The aging cochlea: Towards unraveling the functional contributions of strial dysfunction and synaptopathy

Hearing Research 376 (2019) 111e124

Contents lists available at ScienceDirect

Hearing Research journal homepage: www.elsevier.com/locate/heares

Review Article

The aging cochlea: Towards unraveling the functional contributions of strial dysfunction and synaptopathy € ppl* Amarins N. Heeringa, Christine Ko Cluster of Excellence ‘Hearing4all’ and Research Centre Neurosensory Science, Department of Neuroscience, School of Medicine and Health Science, Carl von Ossietzky University Oldenburg, 26129, Oldenburg, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 September 2018 Received in revised form 1 February 2019 Accepted 26 February 2019 Available online 2 March 2019

Strial dysfunction is commonly observed as a key consequence of aging in the cochlea. A large body of animal research, especially in the quiet-aged Mongolian gerbil, shows specific histopathological changes in the cochlear stria vascularis and the putatively corresponding effects on endocochlear potential and auditory nerve responses. However, recent work suggests that synaptopathy, or the loss of inner hair cellauditory nerve fiber synapses, also presents as a consequence of aging. It is now believed that the loss of synapses is the earliest age-related degenerative event. The present review aims to integrate classic and novel research on age-related pathologies of the inner ear. First, we summarize current knowledge on age-related strial dysfunction and synaptopathy. We describe how these cochlear pathologies fit into the categories for presbyacusis, as first defined by Schuknecht in the ‘70s. Further, we discuss how strial dysfunction and synaptopathy affect sound coding by the auditory nerve and how they can be experimentally induced to study their specific contributions to age-related hearing deficits. As such, we aim to give an overview of the current literature on age-related cochlear pathologies and hope to inspire further research on the role of cochlear aging in age-related hearing deficits. © 2019 Elsevier B.V. All rights reserved.

Keywords: Age-related hearing loss Auditory nerve Endocochlear potential Presbyacusis Ribbon synapse Stria vascularis

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Age-related strial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 2.1. Histopathology of the stria vascularis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 2.2. Decrease of the endocochlear potential and potassium concentration in scala media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 2.3. Where does strial dysfunction fit into Schuknecht's categories of presbyacusis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 2.4. Experimental induction of strial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Age-related synaptopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3.1. Degeneration of ribbon synapses and spiral ganglion neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 3.2. Changes in ribbon synapses that survive in an aging cochlea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 3.3. Where does synaptopathy fit into Schuknecht's categories of presbyacusis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 3.4. Experimental induction of synaptopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 How are auditory nerve responses affected by strial dysfunction and synaptopathy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 4.1. Single-unit auditory nerve fiber responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 4.1.1. Threshold losses in quiet-aged gerbils are likely dominated by strial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 4.1.2. Supra-threshold responses may be more subtly affected . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 4.1.3. Spontaneous rates can be altered by both strial dysfunction and synaptopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 4.2. Compound auditory-nerve responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 4.2.1. Thresholds in quiet-aged gerbils are probably raised due to strial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 4.2.2. Supra-threshold responses are affected by synaptopathy and potentially also by strial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

* Corresponding author. Department of Neuroscience, Carl von Ossietzky University, Carl-von-Ossietzky-Straße 9-11, 26129, Oldenburg, Germany. €ppl). E-mail address: [email protected] (C. Ko https://doi.org/10.1016/j.heares.2019.02.015 0378-5955/© 2019 Elsevier B.V. All rights reserved.

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

112

5.

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations ABR auditory brainstem response ARHL age-related hearing loss BDNF brain-derived neurotrophic factor CAP compound action potential DPOAE distortion-product otoacoustic emission EP endocochlear potential IHC inner hair cell I/O function input-output function [Kþe] endolymphatic potassium concentration NKCC1 Na-K-Cl cotransporter isoform 1 NT-3 neurotrophin-3 OHC outer hair cell OSL osseous spiral lamina SGN spiral-ganglion neuron SR spontaneous rate

1. Introduction Age-related hearing loss (ARHL) affects about two-thirds of people over seventy years of age (Lin et al., 2011), resulting in social isolation and decreased quality of life (Strawbridge et al., 2000). This sensory deficit slowly progresses with age: hearing thresholds increase about 1 dB per year in people aged 60 and over (Lee et al., 2005). Age-related hearing deficits are associated with several histopathological lesions in the inner-ear cochlea. Early histological observations of human temporal bones by Schuknecht, aiming to provide some differential diagnostics, categorized the primary pathological lesions in the aging cochlea as degeneration of the stria vascularis (strial presbyacusis), degeneration of spiral ganglion neurons (SGNs) (neural presbyacusis), and loss of hair cells (sensory presbyacusis) (Schuknecht, 1964). Conductive or mechanical presbyacusis was defined as a hypothetical category based solely on the shape of the audiogram and suggested to be possibly due to alterations in basilar-membrane mechanics (Schuknecht, 1964). Interestingly, there are no significant age-related middle-ear pathologies known in humans (Humes and Dubno, 2010). In the ‘90s, Schuknecht and Gacek (1993) added two further categories: mixed presbyacusis, in which the temporal bone shows a mixture of age-related cochlear histopathologies, and indeterminate presbyacusis, in which the cochlear changes did not adequately explain the hearing deficits. Schuknecht and colleagues suggested that the different cochlear pathologies are associated with dissimilar profiles of hearing loss, as assessed with the audiogram and speechrecognition scores, see (Otte et al., 1978; Pauler et al., 1986, 1988; Schuknecht and Gacek, 1993; Schuknecht et al., 1974), but also see (Suga and Lindsay, 1976). Additional clinical measures, such as otoacoustic emissions and auditory brainstem responses (ABRs), were also more recently proposed for use in diagnosing patients with a specific type of presbyacusis (Dubno et al., 2013; Mills and Schmiedt, 2004; Ueberfuhr et al., 2016). In this review, we relate some of Schuknecht's classical presbyacusis categories to

