Research PaperHidden hearing loss and endbulbs of Held: Evidence for central pathology before detection of ABR threshold increases
Introduction
The consequences of hearing loss are at times baffling. Classic hearing loss has elevated hearing thresholds and loss of sensory receptor cells (Schuknecht, 1974). In contrast, many affected listeners, especially the elderly, exhibit impaired temporal processing and difficulties understanding speech in noise without elevated thresholds or cognitive impairments (Dubno et al., 1984; Fullgrabe et al., 2014; Gordon-Salant, 2005; Gordon-Salant and Fitzgibbons, 1993; Ruggles et al., 2012; Snell and Frisina, 2000). Indeed, the physical substrate of age-related hearing loss in the absence of sensory receptor cell loss has remained “hidden” until quite recently (Liberman and Kujawa, 2017).
The CBA inbred mouse strain and its variants (e.g., CBA/J, CBA/Ca, CBA/CaJ, CBA/CaH) have long been used in hearing research (Berlin, 1963; Ohlemiller et al., 2016; Zheng et al., 1999). These mouse lines show minimal cochlear pathology and stable auditory brainstem response (ABR) thresholds over the first two years of life (Henry and Chole, 1980; Sergeyenko et al., 2013). Consequently, the CBA genotype has been widely accepted as representing normal hearing, and its auditory functions have routinely been compared with those of mice with various forms of hereditary hearing loss (e.g., Connelly et al., 2017; Limb and Ryugo, 2000; Willott, 1984, 1986; Willott et al., 1991). Recently, however, CBA/CaJ mice older than two years of age were observed to exhibit increased ABR thresholds, decreased ABR waveform amplitudes, and increased hair cell losses (Sergeyenko et al., 2013). Moreover, ribbon synapses between auditory nerve fibers and inner hair cells (IHCs) declined in number prior to spiral ganglion cell loss (Kujawa and Liberman, 2009; Stamataki et al., 2006). Thus, even in so-called “normal” hearing mice with a lifetime of non-damaging sound exposure, the cochlea may nonetheless exhibit “hidden” hearing loss as a result of age-related synaptopathy.
The causes of “hidden” and “overt” hearing loss generally originate in the cochlea. There is a growing consensus, however, that many symptoms of hearing loss (e.g., difficulty understanding speech in noise, loudness distortion, and tinnitus) are exacerbated by reactive changes within the central nervous system (Parks et al., 2004). Changes in auditory nerve activity caused by hearing loss have a ripple effect throughout the central auditory pathway (Hashisaki and Rubel, 1989; Kral et al., 2005; Moore and Kowalchuk, 1988; Powell and Erulkar, 1962; Saada et al., 1996; Schwartz and Higa, 1982; Trune, 1982; West and Harrison, 1973), and pathologic changes are likely to first emerge at the cochlear nucleus (CN).
Auditory nerve endings terminate throughout the CN (Muniak et al., 2016), and have been a subject of extensive studies on deprivation-induced changes. In particular, attention has focused on endbulbs of Held (EBs; Held, 1893)—large, axosomatic endings of myelinated auditory nerve fibers that target bushy cells (BCs) in the anteroventral CN (AVCN). These synaptic endings are among the largest synapses in the brain (Ryugo and Spirou, 2009) and show evolutionary conservation across all vertebrates examined to date (Lenn and Reese, 1966; Ryugo and Parks, 2003). They are a crucial structure in the timing pathway, delivering auditory spikes at rapid rates and with high fidelity (Manis et al., 2011). Crucially, EBs have been shown to be structurally and functionally sensitive to changes in levels of spike activity, as would occur with deafness or hearing loss (Connelly et al., 2017; Ngodup et al., 2015; Oleskevich and Walmsley, 2002; Ryugo et al., 1996, 1997, 1998, 2005; Tsuji and Liberman, 1997; Wang and Manis, 2006; Zhuang et al., 2017). Thus, cochlear pathologies have the potential to cascade along ascending neural pathways, consequently producing deficits in central auditory processing (Frisina and Frisina, 1997; Lorenzi et al., 2006).
Age-related synaptopathy between auditory nerve fibers and IHCs (Sergeyenko et al., 2013) would render the affected primary fibers unresponsive to sound. Given the known role of activity on central synapse structure of the auditory nerve, we asked whether there would be a structural correlate of age-related hearing loss expressed centrally in EBs. We first examined our database of historical ABR records for CBA/CaH mice up to one year of age to corroborate and extend recent observations (Sergeyenko et al., 2013). Next, we examined EB morphology in a subset of these mice at specific age-groups. We hypothesized that age-related cochlear synaptopathy, as inferred from reductions in ABR waveform amplitudes, would result in atrophy of EB morphology reminiscent of observations in animals with overt hearing loss (e.g., Connelly et al., 2017; Limb and Ryugo, 2000; Ryugo et al., 1997).
Section snippets
Animals
All procedures were performed in accordance with NHMRC guidelines and approved by the Animal Ethics Committee of the Garvan Institute of Medical Research and St. Vincent's Hospital, UNSW Australia. A total of 22 normal hearing CBA/CaH mice of both sexes (11 male, 11 female) were used in this study. Age at time of study ranged from 4 to 52 wks. Ten mice were of the CBA/CaH genotype. The remaining 12 mice were transgenic CBGlyT2-EGFP mice, which expressed enhanced green flourescent protein (EGFP)
Auditory brainstem response
ABRs were collected from mice beginning at various age points until they were terminated. Most mice used specifically for this study underwent ABR testing at multiple ages (range: 1–45 wks duration between first and last reading; median 9 wks duration). Results were assessed in 4-wk intervals; if a mouse was tested more than once within a specific age group (e.g., 7–10 wks of age) only one sample from that timeframe was included for analysis. Of the 147 additional subjects that were included
Discussion
In this study, we investigated the effects of age-related hearing loss on the morphology of EBs in the AVCN of mice. It was hypothesized that “hidden” hearing loss—a net reduction in sound-evoked activity in the auditory nerve in the absence of threshold shifts (Liberman and Kujawa, 2017)—would result in measurable changes in EB structure, producing a moderate version of EB pathology observed in mice with progressive acquired hearing loss (Connelly et al., 2017) or congenital deafness (Limb and
Role of authors
The authors had full access to all the data in the study and take responsibility for the integrity of the data and accuracy of the analysis. Study concept and design: MAM, FEA, DKR. Acquisition of data: FEA. Analysis and interpretation of data: MAM, FEA, DKR. Drafting and revising the manuscript: MAM, FEA, DKR.
Declarations of interest
None.
Acknowledgements
The authors thank Catherine Connelly, Kiera Grierson, Giedre Milinkeviciute, and Kirupa Suthakar for sharing their ABR data, Timothy Peters for providing statistical advice, and the reviewers for their constructive comments on the manuscript. This work was supported by grants from the National Health and Medical Research Council (NHMRC; Grant nos.1080652 and 1081478), the Oticon Foundation (Grant no. 12-1540), the Walker Family Foundation, and gifts from Alan and Lynne Rydge, Haydn and Sue Daw,
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Authors contributed equally to this work.