Atoh1-Cre TG mice [25], Rac1flox/flox mice [26], and Rac3−/− mice [27] were backcrossed to generate Atoh1-Cre;Rac1flox/flox mice (Atoh1-Cre−/−;Racflox/flox as a control, Atoh1-Cre+/−;Rac1flox/flox as Rac1-KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− mice (Atoh1-Cre+/−;Rac1flox/flox;Rac3−/− as Rac1/Rac3-DKO) [15]. CAG-STOPflox-tdTomato (Ai9) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and backcrossed to generate Atoh1-Cre;tdTomato mice, which were used to examine the function of the Atoh1 promoter in cochleae. The efficacy of Atoh1-Cre mice in cochleae has been previously reported [25, 28].

Offspring were genotyped via PCR using the following primer pairs: Atoh1-Cre (5′-GCATACCTGGAAAATGCTTC-3′ and 5′-CCAGTGAAACAGCATTGCTG-3′), Rac1flox (5′-ATTTTCTAGATTCCACTTGTGAAC-3′ and 5′-ATCCCTACTTCCTTCCAACTC-3′), Rac3− (5′-CATTTCTGTGGCGTCGCCAAC-3′ and 5′-TTGCTGGTGTCCAGACCAAT-3′), Rac3+ (5′-CATTTCTGTGGCGTCGCCAAC-3′ and 5′-CACGCGGCCGAGCTGTGGTG-3′), and tdTomato (5′-GGCATTAAAGCAGCGTATCC-3′ and 5′-CTGTTCCTGTACGGCATGG-3′).

Mice were housed under specific pathogen-free conditions using an individually ventilated cage system (Techniplast, Tokyo, Japan). Both male and female mice were included in analyses unless otherwise indicated (mice younger than 1 week were not differentiated based on sex). Age- and sex-matched siblings were used as controls.

In situ hybridization (ISH) was performed using the cochleae from P6 WT mice (CLEA Japan, Tokyo) as previously described [28]. Briefly, the following 45 bp DNA probes labeled at their 5′-end with digoxigenin-11-dUTP were used: Rac1 (antisense [nucleotides 346–390 from ATG]: 5′-GAGCAGGCAGGTTTTACCAACAGCTCCGTCTCCCACCACCACACA-3′ and sense: 5′-TGTGTGGTGGTGGGAGACGGAGCTGTTGGTAAAACCTGCCTGCTC-3′), and Rac3 (antisense [155–199]: 5′-CAGCAGGCACGTCTTCCCCACGGCACCATCGCCAACCACCACGCA-3′ and sense: 5′-TGCGTGGTGGTTGGCGATGGTGCCGTGGGGAAGACGTGCCTGCTG-3′). ISH signals were detected immunohistochemically; the staining intensities of the sense and antisense probes were compared using a digital image analyzer (WinRoof version 7.0, Mitani Corp., Tokyo, Japan).

DNA microarray analysis was performed as previously described [26]. Total RNA was extracted from the cochleae of five P6 WT mice using a NucleoSpin RNA kit (MACHEREY–NAGEL GmbH & Co. KG, Düren, Germany). Gene expression profiles were examined using the SurePrint G3 Mouse GE 8 × 60 K Microarray Kit (Agilent Technologies, Santa Clara, CA, USA).

RT-PCR was performed with 1 µg of total RNA obtained from 14 membranous cochleae and vestibules of 4-week-old WT mice using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), as previously described [26]. The following primer pairs were used: 5′-GCAGACAGACGTGTTCTTAATTTGC-3′ and 5′-CAACAGCAGGCATTTTCTCTTCC-3′ for Rac1 (predicted product size: 358 bp); 5′-GGAGGACTATGACCGCCTC-3′ and 5′-GCGCTTCTGCTGTCGTGTG-3′ for Rac2 (379 bp); and 5′-CCCACACACACCCATCCTTC-3′ and 5′-CAGTGCACTTCTTGCCTGGC-3′ for Rac3 (257 bp).