121 121 121 121

experimental animal work, in order to foster future developments of improved diagnostic tools. ARHL is often referred to as presbyacusis, and these terms have been used interchangeably. Presbyacusis has been previously defined as the accumulation of all kinds of trauma over the lifespan that lead to hearing loss (Gates and Mills, 2005). When referring to studies on aged humans, this is inevitable. However, the focus of this review is on cochlear pathologies that result from natural aging processes (Caspary and Llano, 2018), in controlled animal studies. We define natural aging as degenerative processes, compared to young, healthy cochleae, that occur independently of external (ototoxic or traumatic) events in quiet-aged animals. Strial and neural presbyacusis are probably the most directly related to aging processes, whereas sensory presbyacusis is likely predominantly the result of (cumulative) noise exposure (Schmiedt et al., 1990; Schuknecht and Gacek, 1993). Therefore, this review focuses on strial and neural pathologies and will not discuss loss of hair cells. Strial dysfunction has often been observed in cochleae of aged humans and animals and is an important cause of ARHL (Schmiedt, 2010; Schuknecht and Gacek, 1993). Therefore, a large body of animal research has been devoted to this phenomenon, as reviewed for the mouse, gerbil, and rat in (Ohlemiller, 2009; Schmiedt, 2010; Syka, 2010), respectively. Histopathologies of the different cell types in the stria vascularis have been thoroughly characterized, as has the resulting reduction in the endocochlear potential (EP) and some of the corresponding changes in hearing sensitivity (Schmiedt, 2010). However, recent research suggests that synaptopathy, i.e. the loss of synapses between the inner hair cells (IHCs) and auditory nerve fibers, is also an important part of cochlear aging processes. Based on observations on mice, it is now believed that the loss of afferent synapses is the earliest age-related degenerative event and, importantly, is typically not evident in the threshold audiogram (Kujawa and Liberman, 2015; Liberman, 2017; Sergeyenko et al., 2013). This review is aimed at integrating classic and novel research on age-related pathologies of the stria vascularis and primary afferent neurons, and at identifying urgent, unresolved questions regarding the functional consequences for auditory nerve coding. The Mongolian gerbil is an attractive model for studying agerelated cochlear deficits and linking the findings to functional consequences (and human presbyacusis), because of a unique combination of traits. First, gerbils have a moderately short lifespan of around 36 months (Cheal, 1986), making aging studies experimentally feasible. Second, the gerbil has good low-frequency hearing sensitivity, similar to humans (Ryan, 1976). Third, the gerbil is readily trained for auditory discrimination tasks (Hamann et al., 2004), providing a bridge to human psychophysics. Fourth, quiet-aged gerbils show characteristics of strial and neural presbyacusis, but no confounding IHC loss and only minor outer hair cell (OHC) loss (Tarnowski et al., 1991) (Fig. 2C). Fifth, experimentally induced synaptopathy can be precisely controlled in its extent in the gerbil, by applying different concentrations of ouabain to the cochlear round window (Bourien et al., 2014). Sixth, there is already a large body of research on linking strial presbyacusis to cochlear function in the quiet-aged gerbil, primarily carried out by Schmiedt and colleagues. And last, currently all single-unit auditory-nerve

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

113

recordings in aged animals that provide an essential link between age-related cochlear pathologies and functional consequences, were obtained in quiet-aged gerbils. Therefore, this review will focus on studies of the quiet-aged gerbil. Studies in other species will not be exhaustively reviewed but are included where they add salient information, particularly for the more recent research field of cochlear synaptopathy. For studying the contribution of genetic variation, which plays an important role in the manifestation of natural cochlear aging, inbred strains of mice are evidently more suitable animal models than the gerbil (Ohlemiller, 2018). We address and review the following questions: 1. What is the state of current knowledge on the relative roles of strial dysfunction and synaptopathy in age-related hearing deficits? 2. Where do these pathologies fit into the classic Schuknecht categories? 3. How do these pathologies affect sound coding by the auditory nerve? 4. How can these pathologies be experimentally examined in isolation to study their specific contributions to age-related hearing deficits? Below, we first provide a summary of the literature on agerelated strial dysfunction and age-related synaptopathy. Note that the causes of age-related strial dysfunction and synaptopathy, and compounding risk factors, such as genetic predispositions, oxidative stress, exposure to noise and ototoxic compounds, etc. have been extensively reviewed elsewhere and will not be addressed here (Fetoni et al., 2011; Kujawa and Liberman, 2015; Liberman, 2017; Moser and Starr, 2016; Ohlemiller, 2009; Ruan et al., 2014; Wang and Puel, 2018; Yamasoba et al., 2013). We then discuss, separately for strial dysfunction and synaptopathy, how these disorders fit into Schuknecht's categories for presbyacusis and how they can be experimentally induced. Subsequently, we discuss how single-unit and compound auditory nerve responses are (hypothetically) affected by age-related strial dysfunction and synaptopathy. Finally, we summarize the main conclusions and future research avenues. 2. Age-related strial dysfunction The stria vascularis is a highly vascularized tissue located in the lateral wall of the cochlea. It provides the high EP and potassium concentration of the endolymph [Kþe] in scala media (Fig. 1A). Both a high EP (~þ80 mV) and a high [Kþe] (~180 mM) are necessary for proper cochlear amplification and signal transduction (Mistrik and Ashmore, 2009; Ruggero and Rich, 1991). Ion channels, cotransporters, and electrogenic pumps located in the stria vascularis are crucial for generating and maintaining these high EP and [Kþe] and include a Na-K-Cl cotransporter (NKCC1), a Na-K-ATPase, and several potassium and chloride channels (Diaz et al., 2007; Ohlemiller, 2009; Rickheit et al., 2008) (Fig. 1B). It is important to note that the EP and [Kþe], although linked, are not the result of exactly the same processes in the stria vascularis. It has been shown that the marginal cells are primarily responsible for the [Kþe], but contribute only a few mV to the EP (Wangemann et al., 1995). On the other hand, the intrastrial space already maintains a high positive potential, suggesting that the EP is generated by intermediate and basal cells (Nin et al., 2008; Salt et al., 1987) (Fig. 1B). Furthermore, following pharmacological blocking of the NKCC1 cotransporter or anoxia, the EP dropped much more rapidly and recovered faster than the [Kþe] (Rybak and Morizono, 1982; Salt and Konishi, 1979). Below, we first briefly review the literature describing age-

Fig. 1. Schematic cross-section of the cochlea and stria vascularis. A) The stria vascularis, indicated in dark green, is located in the lateral wall of the cochlea. The red arrow represents the direction and approximate route of Kþ cycling. Endolymph in the scala media, with the corresponding high EP and [Kþe], is represented in light blue. Greek numbers in the lateral wall indicate locations of the specific fibrocyte types I-V. B) Schematic magnification of the dashed rectangle in panel A, in which the location of the basal, intermediate, and marginal cell types of the stria vascularis are shown. The subcellular locations of crucial ion channels, the cotransporter NKCC and the Na-KATPase are also indicated. [Kþ] and the intrastrial- and endocochlear potential are indicated in red lettering. Reprinted from Hibino et al. (2010) (their Fig. 1), by permission from Springer Nature.

related histopathologies in the different cell types of the stria vascularis (Table 1). The causes for these pathologies, such as ROS accumulation, mitochondrial DNA damage, genetic predispositions, inflammatory factors, etc., have been extensively reviewed elsewhere (Ruan et al., 2014; Fetoni et al., 2011; Ohlemiller, 2009). Next, we review how these histopathologies affect the EP and [Kþe]. Subsequently, we discuss where strial dysfunction fits into Schuknecht's categories of presbyacusis. This section closes with a review of how strial dysfunction can be deliberately induced to rigorously study its consequences in animal models.

