure of 98°C for 1 h, followed by neutralization (40 mM Tris-HCl; Sigma) for 5 min at room temperature to harvest gDNA in the supernatant. Allele-specific PCR was used to determine the genotypes of the mice using the vendor’s protocols. The primers included: Nefl^CreER/CreER: common, ATT ATT ATT GTA AAC ATC TGT GTG ATT CA; mutant forward, CGC ATA GAA ATT GCA TCA ACG CAT; and wild type reverse, AGA GGA GCA GGT GGC TAA GAA GAA AGA; Ai9: wild type forward, AAG GGA GCT GCA GTG GAG TA; wild type reverse, CCG AAA ATC TGT GGG AAG TC; mutant forward, CTG TTC CTG TAC GGC ATG G; and mutant reverse, GGC ATT AAA GCA GCG TAT CC; iDTR: common, AAA GTC GCT CTG AGT TGT TAT; mutant, GCG AAG AGT TTG TCC TCA ACC; and wild type reverse, GGA GCG GGA GAA ATG GAT ATG.

Nefl^CreER/CreER mice were crossed with the reporter strain Ai9 to obtain heterozygous Nefl^CreER/+;Ai9^+/– offspring for the characterization of Cre activity in the cochlea. Tamoxifen or vehicle was administered to the dam via gavage on P1 for 4–5 days, and the pups received treatment via feeding. Tamoxifen (T5648, Sigma) was dissolved in corn oil (C8267, Sigma) at 10 mg/ml. Either tamoxifen (4 mg/40 g body weight) or corn oil was administered daily via oral gavage for 4–5 consecutive days (Koundakjian et al., 2007; Fang et al., 2012). Treated mice were followed up and euthanized on P10, P14, P21, and P28 for histology and immunofluorescence study to determine the Cre activity. Nefl^CreER/CreER mice were crossed with iDTR mice to obtain heterozygous Nefl^CreER/+;iDTR^+/– mice in order to determine the SGN degeneration. For the DT treatment, Nefl^CreER/+;iDTR^+/– mice were treated with tamoxifen as above, and a single dose of DT (List Biology Laboratories #150, 10 ng/g body weight, i.p.) was administered on P7, P14, or P21 (Figure 1).

Animals received hearing tests at 4 and 5 weeks old prior to euthanasia. Distortion product otoacoustic emission (DPOAE) and ABR tests were used to study the function of outer hair cells and the auditory system using the RP2.1 and RZ6 systems [Tucker-Davis Technology (TDT), Alachua, FL, United States] (Hu et al., 2009; Zhang et al., 2011; Deng et al., 2019). The TDT System 3 software was applied for signal generation and auditory response collection. The ABR stimulation level ranged from 5 to 90 dB sound pressure level (SPL) in 5-dB steps using 8, 16, 24, and 32 kHz pure tone and click sound. The threshold was determined as the lowest stimulation decibel SPL that generated a wave II amplitude larger than 0.2 mV. At 16 and 24 kHz, the configuration of DPOAE was set as F2/F1 = 1.2 and L1 = L2 + 10 dB. L1 ranged from 10 to 80 dB SPL in 5-dB SPL steps. The DPOAE threshold was determined as the lowest level of DPOAE responses (dp) of at least 10 dB above the noise floor.

Mice were anesthetized with CO₂, followed by heart perfusion using saline (0.9% NaCl) and 4% paraformaldehyde (PFA; 158127, Sigma). The cochlear tissues were rapidly dissected and perfused with PFA. The dissected cochlear tissue was decalcified in 0.1 M EDTA for 1–2 weeks until the tissues softened, followed by surface preparation or cryosection at 10-μm thickness using our published methods (Hu et al., 2004; Zhang et al., 2011; Deng et al., 2019; Deng and Hu, 2020). Immunofluorescence was used for the detection of neuronal and hair cell proteins using our published methods (Hu et al., 2004; Zhang et al., 2011; Deng et al., 2019; Deng and Hu, 2020). The primary antibodies included anti-Nefl (1:200; sc-20012, Santa Cruz Biotechnology, Dallas, TX, United States), anti-beta III tubulin (TUJ1, 1:1,000; ab-2313564, Aves Labs, Tigard, OR, United States), and anti-myosin VIIa [1:200; 138-1-C, Developmental Studies Hybridoma Bank (DSHB), Iowa, City, IA, United States, and 25-6790, Proteus, Ramona, CA, United States]. Secondary antibodies were Alexa Fluor-488 (715-546-150), Cy3 (711-165-152), or Alexa Fluor-647 (703-606-155) conjugated antibodies (1:500; all from Jackson ImmunoResearch, West Grove, PA, United States). Leica SPE confocal microscope and DM2500 upright epifluorescence microscopes were used for observation and imaging.

