Background

Hearing impairment is a widespread sensory deficiency suffered by millions worldwide, of all ages and gender. Effects sizes can range from mild to severe. It can be congenital or develop later in life, often in a frequency-dependent age-related progressive manner. Numerous and varied pathologies can contribute to or be responsible for hearing loss (Davis 1995; Fortnum et al., 2001). Environmental conditions, such as noise exposure or drug-toxicity, can also have profound effects on hearing ability. Genetics is known to play many crucial roles in the early development of hearing and in the maintenance of hearing function into old age, as well as in predisposing the auditory system towards pathology or protecting the system against pathological decline. With the development of molecular methods to target the function of specific genes in the mouse and the emergence of phenotyping programs to investigate the systemic roles of these genes, it became necessary to develop a sensitive, rapid, robust and reproducible method to screen hearing function in these mutant animals.

Measurement of the Preyer Reflex (an acoustic startle response) can be used to quickly test very high numbers of animals, but it can detect only severe hearing loss and may be affected by motor system deficiencies in mutant mice. Measurements of otoacoustic emissions can be performed quickly and non-invasively, but they do not detect as many pathological conditions as measures of the output of the cochlea as a whole. The Auditory Brainstem Response (ABR), an electrophysiological response incorporating auditory nerve and auditory brainstem activity, is considered the ideal method to assess hearing function across large numbers of mice.

Table 1. Comparison of methods of functional assessment of hearing in small mammals


Methods considered are listed across Table 1: ABR, Auditory Brainstem Response. Startle, Startle Response (such as Preyer Reflex, Pre-Pulse Inhibition, etc.). OAE, Oto-Acoustic Emissions. MEMR, Middle Ear Muscle Reflex. Tymp, Tympanometry. ECoG, Electrocochleography. EP, Endocochlear Potential. The usefulness of a particular method to assess a particular function is indicated as a binary Yes/No choice. Yes, indicates that a test may give results to aid identification/diagnosis of a particular problem. No, indicates the test may not useful for this purpose. Middle Ear Conductive HL, is the test helpful to detect a conductive hearing loss, such as otitis media. IHC dysfunction, to detect Inner Hair Cell dysfunction. OHC dysfunction, to detect Outer Hair Cell dysfunction. SV dysfunction, to detect dysfunction of the Stria Vascularis. Inner Ear developmental problems, to detect early developmental issues affecting the inner ear. Age-Related HL, helpful to detect aspects of progressive hearing loss, if appropriate frequency stimuli are used. Involves other neural systems, can be influenced by dysfunction of other neural systems, such as descending inputs to the cochlea, motor systems, sensori-motor integration, etc. Assessment of cochlear sensitivity, to assess thresholds as an estimate of the sensitivity of cochlear function. Assessment of CAS function, to detect dysfunction within the Central Auditory System. High-Throughput, is the test amenable to high-throughput screening (e.g., simple and quick to set-up, short recording time, able to detect a broad spectrum of auditory dysfunction, etc.). The auditory brainstem response (ABR) is considered the best overall method to detect the widest spectrum of hearing impairment pathologies in a high-throughput screen where very large numbers (1000’s–10,000’s) of individual animals need to be assessed.

The method described here was adopted as the standard protocol used as a screen of hearing function by the International Mouse Phenotyping Consortium (IMPC, https://www.mousephenotype.org) at numerous research centers worldwide (summarized in Bowl et al., 2017). Detailed analyses of the data produced using this method at the Wellcome Sanger Institute have been published (White et al., 2013; Ingham et al., 2019). Whilst ABRs are commonly used to examine auditory function across a range of species, this method is optimized for rapid measurements in the mouse.

The ABR method, and our customized software, has been used in many secondary phenotyping studies to contribute to more detailed characterization of mutant phenotypes identified in the high-throughput screen (Ingham et al., 2019). The description presented here has been updated to reflect currently available digital signal processing technology. The method can easily be modified to perform evoked potential studies tailored towards more bespoke data collection and analysis requirements of individual studies (e.g., Chen et al., 2014; Buniello et al., 2016; Ingham et al., 2016) and can be used to assess more complex aspects of auditory function. For example, we can measure frequency tuning curves (Ebrahim et al., 2016), forward masking functions (Kuhn et al., 2011), and other measures of auditory temporal processing, based on assessment of ABR wave 1 amplitude (and latency). With training and experience, experimenters can use this system to generate high quality ABR recordings and produce reproducible measurements of threshold sensitivity, waveform shape and peak amplitude and latency, to give mean values with low associated variance helping to increase the statistical power of the method (e.g., Ingham et al., 2017).