2.1. Histopathology of the stria vascularis In the lateral wall of the gerbil cochlea, the most common finding with increasing age is a decrease in the area or volume of the stria vascularis (Gratton and Schulte, 1995; Sakaguchi et al., 1998; Schulte and Schmiedt, 1992). This strial degeneration starts in the most apical and most basal part of cochlea and extends with age towards the middle turns. Marginal cells lose their primary and secondary processes, which contain the many mitochondria that provide energy for the numerous Na-K-ATPase electrogenic pumps

114

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

of these cells (Spicer and Schulte, 2005). Immunoreactivity for NKCC1 and Na-K-ATPase in the stria vascularis deteriorates in a parallel fashion (Sakaguchi et al., 1998; Schulte and Schmiedt, 1992). Similar findings have been reported in aged rats (Buckiova et al., 2007; Mannstrom et al., 2013) and mice (Han et al., 2016; Saitoh et al., 1995). It is not yet known if the loss of these proteins triggers strial atrophy or vice versa. The stria vascularis, as its name suggests, is highly vascularized and its capillaries are also subject to age-related declines. Starting again at the extreme ends of the cochlea, areas with few or no capillaries can be found, and these areas expand to the middle regions with increasing age (Gratton and Schulte, 1995). This process is preceded by decreases in capillary diameter and a thickening of the basement membrane of the capillaries (Thomopoulos et al., 1997). A decrease in total area of the strial capillaries with age is associated with the previously described strial atrophy (Gratton et al., 1997). Age-related, reduced vascularization has also been described in other rodents (Buckiova et al., 2006; Han et al., 2016; Ohlemiller et al., 2010). 2.2. Decrease of the endocochlear potential and potassium concentration in scala media The histopathologies in the stria vascularis are conceivably causally linked to changes in the endolymph of the scala media. Indeed, aged gerbils typically have a decreased EP (Schmiedt, 2010; Schulte and Schmiedt, 1992), which correlates with strial atrophy (Schulte and Schmiedt, 1992) (Fig. 2A) and with degeneration of the microvasculature in the stria vascularis (Gratton et al., 1996). These correlations suggest that the EP reduction is primarily due to strial dysfunction and not, for example, the result of a reduced resistance between cochlear compartments. Furthermore, application of salient antagonists, such as furosemide and ouabain that block NKCC1 and Na-K-ATPase, respectively, result in an EP reduction in healthy young individuals (Nin et al., 2008; Rybak et al., 1984; Sewell, 1984c). A reduction in EP results directly in elevated hearing thresholds. The first line of evidence for this comes from experiments using furosemide. An EP decline following furosemide application correlates tightly with the thresholds of auditory nerve single units (Evans and Klinke, 1982; Sewell, 1984c). Similarly, EP reduction, either due to chronic intracochlear furosemide infusions or due to aging, correlates with threshold shifts measured using the compound action potential (CAP) of the auditory nerve, especially at frequencies above 4 kHz (Schmiedt et al., 2002a) (Fig. 3A). Additionally, it has been shown that directly injecting current into scala media of gerbils with ARHL significantly improved the animal's CAP thresholds (Schmiedt, 1993) (Fig. 3B), showing conclusively that EP reduction in aged gerbils is a direct cause of deteriorated thresholds of hearing. In contrast to the EP, [Kþe] does not decline significantly with aging and, although related, there is no immediate correlation between EP and [Kþe] decline (Schmiedt, 1996). It was suggested that because EP reduction decreases the energy needed to pump Kþ into the scala media, it is possible to maintain a high [Kþe] even in the face of an already compromised function of the stria vascularis. However, once the EP falls below a threshold value of þ60 mV in Fig. 2. Schuknecht's presbyacusis categories in the cochlea of quiet-aged gerbils. A) Evidence for age-related strial dysfunction. Strial degeneration is correlated to mean EP decrease in quiet-aged gerbils; replotted Fig. 5 of (Schulte and Schmiedt, 1992). Inset: Mean EP (±STD) is significantly decreased in quiet-aged (orange) compared to young gerbils (black); replotted part of Fig. 2 in (Schmiedt, 1996). B) Evidence for age-related synaptopathy (neural presbyacusis). Mean number of ribbon synapses per IHC (±STD) in young (black) and quiet-aged gerbils (orange), as a function of approximate position along the cochlea, and the overall means (*p < 0.05, **p < 0.01 by Mann Whitney U test); replotted Fig. 4 of (Gleich et al., 2016). C) Evidence for minimal age-related hair-

cell loss (sensory presbyacusis). Mean percentage of IHCs (triangles) and OHCs (circles) still present in quiet-aged gerbils, compared to young gerbils, as a function of frequency along the cochlea. IHC loss is minimal; mean OHC loss does not exceed 25% compared to young gerbils. Note that the criterion for sensory presbyacusis, as defined by (Schuknecht and Gacek, 1993), is the presence of any total loss of hair cells starting in the basal end of the cochlea that extends into the lower (speech) frequency part of the cochlea; replotted Fig. 7 of Tarnowski et al. (1991).

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

115

Table 1 Overview of putative cochlear pathologies and abnormal auditory nerve fiber responses in quiet-aged gerbils. Presbyacusis category

Experimental finding

Strial presbyacusis Strial presbyacusis Strial presbyacusis Strial presbyacusis Strial presbyacusis Strial presbyacusis Neural presbyacusis Neural presbyacusis Mixed neural and strial presbyacusis Mixed neural (and strial?) presbyacusis Synaptopathic presbyacusis?