In the quantitative study, the ABR and DPOAE data were analyzed using two-way analysis of variance (ANOVA) with post-hoc tests. The two factors were treatment type (DT and vehicle) and treatment time (P7, P14, and P21). Post-hoc tests were used to compare the vehicle and DT treatments in the P7, P14, and P21 groups. For cell counting, the cells and the area were calculated using the cell count and measurement modules of ImageJ software (NIH) using our published methods (Li et al., 2016; Hu et al., 2017, 2019, 2021; Liu et al., 2018). For SGN cell counting, the SGN area was determined, and Nefl-positive cells were calculated for the P7, P14, and P21 groups. The average cell number per 10⁴ μm² was calculated for data analysis. All three cochlear turns were analyzed for the generation of data. For hair cell counting, surface preparation was performed to expose the hair cell epithelium, and myosin VIIa-positive cells were calculated at 100-μm distance for each animal using our published methods (Liu et al., 2018; Deng et al., 2019). Six animals were included in each group for statistical analysis of the ABR, DPOAE, and SGN cell counts, and five cochlear basilar membranes were dissected per group for analysis of the number of hair cells. A p-value of 0.05 was considered as the criterion of statistical significance.

Nefl is expressed in developing and mature neurons, including SGNs (Sun et al., 2018). The Nefl^CreER/CreER mouse strain possessing a tamoxifen-inducible Cre cassette knocked into the Nefl gene was used in this study (Rotolo et al., 2008). To determine the Nefl-mediated CreER activity, the Cre reporter transgenic mouse strain Ai9 that has a LoxP-flanked STOP cassette preventing the transcription of a ubiquitous CAG promoter-driven tdTomato fluorescence was used. Tamoxifen or vehicle was administered to the dam via gavage on P1 for 4–5 days (Figure 1). The heterozygous Nefl^CreER/+;Ai9^+/– pups received treatment via feeding and were followed up and euthanized on P10. It was observed that SGNs expressed both CreER-mediated tdTomato fluorescence and Nefl immunofluorescence, suggesting the CreER activity of Nefl^CreER/+ at the neonatal stage (Figure 2A). It is known that SGNs develop during the postnatal period and mature around P21–28 (Shrestha et al., 2018; Sun et al., 2018), so we opted to determine the CreER activity during postnatal development. The dam was treated with tamoxifen or vehicle by gavage from P1 to P5 for 4–5 days, and the offspring received tamoxifen via feeding, followed by euthanasia on P14, P21, and P28. Robust tdTomato fluorescence was observed in SGNs from P14 to P28 in tamoxifen-treated Nefl^CreER/+;Ai9^+/– offspring, which overlapped with the Nefl immunofluorescence (Figures 2B–D). In vehicle-treated Nefl^CreER/+;Ai9^+/– offspring, only Nefl immunofluorescence was observed without tdTomato fluorescence (Figures 2B–D). This experiment suggests that Nefl^CreER/+ mouse SGNs possess the inducible CreER activity during the postnatal development period.

To generate an SGN damage model, the iDTR knock-in mouse strain was used. iDTR mice had the simian DTR insertion at the ROSA26 locus that is blocked by an upstream LoxP-flanked STOP sequence (Buch et al., 2005). When bred with Cre-recombinase-expressing Nefl^CreER/CreER mice, the STOP sequence was deleted in Nefl-Cre-expressing SGNs to allow DTR expression. Following DT treatment, iDTR-expressing SGNs were susceptible to ablation.

