The reticular lamina and basilar membrane vibrations in the transverse direction in the basal turn of the living gerbil cochlea.

The prevailing theory of cochlear function states that outer hair cells amplify sound-induced vibration to improve hearing sensitivity and frequency specificity. Recent micromechanical measurements in the basal turn of gerbil cochleae through the round window have demonstrated that the reticular lamina vibration lags the basilar membrane vibration, and it is physiologically vulnerable not only at the best frequency but also at the low frequencies. These results suggest that outer hair cells from a broad cochlear region enhance hearing sensitivity through a global hydromechanical mechanism. However, the time difference between the reticular lamina and basilar membrane vibration has been thought to result from a systematic measurement error caused by the optical axis non-perpendicular to the cochlear partition. To address this concern, we measured the reticular lamina and basilar membrane vibrations in the transverse direction through an opening in the cochlear lateral wall in this study. Present results show that the phase difference between the reticular lamina and basilar membrane vibration decreases with frequency by ~ 180 degrees from low frequencies to the best frequency, consistent with those measured through the round window. Together with the round-window measurement, the low-coherence interferometry through the cochlear lateral wall demonstrates that the time difference between the reticular lamina and basilar membrane vibration results from the cochlear active processing rather than a measurement error.

CRISPR/Cas9 editing of the MYO7A gene in rhesus macaque embryos to generate a primate model of Usher syndrome type 1B.

Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.

Optimizing Auditory Brainstem Response Acquisition Using Interleaved Frequencies.

Auditory brainstem responses (ABRs) require averaging responses to hundreds or thousands of repetitions of a stimulus (e.g., tone pip) to obtain a measurable evoked response at the scalp. Fast repetition rates lead to changes in ABR amplitude and latency due to adaptation. To minimize the effect of adaptation, stimulus rates are sometimes as low as 10 to 13.3 stimuli per second, requiring long acquisition times. The trade-off between reducing acquisition time and minimizing the effect of adaptation on ABRs is an especially important consideration for studies of cochlear synaptopathy, which use the amplitude of short latency responses (wave 1) to assess auditory nerve survival. It has been proposed that adaptation during ABR acquisition can be reduced by interleaving tones at different frequencies, rather than testing each frequency serially. With careful ordering of frequencies and levels in the stimulus train, adaptation in the auditory nerve can be minimized, thereby permitting an increase in the rate at which tone bursts are presented. However, widespread adoption of this stimulus design has been hindered by lack of available software. Here, we develop and validate an interleaved stimulus design to optimize the rate of ABR measurement while minimizing adaptation. We implement this method in an open-source data acquisition software tool that permits either serial or interleaved ABR measurements. The open-source software library, psiexperiment, is compatible with widely used ABR hardware. Consistent with previous studies, careful design of an interleaved stimulus train can reduce ABR acquisition time by more than half, with minimal effect on ABR thresholds and wave 1 latency, while improving measures of wave 1 amplitude.