Degeneration of the stria vascularis (Gratton and Schulte, 1995; Schulte and Schmiedt, 1992) Marginal cell malformation (Spicer and Schulte, 2005) Reduced expression of NKCC1 and Na-K-ATPase in stria vascularis (Sakaguchi et al., 1998; Schulte and Schmiedt, 1992) Vascular degeneration and malformation (Gratton and Schulte, 1995; Gratton et al., 1997; Thomopoulos et al., 1997) Decreased EP (Schmiedt, 1996; Schulte and Schmiedt, 1992) Single-unit and compound auditory nerve threshold elevations (Mills et al., 1990; Schmiedt et al., 1990) Reduced number of synaptic ribbons per IHC (Gleich et al., 2016) Degeneration of nerve fibers in OSL and spiral ganglion (Keithley et al., 1989; Suryadevara et al., 2001) Single-unit SR abnormalities (Heeringa et al., 2018; Schmiedt et al., 1996)

Synaptopathic presbyacusis?

Reduced CAP or ABR wave I amplitude (Hellstrom and Schmiedt, 1990) Structural changes in afferent synapses that survive in the aged ear (Stamataki et al., 2006) e shown in C57BL/6J mice, remains to be determined for quiet-aged gerbil Relocation of lateral efferents from afferent terminal to IHC somata (Lauer et al., 2012) e shown in C57BL/6J mice, remains to be determined for quiet-aged gerbil

D

normal, young

young, ouabain-treated

Fig. 3. Effects of strial dysfunction and synaptopathy on auditory nerve responses. A) Mean CAP threshold shifts in three groups of quiet-aged gerbils (shown in orange) and furosemide-treated gerbils (shown in grey). Horizontal dashed line indicates a best fit to the furosemide data for frequencies <4 kHz; diagonal dashed line indicates a best fit to the furosemide data above 4 kHz and has a slope of 8.4 dB/octave; replotted Fig. 7 of Schmiedt et al. (2002a), republished with permission of the Society of Neuroscience; permission conveyed through Copyright Clearance Center, Inc. B) Effects of current injection into the scala media of a quiet-aged gerbil on CAP threshold and amplitude in response to a 4 kHz tone burst. Circles indicate the baseline CAP I/O function of the old gerbil, EP ¼ 41 mV. Diamonds indicate the I/O function during a 10 mA current injection into scala media of the first turn (base) of the cochlea. Note that threshold improved by > 20 dB and the slope of the I/O function became steeper; replotted part of Fig. 6.8 in Schmiedt (1993), republished with permission of Taylor and Francis Group LLC Books; permission conveyed through Copyright Clearance Center, Inc. C) The simultaneous decrease in the EP (black solid line) and auditory nerve SR (magenta circles), as well as auditory nerve threshold elevation (blue triangles), measured in the same ear of a cat following a brief intravenous injection of furosemide. The time of injection is indicated by the grey bar; replotted and combined Fig. 3 of Sewell (1984a), republished with permission of John Wiley and Sons Inc., permission conveyed through Copyright Clearance Center, Inc., and Fig. 1 of Sewell (1984c). D) Auditory-nerve fiber distribution based on SR, in young gerbils that were untreated (left) and that were treated with 33 mM ouabain application to the cochlear round window (right). Note that the population of low-SR fibers is most drastically reduced by the ouabain treatment. Reprinted from Fig. 5 of Bourien et al. (2014), by permission of The American Physiological Society.

116

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

aged gerbils, [Kþe] also declines (Schmiedt, 1996). Age-related reduction of [Kþe] and its effects on hearing have not been studied as thoroughly as age-related EP reduction. 2.3. Where does strial dysfunction fit into Schuknecht's categories of presbyacusis? Human temporal bone studies have shown similar pathologies of the stria vascularis, as summarized above and in Table 1 for gerbil studies. Strial atrophy, measured as a reduction of strial volume, has been frequently observed in cochleae of aged humans (Ishiyama et al., 2007; Johnsson and Hawkins, 1972b; Schuknecht, 1964; Schuknecht et al., 1974). The degree of strial atrophy correlates well with age and with hearing loss at frequencies between 0.5 and 4 kHz (Ishiyama et al., 2007; Pauler et al., 1988); frequencies > 4 kHz are typically not evaluated in humans. As in gerbils, strial atrophy begins in the most apical and most basal (hook) regions of the cochlea and progresses towards the middle regions (Johnsson and Hawkins, 1972b; Schuknecht, 1964). Furthermore, marginal cell processes shrink and are eventually completely disappeared, and edema is seen in the marginal and intermediate cell layers (Schuknecht et al., 1974). Thickening of the basement membrane around strial capillaries has also been reported (Johnsson and Hawkins, 1972a; Schuknecht et al., 1974). This phenotype was termed strial presbyacusis by Schuknecht (1964). Having a strial volume loss greater than 30% has been used as a criterion to be classified with strial presbyacusis and this associates with a uniform, flat hearing loss across frequencies (Pauler et al., 1988; Schuknecht and Gacek, 1993). We suggest, as suggested by Schuknecht, that strial presbyacusis is an important result of aging processes in the cochlea of humans and animals. However, in humans, and possibly gerbil, it is also typically only one component of a mixed presbyacusis (Gates and Mills, 2005; Schuknecht and Gacek, 1993). Quiet-aged gerbils show similar histopathologies of the stria vascularis, suggesting a valid model for strial presbyacusis (Table 1). Since the stria vascularis is responsible for generating and maintaining the EP and because the age-related pathologies in the gerbil correlate with a pronounced EP reduction, it is highly likely that humans with strial presbyacusis also have a decreased EP. However, EP reduction has never been shown experimentally in aged humans. 2.4. Experimental induction of strial dysfunction Strial dysfunction can be induced by administration of loop diuretics - pharmacological compounds that affect ion transport that are used to treat patients with kidney conditions. Because of similar ion-transport proteins in the kidney and the stria vascularis, loop diuretics also affect the EP (Ikeda et al., 1997). Furosemide, a reversible antagonist of NKCC cotransporters, is frequently used to induce strial dysfunction in laboratory animals. Correspondingly, at high systemic furosemide concentrations, patients treated with this drug often develop (usually transient) hearing loss and/or tinnitus (Ikeda et al., 1997). Structural changes in the stria vascularis following furosemide administration include enlargements of intracellular spaces (strial edema), swelling of the marginal cells and their processes, and shrinkage and atrophy of intermediate cells (Forge, 1976; Pike and Bosher, 1980). Concurrently, the EP decreases and thresholds measured by the CAP and in single auditory nerve fibers are elevated as a function of the systemic furosemide concentration (Evans and Klinke, 1982; Sewell, 1984a, 1984c). However, the [Kþe] does not follow the same time course; its decline and recovery is slower and lags behind the EP changes following furosemide injection (Rybak and Morizono, 1982),