Homozygous Nefl^CreER/CreER and iDTR mice were crossbred to obtain heterozygous Nefl^CreER/+;iDTR^+/– offspring, which were treated with tamoxifen on P1–P5, followed by DT or vehicle injection on P7, P14, and P21 (Figure 1). Click and pure tone ABR tests were performed at 4 and 5 weeks old (Figure 3). It was found in the P7 treatment group that all DT-treated mice did not have an ABR response waveform following 90-dB SPL click stimulation (> 90 dB SPL) at 4 weeks old, whereas the threshold of vehicle-treated mice was 22.5 ± 4.2 dB SPL (mean ± SD) (Figure 3E). The click ABR threshold was similar a week later, at 5 weeks old: no response at 90 dB SPL in the DT group and remained normal (24.1 ± 3.8 dB SPL) in the vehicle group (Figure 3E1). In the P14 treatment group, the click ABR thresholds were 23.3 ± 6.1 and 85.8 ± 8.0 dB SPL for the vehicle and DT groups at 4 weeks old and were 22.5 ± 4.2 and > 90 dB SPL at 5 weeks old, respectively. In the P21 group, the click ABR thresholds for the vehicle and DT groups were 25.8 ± 11.1 and 76.3 ± 16.0 dB SPL at 4 weeks old and were 28.3 ± 9.3 and 83.8 ± 9.5 dB SPL at 5 weeks old, respectively (Figures 3E,E1). In the statistical analysis, two-way ANOVA and post-hoc tests were performed with two factors: treatment types (vehicle and DT) and treatment ages (P7, P14, and P21; n = 6 mice per group). In the test at 4 weeks old, the overall effects of treatment type and treatment age were not statistically significant [F_{(2, 30)} = 1.595, p = 0.2196]. The effect of treatment age was also not statistically significant [F_{(2, 30)} = 0.3451, p = 0.7109]. However, the effect of treatment type (vehicle vs. DT) was statistically significant [F_{(1, 30)} = 459.7, p < 0.0001]. In the post-hoc test of the comparison of the vehicle and DT groups, significant differences were observed in the P7, P14, and P21 groups (p < 0.0001 for all three groups). In the test at 5 weeks old, the overall effects of treatment type and treatment age were not statistically significant [F_{(2, 30)} = 2.831, p = 0.0748]. The effect of treatment age was also statistically insignificant [F_{(2, 30)} = 0.09132, p = 0.9130]. However, the effect of treatment type was statistically significant [F_{(1, 30)} = 1197, p < 0.0001]. In the post-hoc test of the comparison of the vehicle and DT groups, P7, P14, and P21 groups had significant differences (p < 0.0001 for all three groups).

In pure tone ABR, responses to the 16-kHz stimulation were analyzed (Figures 3F,F1). In the P7 group, the thresholds of the vehicle and DT groups were 29.2 ± 4.9 and 88.3 ± 2.6 dB SPL at 4 weeks old and were 34.2 ± 5.8 and > 90 dB SPL at 5 weeks old, respectively. For the P14 group, these thresholds became 32.5 ± 8.2 and 83.3 ± 5.2 dB SPL at 4 weeks old and 38.3 ± 13.3 and > 90 dB SPL at 5 weeks old, respectively. For the P21 group, the threshold values were 32.5 ± 7.6 and 82.5 ± 9.6 dB SPL at 4 weeks old and were 35.0 ± 5.5 and 86.3 ± 4.8 dB SPL at 5 weeks old, respectively. In the statistical analysis, two-way ANOVA and post-hoc tests were performed with the two factors: treatment type (vehicle and DT) and treatment age (P7, P14, and P21; n = 6 mice per group). In the test at 4 weeks old, the overall effects of treatment type and treatment age were not statistically significant [F_{(2, 30)} = 2.500, p = 0.0990]. The effect of treatment age was also statistically insignificant [F_{(2, 30)} = 0.2880, p = 0.7518]. However, the effect of treatment type (vehicle vs. DT) was statistically significant [F_{(1, 30)} = 407.2, p < 0.0001]. In the post-hoc test of the vehicle and DT groups, significant differences were observed in the P7, P14, and P21 groups (p < 0.0001 for the three groups). In the test at 5 weeks old, the overall effects of treatment type and age were not statistically significant [F_{(2, 30)} = 0.3387, p = 0.7154]. The effect of treatment age was statistically insignificant [F_{(2, 30)} = 0.6290, p = 0.5400]. However, the effect of treatment type was statistically significant [F_{(1, 30)} = 594.6, p < 0.0001]. In the post-hoc comparison of the vehicle and DT groups, the P7, P14, and P21 groups showed significant differences (p < 0.0001).