Rac1 and Rac3 in the pEGFP(C1) vector (Takara Bio, Kusatsu, Japan; termed EGFP-Rac1 and EGFP-Rac3, respectively) have been previously described [13]. Organotypic OC explant cultures were prepared from WT P4 rats as previously described [28]. For transfection, a Helios Gene Gun (Bio-Rad Laboratories, Hercules, CA, USA) and Helios Gene Gun Diffusion Screen (165–2475) were used, which reduce tissue damage owing to the high concentration of gold particles in the center of the shot. Gold particles (1.0 μm diameter) were coated with the plasmids at a ratio of 2 μg plasmid to 1 mg gold particles and precipitated onto the inner wall of Tefzel tubing, which was cut into individual cartridges containing 1 μg of the plasmid. The next day (ex vivo day 1), the samples were bombarded with gold particles from one cartridge per culture using 110 psi helium pressure, as previously described [29]. The explants were fixed 24 h after transfection with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), counterstained with Alexa568-conjugated phalloidin, and observed under an LSM700 confocal microscope (Carl Zeiss, Oberkochen, Germany).

OCs from P2 Rac1-fluorescence resonance energy transfer (FRET) biosensor TG mice [30] were dissected in Leibovitz’s L-15 medium (Invitrogen), attached to 3.5-mm Cell-Tak coated dishes (150 µg/µL; BD Biosciences) and maintained in Dulbecco’s modified Eagle medium/F-12 supplemented with 10% fetal bovine serum. FRET imaging under a two-photon excitation microscope was performed as previously described [28]. Samples were maintained in an incubation chamber (Tokai Hit, Nagoya, Japan) and imaged using a BX61WI/FV1000 upright microscope equipped with a × 60 water-immersion objective (LUMPlanFLN; Olympus, Tokyo, Japan) connected to a Mai Tai DeepSee HP Ti:sapphire laser (Spectra Physics, Mountain View, CA, USA). FRET/CFP images were acquired and analyzed using MetaMorph (Universal Imaging, West Chester, PA, USA) and Imaris software (Bitplane AG, Zürich, Switzerland) and represented using the intensity-modulated display mode, in which eight colors from red to blue are used to represent the FRET/CFP ratio.

Auditory brainstem responses (ABRs) were obtained under anesthetization with a mixture of medetomidine, midazolam, and butorphanol (intraperitoneal injection, 0.3, 4.0, and 5.0 mg/kg, respectively) on a heating pad, as previously described [31]. Briefly, ABR waveforms using sound stimuli of clicks or tone bursts at 8, 16, 24, or 32 kHz were recorded and averaged. ABR waveforms were recorded using elicitation sound that ranged from 100 to 5 dB SPL, and the thresholds (dB SPL) were defined by decreasing the sound intensity by 5 dB intervals until the lowest sound intensity level was reached, resulting in a recognizable ABR wave pattern (primarily judged by recognition of wave III).

NE experiments were performed as previously described [31]. Briefly, 6-week-old control and Rac1/Rac3-DKO mice were anesthetized and exposed to 110 dB SPL octave-band noise centered at 8 kHz for 1 h inside a sound chamber. These NE conditions cause a permanent threshold shift in WT mice [31]. ABR thresholds (dB SPL, at 4, 12, and 20 kHz) were measured immediately before NE and were measured sequentially after NE on day 0 and days 2, 7, and 14. NE-induced hearing deterioration was evaluated using the ABR threshold shift, calculated based on differences in the ABR threshold before and after NE.

To examine cochlear whole mounts, surface preparations, and cryostat sections, tissues were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, as previously described [31]. Samples for surface preparations and cryostat sections were decalcified in 0.12 M ethylenediaminetetraacetic acid for 1 week at 4 °C or for 2 days at 23 °C. After permeabilization with phosphate-buffered saline containing 0.3% Triton X-100, the samples were incubated with Alexa Fluor 488-labeled phalloidin (Invitrogen) with/without DAPI for 1 h at 23 °C. The stained tissues were mounted in Prolong anti-fade (Invitrogen) with a coverslip and observed using an LSM700 confocal microscope.

Scanning electron microscopy (SEM) analysis was performed as previously described [32]. Freshly dissected cochleae of 13-week-old WT, Rac1-KO, and Rac1/Rac3-DKO mice were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 h, followed by post-fixation with 1% osmium tetroxide in H2O for 1 h at 23 °C. Tissues were dehydrated using a graded ethanol series, followed by tert-butyl alcohol, and dried in a vacuum freeze dryer (VFD-30; Ulvac Inc., Tokyo, Japan). Dried tissues, mounted on stages, were sputter coated with gold in an Ion Sputter MC1000 (Hitachi High-Tech Corp., Tokyo, Japan) and observed using a TM3030Plus scanning electron microscope (Hitachi High-Tech).

Blinded data analysis was performed by two otologists or scientists. Statistical analyses were performed with Prism 7.0 software (GraphPad Software Inc., La Jolla, CA, USA) using two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Statistical significance was set at P < 0.05.