suggesting that furosemide primarily targets the EP and that the effects on [Kþe] are secondary. The above-mentioned effects of furosemide on the stria vascularis, the EP, the [Kþe], and hearing thresholds were obtained following intravenous or intraperitoneal injections and the effects were typically fast, transient, and very drastic (see solid line in Fig. 3C for an example of the EP measured after an intravenous furosemide injection). In an attempt to produce a milder but longlasting effect more similar to the situation in aged cochleae, Schmiedt and colleagues developed a model for strial presbyacusis by chronically infusing furosemide onto the round window of the gerbil cochlea. After 7 days of such infusion, EP, CAP thresholds, and amplitudes of distortion product otoacoustic emission (DPOAE, reflecting OHC integrity and function) reached a level similar to that in aged gerbils (Schmiedt et al., 2002a). However, in contrast to quiet-aged gerbils, strial edema and atrophy of intermediate cells was only seen in the hook region of the cochlea, closest to the location of the furosemide infusion (Lang et al., 2010). Another factor to consider when using furosemide to induce strial dysfunction is its direct effect on OHCs. Using DPOAE and simultaneous EP recordings, Mills et al. (1993) showed that the recovery of the DPOAE amplitude follows a different, faster, time course than that of the EP. This suggests an adaptive mechanism of OHC function upon EP decline. Furthermore, OHC motility in vitro is also directly sensitive to furosemide (Santos-Sacchi et al., 2001). The extent of these effects is likely dependent on the administration route for furosemide: whereas intravenous injection will mainly target the stria vascularis and in turn the EP, intracochlear infusion may have larger, direct effects on OHC motility. In summary, chronic furosemide application mimics typical agerelated effects on EP and hearing thresholds. However, lateral-wall histopathologies resulting from this protocol are not consistent with the typical, age-related changes observed. Furthermore, adaptive effects, especially in OHCs, may confound the outcomes and result in discrepancies between different studies, and especially between chronically furosemide-treated and quiet-aged experimental groups. Therefore, furosemide, and probably other loop diuretics, do not directly mimic strial presbyacusis and should be used and interpreted with care. The observed variation in the degree and time of onset of strial dysfunction in both humans and gerbils implies the involvement of genetic factors, as reviewed previously (Ohlemiller, 2006, 2009). Indeed, studies using different strains of mice or rats revealed differences in the development and manifestation of strial presbyacusis and ARHL (Bielefeld et al., 2008; Fetoni et al., 2011; Ohlemiller and Gagnon, 2004; Ohlemiller et al., 2010; Saitoh et al., 1995). A table comparing ARHL in different rodent species and strains can be found in Fetoni et al. (2011). Studies in mice with specific mutations have revealed a number of genes involved in strial dysfunction, for example genes responsible for proper functioning of potassium channels (Diaz et al., 2007; Mustapha et al., 2009; Ohlemiller et al., 2009). Furthermore, there are mouse strains available that model specific aspects of human strial presbyacusis, such as higher incidences of strial presbyacusis in post-menopausal women vs. men, and in people with albinism (Cable et al., 1993; Guimaraes et al., 2004; Ohlemiller, 2009). Therefore, using specific (mouse) strains that have a genetic predisposition or genetic mutation for developing strial dysfunction is another effective approach to study strial presbyacusis. 3. Age-related synaptopathy At the IHC-auditory nerve fiber synapse, also called the ribbon synapse, acoustic information is transformed into a neural spiking signal that is carried to the central auditory system (Fig. 4). The

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

117

Fig. 4. Schematic overview of ribbon synapses. A) Cross section through the organ of Corti, with the location of the OHCs and IHCs indicated. The ribbon synapses are located at the basal poles of the hair cells and are juxtaposed to the peripheral terminals of type I afferent auditory nerve fibers. B) Schematic magnification of the region framed by the rectangle in panel A. Two representative ribbon synapses are drawn, located at the base of the IHC, and highlight differences between synapses located at the IHC's pillar and modiolar sides, mostly based on classic cat data. On the modiolar side, larger presynaptic ribbons are juxtaposed to auditory nerve fibers with smaller axonal diameters, lower SRs, and higher thresholds, and vice versa on the pillar side. Reprinted from (Liberman, 2017), under the Creative Commons Attribution License.

presynaptic part, i.e. the IHC active zone, consists of a ribbon at which numerous vesicles, filled with the excitatory neurotransmitter glutamate, are anchored (Nouvian et al., 2006). The postsynaptic density comprises mostly AMPA-type receptors that bind the released glutamate (Puel, 1995). The complex molecular makeup of the pre- and post-synaptic structures gives rise to the unique abilities of type I auditory neurons to accurately encode rapid temporal acoustic fluctuations (Nouvian et al., 2006; Reijntjes and Pyott, 2016). In the mammalian cochlea, each auditory afferent makes only one synaptic connection with an IHC, whereas each IHC is innervated by multiple afferents. Around 10e25 auditory nerve fibers connect to one IHC in the normal-hearing gerbil ear, depending on its location along the cochlea (Meyer et al., 2009; Zhang et al., 2018). Certain morphological characteristics of the ribbon synapse correlate with morphological and physiological properties of the apposed auditory nerve fiber. In cats, synapses on the pillar side of the IHC have smaller ribbons, which are less complex and have fewer vesicles, and are connected, on the postsynaptic side, to afferents with a larger axonal diameter, compared to synapses located on the modiolar side (Fig. 4B). Furthermore, these fibers connecting on the pillar side of the IHC tend to have lower thresholds and higher spontaneous rates (SRs) than fibers connecting on the modiolar side (Liberman, 1982; Merchan-Perez and Liberman, 1996). In addition, it has been shown in mice that the larger ribbons on the pillar side are connected to afferent terminals with smaller postsynaptic glutamate receptor patches, and vice versa (Liberman et al., 2011). However, these correlations do not hold for all mammalian species. In gerbils, the smaller ribbons on the modiolar side of the IHC are associated with smaller glutamate receptor patches (Zhang et al., 2018). It is important to keep in mind that to date, a direct correlation between fiber physiology and morphology has only been shown for the cat (Liberman, 1982; Liberman and Oliver, 1984; Merchan-Perez and Liberman, 1996) and guinea pig (Gleich and Wilson, 1993; Tsuji and Liberman, 1997). Furthermore, these correlations applied to presynaptic ribbon size, contact position on the IHC, and axon diameter, but not to the size of the postsynaptic terminal or the postsynaptic density comprising the glutamate receptors (the latter was not investigated). Very recently, characterization of the molecular profiles of type I afferent fiber subtypes has begun (Petitpre et al., 2018; Shrestha et al., 2018; Sun et al., 2018).