Immunofluorescence using anti-Nefl and anti-TUJ1 antibodies was conducted to study the morphology and protein expressions of SGNs for the Nefl^CreER/+;iDTR^+/– offspring at the end of the experiment. It was observed that SGNs expressed neuronal proteins Nefl and TUJ1 in the P7, P14, and P21 groups (Figures 4A–C). In the quantitative study, the number of Nefl-expressing cells was consistent in vehicle-treated groups, whereas it decreased in the P7, P14, and P21 groups treated with DT (Figure 4D). In vehicle-treated mice, the average numbers of Nefl-expressing cells per 10⁴ μm² were 30.6 ± 5.0, 31.1 ± 4.3, and 31.4 ± 5.0 for the P7, P14, and P21 groups, respectively. In DT-treated groups, the average numbers of Nefl-expressing cells per 10⁴ μm² were 12.9 ± 3.0, 19.0 ± 3.1, and 17.9 ± 2.8 for the P7, P14, and P21 groups, respectively. In the statistical analysis, two-way ANOVA and post-hoc tests were performed with two factors: treatment type (vehicle and DT) and treatment age (P7, P14, and P21; n = 6 mice per group). The overall and the treatment age effects were not statistically significant [F_{(2, 30)} = 3.065, p = 0.0916, and F_{(2, 30)} = 1.677, p = 0.2355, respectively]. However, the effect of treatment type (vehicle vs. DT) was statistically significant [F_{(1, 30)} = 80.64, p = 0.0003]. In the post-hoc comparison of the vehicle and DT groups, significant differences were observed (p = 0.0002, 0.0042, and 0.0018 for the P7, P14, and P21 groups, respectively) (Figure 4).

To evaluate the hair cell function of the Nefl^CreER/+;iDTR^+/– offspring, DPOAE was performed. The DPOAE thresholds were determined in the P14 and P21 groups at 4 weeks old (Figure 5A). In the statistical analysis using two-way ANOVA, the overall and individual effects of treatment type (vehicle vs. DT) and treatment age (P14 vs. P21; n = 6 mice per group) at 16-kHz stimulation were not statistically significant (p > 0.05): p = 0.9108, 0.6062, and 0.1360 for the overall, treatment type, and treatment age effects, respectively (Figure 5A1). In the analysis of the 24-kHz stimulation, these numbers became p = 0.2640, 0.1601, and 0.9175, respectively (Figure 5A2). These data suggest that the DPOAE thresholds were not significantly different between the DT and vehicle groups.

In the hair cell protein expression study, anti-myosin VIIa antibodies were used to identify hair cells using the basilar membrane surface preparation for the P14 group (Figure 5B). In the quantification study, the average numbers of inner hair cells were 11.9 ± 0.96 and 11.1 ± 1.5 per 100 μm for the vehicle and DT groups, whereas the numbers for outer hair cells were 35.4 ± 2.87 and 33.4 ± 4.42, respectively (Figure 5B1). Statistical analysis showed no significant difference between the vehicle and DT groups (ANOVA, n = 5 mice per group): the p-values were 0.9138 and 0.4626 for inner and outer hair cells, respectively.

These data show that hair cell number and function were not statistically different between the DT and vehicle groups, suggesting no hair cell damage was observed in this animal model.