In the sections below, we review recent literature relating to age-related synapse loss or synaptopathy. Since the idea of quantifying ribbon synapses is relatively recent, studies of age-related ribbon synapse loss are still scarce. Therefore, we also discuss the literature on age-related SGN loss, i.e. neuropathy or neural presbyacusis, a likely result of synaptopathy. Next, we discuss possible age-related deficits in the remaining ribbon synapses, i.e. those that survive and are still present in an aging cochlea. Then, we discuss where synaptopathy fits into Schuknecht's categories of presbyacusis and finish by reviewing how synaptopathy can be experimentally induced. 3.1. Degeneration of ribbon synapses and spiral ganglion neurons In aged gerbils, a mild loss in the number of ribbons per IHC of about 20%, which is most pronounced at the apex, was reported (Gleich et al., 2016) (Fig. 2B). Correspondingly, in an earlier study in which nerve fibers were counted in the osseous spiral lamina (OSL), it was shown that the number of nerve fibers per IHC decreased in aged gerbils (Suryadevara et al., 2001). An age-related decrease in ribbon synapses per IHC is also apparent in rats and €hrle et al., 2016; mice (Cai et al., 2018; Jiang et al., 2015; Mo Sergeyenko et al., 2013). Synapse loss is typically expressed by number of functional ribbon synapses per IHC, defined as colocalized pre- and postsynaptic label in immunohistological preparations. In quiet-aged gerbils, IHC loss is very rare (Adams and Schulte, 1997; Tarnowski et al., 1991). Indeed, the SGN degeneration in quiet-aged gerbils is of the order of a 15e25% loss, and thus comparable to the abovementioned ribbon synapse loss per IHC in old gerbils (Keithley et al., 1989; Schmiedt, 2010). However, in some mouse and rat models of ARHL, as well as in aged humans, IHCs also degenerate (Hequembourg and Liberman, 2001; Popelar et al., 2006; Schuknecht, 1964; Sergeyenko et al., 2013). When this is the case, the number of ribbons per IHC underestimates the total extent of nerve-fiber loss. The total number of synapses, number of fibers in the OSL, or number of SGNs would then be a more accurate measure of nerve-fiber loss. In aged mice, SGN degeneration is more severe than in gerbils, typically above 30%, but up to 60%, depending on the mouse strain (Dazert et al., 1996; Hequembourg and Liberman, 2001; Ohlemiller and Gagnon, 2004; Sergeyenko et al., 2013).

118

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

Synapse loss is also observed as a result of mild noise exposure, associated with a temporary threshold shift (Kujawa and Liberman, 2009). This raises the question as to whether age-related synapse loss is the result of cumulative, mild acoustic trauma over the lifespan. Synaptopathic noise exposure early in life, associated with no permanent threshold shift, exacerbates ARHL (Fernandez et al., 2015). However, synapse loss is also seen following tympanic membrane removal (Liberman et al., 2015), suggesting that noise exposure is not necessary for synapse loss and that other factors, such as regular activation of the afferent and efferent system, may be important for maintenance of the afferent synapses. Through such a mechanism, age-related strial dysfunction may itself be involved in the development of age-related synaptopathy. Although such an interaction has not yet been demonstrated, the correlation between extent of EP reduction and degree of alterations in afferent fiber morphology observed in aged gerbils (Suryadevara et al., 2001) suggests that this is worth exploring. 3.2. Changes in ribbon synapses that survive in an aging cochlea Excitotoxicity may be an important mechanism involved in the loss of ribbon synapses with age. Triggered by a variety of factors, excessive glutamate in the synaptic cleft induces prolonged depolarization and results in large ion influxes and subsequent water influx into the afferent post-synaptic fiber. This leads to swelling and retraction of the afferent terminal (Puel, 1995). These retracted terminals can either regenerate and form a new synapse with the IHC or remain permanently separated, eventually leading to SGN loss (Puel et al., 1995; Sergeyenko et al., 2013; Shi et al., 2016). Whether an afferent fiber regenerates or not depends, among other things, on exposure to neurotrophic signaling from the cochlea, i.e. fiber growth-promoting substances, such as neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) (Wan and Corfas, 2015). The role of neurotrophic factors in age-related synaptopathy has not yet been determined. Interestingly, in aged mice, the auditory nerve terminals that remain on IHCs show structural changes that suggest excitotoxicity and regeneration (Stamataki et al., 2006). The olivocochlear efferent system may also have a significant role in age-related synaptic changes. The normal, axodendritic innervation of the afferent terminals by the lateral olivocochlear efferents protects against age-related synapse loss: upon damaging the efferent pathways, more synapse loss was observed over time (Liberman et al., 2014). Furthermore, in aged mice, fewer lateral efferent axons are connected to the remaining surviving afferent terminals. Instead, some lateral efferents make new connections directly with the IHC (Lauer et al., 2012). This suggests that reduced axodendritic innervation by the lateral efferents may accelerate or even initiate age-related synapse loss e or, vice versa, that loss of afferent synapses entails a relocation of the efferent contacts. 3.3. Where does synaptopathy fit into Schuknecht's categories of presbyacusis? Recent studies have begun to quantify synapse loss in human temporal bones. The number of ribbons per IHC decreased with increasing age, with only 2e7 ribbons/IHC remaining in the oldest individual, depending on the cochlear location (Viana et al., 2015). Furthermore, peripheral axon counts, at the level of the OSL, decreased linearly with age and were found to be three times as high as the IHC degeneration, reaching losses of more than 60% in the majority of people aged over 60 (Wu et al., 2018). A large study of 100 temporal bones from humans aged between 0 and 100 years old showed that SGN counts similarly decrease linearly with age, at a rate of ~1000 neurons per decade (Makary et al., 2011). At 100

years of age, the average SGN loss compared to neonatal is about 30%. Since the people included in these studies had no significant hair-cell loss and no history of otologic disease, these data suggest that age-related synaptopathy occurs in humans as well and, furthermore, that age-related auditory nerve degeneration starts peripherally, at the synapse, and progresses slowly towards the SGN cell body. Neural presbyacusis was defined by Schuknecht and Gacek (1993) as a primary loss of SGNs of at least 50%, in the absence of any apparent pathology in the IHCs and OHCs. This means that in order to be diagnosed with neural presbyacusis, cochleae should show considerably more SGN loss over and above the inevitable age-related loss of SGNs described above. The criterion of 50% SGN loss was chosen based on studies showing that word discrimination was not clearly affected until SGN counts dropped below 50% (Otte et al., 1978; Pauler et al., 1986). Importantly, this discrimination deficit was typically not accompanied by audiometric threshold loss (Pauler et al., 1986). Thus, we suggest that Schuknecht's neural presbyacusis is equivalent to what is now often referred to as “hidden hearing loss” (Schaette and McAlpine, 2011). The original word-discrimination tests may have been the first tentative evidence for this phenomenon and efforts to identify more and/or better metrics are currently underway (Plack et al., 2014). The specific criterion of 50% reduction in SGN counts used to diagnose neural presbyacusis may thus not hold but the principal phenomenon was already recognized in Schuknecht's typology. Indeed, preceding the introduction of the classic presbyacusis categories, Schuknecht and Woellner (1955) had already experimentally demonstrated the phenomenon of neural presbyacusis (hidden hearing loss) in cats. Based on the loss of synapses, peripheral axons, and SGNs shown in human temporal bone and animal studies, we suggest that age-related ribbon synapse loss is an early stage of neural presbyacusis (Table 1). In mice, the rate of ribbon loss was similar to the rate of SGN loss over the lifespan, with the total percentage of SGN loss lagging by about 40 weeks (Sergeyenko et al., 2013). This strongly suggests that age-related ribbon synapse loss caused SGN loss. Furthermore, when the peripheral and central axons of SGN were counted in humans with presbyacusis, there was a systematically higher count of central fibers compared to peripheral fibers in the OSL (Felix et al., 1990). The difference between peripheral and central fiber counts was especially pronounced in humans classified with neural presbyacusis. This also suggests a retrograde degeneration starting at the peripheral end of the afferent auditory neuron. While the sequence of afferent neurodegenerative events thus appears to be clear, there is evidence that onset and time course relative to the lifespan may differ across species or even populations (Altschuler et al., 2015; Buckiova et al., 2006; Cai et al., € hrle et al., 2016; Sergeyenko et al., 2013; Zhang et al., 2018; Mo 2016). The synapses remaining in aged ears, however, may not be the same as those of young cochleae. Indeed, it has been shown that the morphology of ribbon synapses in aged mice is altered compared to those of young mice (Lauer et al., 2012; Stamataki et al., 2006). Evidence for regeneration of synapses after insults (Puel et al., 1995; Wan et al., 2014) suggests that in the face of age-related synapse loss, partial regeneration of synapses may be ongoing and could underlie the observed changes in synapse morphology. More research is needed to elucidate the effects of such more subtle synaptic changes on audiograms and auditory processing. Importantly, this putative type of age-related synaptopathy may be distinct from a mere loss of neural elements and may thus define an additional category of presbyacusis, which we tentatively term synaptopathic presbyacusis (Table 1).

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

3.4. Experimental induction of synaptopathy Currently the most common way to induce synaptopathy and SGN degeneration is a moderate noise exposure that induces a temporary but no permanent threshold shift (Liberman and Kujawa, 2017). An alternative method that does not involve acoustic trauma is to apply ouabain, which is a Na-K-ATPase antagonist, to the round window of the cochlea. Ouabain increases intracellular [Ca2þ] and [Naþ] and decreases intracellular [Kþ] in neuronal tissue, resulting in the activation of pro-apoptotic proteins such as cytochrome c and caspase-3 (Xiao et al., 2002). Due to its antagonistic action on the Na-K-ATPase pump, large concentrations of ouabain on the round window (~1 mM) result in an EP reduction (Rybak et al., 1984). This reduction is transient and the EP recovers rapidly within 2 h after removal of ouabain. Cochlear afferents, however, permanently degenerate in massive numbers following application of 1 mM of ouabain (Schmiedt et al., 2002b; Yuan et al., 2014). Ouabain selectively affects the IHC-innervating type I afferents and not the OHC-innervating type II afferents (Lang et al., 2005). Recent experiments have established that, in the gerbil, brief application of very low doses of ouabain (e.g. 33 mM) on the round window cause a much more moderate effect, offering the opportunity to carefully titrate the induced synaptopathy (Bourien et al., 2014). Furthermore, in gerbil, the type-I afferents with low SRs are the most vulnerable and degenerate first with low doses of ouabain (Bourien et al., 2014) (see also Fig. 3D). Several factors are relevant when using ouabain to model agerelated synaptopathy. First, SGN degeneration occurs much more rapidly after ouabain treatment compared to age-related synaptopathy. Apoptotic markers are detected in the ganglion as early as 3 h after ouabain treatment, whereas SGN loss occurs about 40 weeks after synapse loss (Lang et al., 2005; Sergeyenko et al., 2013). Second, as mentioned above, during age-related synaptopathy, cochlear innervation by lateral olivocochlear efferents changes from being axodendritic to partly axosomatic, innervating the IHC soma directly (Lauer et al., 2012). Interestingly, ouabain treatment results in the appearance of giant nerve terminals near the apex of the IHC soma. These giant terminals do not have a juxtaposed IHC ribbon. It has therefore been speculated that they are lateral efferent terminals (Yuan et al., 2014). This would suggest that efferent plasticity is similar between ouabain-induced and agerelated synaptopathy. Since efferent innervation of the afferents protects against synapse loss (Liberman et al., 2014; Reijntjes and Pyott, 2016), such a relocation of efferents in both age-related and ouabain-induced synaptopathy may accelerate the synapse loss. Third, it is not known whether age-related synaptopathy in gerbils is similarly selective, first affecting low-SR afferent fibers, as known from ouabain- or noise-induced synaptopathy (Bourien et al., 2014; Furman et al., 2013). In mice, there are potentially conflicting results about age-related effects on specific types of auditory nerve fibers. Age-related synapse loss in C57BL/6J mice showed no differential distribution along the pillar-modiolar dimension (Stamataki et al., 2006), which is the typical dimension along which auditory nerve fiber threshold and SR varies. This suggests that age-related synaptopathy may not be selective for a certain type of synapse. However, a recent study on CBA/CaJ mice that molecularly differentiated synapse types showed selective age-related degeneration of one particular type of synapse (type Ic), that matched the anatomical features of low-SR high-threshold fibers (Shrestha et al., 2018). Taken together, we suggest that ouabain could be used to accurately model age-related synaptopathy, although more research is needed to establish crucial details. Because of the importance of neurotrophic support for SGN survival, animal models in which trophic support in the cochlea is disrupted could also be used for the experimental induction of

119

synaptopathy. In temporal bones of aged humans, it was observed that the presence of pillar and Deiters cells, both supporting cells located in the organ of Corti, determined whether myelinated radial fibers in the OSL persisted or not (Johnsson and Hawkins, 1972c), suggesting that supporting cells play a crucial role in SGN survival. It was later discovered that these supporting cells produce NT-3 and BDNF, which are both important neurotrophic factors for SGN survival (Stankovic et al., 2004; Sugawara et al., 2007; Wan et al., 2014). NT-3, specifically, is involved in the regeneration of ribbon synapses following acoustic overexposure (Wan et al., 2014). Neurotrophin knockout models are another effective approach to study age-related synaptopathy and SGN loss and the functional consequences of regenerated synapses.

4. How are auditory nerve responses affected by strial dysfunction and synaptopathy? In this section, we discuss how each aspect of auditory nerve responses might be affected by strial dysfunction and synaptopathy. Auditory nerve responses can be assessed by directly recording action potentials from single fibers (single-unit auditory nerve fiber responses) or by recording a compound response using either subcutaneous electrodes (ABR) or an electrode on the round window of the cochlea (CAP). The results of these two ways of measuring auditory nerve activity have different implications and can point to different histopathological processes, and are therefore discussed in separate sections below (subsections 4.1 and 4.2, respectively). Whereas ABR and CAP data are abundantly available for both animals and humans with ARHL, there are only few publications reporting single-unit auditory nerve data from old animals, all of which derive from studies in quiet-aged gerbils.

4.1. Single-unit auditory nerve fiber responses 4.1.1. Threshold losses in quiet-aged gerbils are likely dominated by strial dysfunction In quiet-aged gerbils, thresholds at the best frequency of singleunit auditory nerve fibers are elevated (Schmiedt et al., 1990). Synapse loss could explain this only if it specifically targeted synapses from fibers with low thresholds, leaving high-threshold fibers unaffected. However, studies with noise- and ouabain-induced synaptopathy showed specific degeneration of low-SR fibers (Fig. 3D), which typically have high thresholds, thus leaving the low-threshold fibers unaffected (Bourien et al., 2014; Furman et al., 2013). Correspondingly, with noise-induced synapse loss, the thresholds of the surviving auditory nerve fibers are not elevated compared to controls (Furman et al., 2013). For strial dysfunction, the effects on auditory-nerve fiber threshold are clearer. Furosemide-induced strial dysfunction, and concurrent EP reduction, reduces the hair cells’ receptor potentials and thus results in single-unit threshold elevation. Importantly, the reduced receptor drive impairs both IHC and OHC function and thus acts two-fold to elevate thresholds: via a direct effect on IHC and via reduced cochlear amplification by OHCs (Ruggero and Rich, 1991). Both intravenous and intracochlear furosemide administration results in elevated thresholds of auditory nerve fibers (Evans and Klinke, 1982; Lang et al., 2010; Sewell, 1984c) (Fig. 3C). In fact, both age-related and furosemide-induced threshold shifts similarly and selectively affect the tip, and much less the low-frequency tail of the tuning curve (Schmiedt et al., 1990; Sewell, 1984c). Therefore, age-related single-unit auditory nerve fiber threshold shifts can be explained by strial dysfunction and concurrent EP reduction (Table 1).

120

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

4.1.2. Supra-threshold responses may be more subtly affected Studies on supra-threshold, single-unit auditory nerve activity in quiet-aged gerbils examined responses to pure tones in quiet, two-tone suppression, the slopes of rate-level functions (Hellstrom and Schmiedt, 1991; Schmiedt et al., 1990), and temporal coding in response to noise stimuli (Zhang et al., 2017). None of the measures showed any differences from those recorded in young gerbils, suggesting that supra-threshold responses of single unit auditory nerve fibers remain unaltered in quiet-aged gerbils, at least when evaluated at comparable sensation levels (i.e. adjusting stimulus levels to account for threshold shifts). Unfortunately, there are no comparable data from animals with experimentally induced strial dysfunction or synaptopathy. However, since these data were recorded in quiet-aged gerbils, in which both strial dysfunction and synaptopathy have been demonstrated by independent studies, it is likely that neither of these processes severely affects those suprathreshold responses. Data from guinea pigs with noise-induced synaptopathy confirms this, showing no effects on further suprathreshold measures, including tuning sharpness (Q10dB), dynamic range of the rate-level function, and first-spike latency and its variance (Furman et al., 2013). As discussed above for single-unit thresholds, the only way synapse loss is likely to affect suprathreshold responses is by specifically targeting synapses of fibers with certain physiological characteristics, leaving a biased sample unaffected. More research is needed to further investigate this hypothesis. On the other hand, furosemide-induced EP reduction can result in an altered shape of the rate-level function but does not affect maximum evoked activity (Evans and Klinke, 1982; Sewell, 1984b). This highlights the fact that findings may be highly dependent on the protocols used. Based on the limited data available, it is premature to conclude that neither age-related strial dysfunction nor synaptopathy affects any supra-threshold responses of the surviving auditory nerve single units. Future research should benefit from testing specific computational model predictions (Carney, 2018; Marmel et al., 2015; Saremi and Stenfelt, 2013). 4.1.3. Spontaneous rates can be altered by both strial dysfunction and synaptopathy Studies on SR in aged gerbils have shown somewhat conflicting results. There appears to be a loss of low-SR fibers among units with high best frequencies (>6 kHz) (Schmiedt et al., 1996). However, recent results from our lab showed a general decrease of SR, especially for single units with low best frequencies (Heeringa et al., 2018). A disproportionate loss of low-SR fibers is typical for both noise- and ouabain-induced synaptopathy (Fig. 3D) (Bourien et al., 2014; Furman et al., 2013), leading to the expectation that overall, mean spontaneous activity should increase, rather than decrease. However, as discussed above, it is not known for gerbils whether age-related synaptopathy has a similar specificity for low-SR fibers. Furthermore, lateral olivocochlear efferents may normally be involved in regulating spiking rates of afferents (Liberman, 1990; Ruel et al., 2001; Zheng et al., 1999), such that changes in efferent innervation in aging animals (see section 3.2) might also modify SRs in unknown ways. Furosemide-induced EP reduction, on the other hand, is clearly correlated with a decrease in SR within a single fiber (Fig. 3C) (Sewell, 1984a). In summary, EP reduction and synaptopathy may affect SR differentially and in various ways (Table 1). The relative predominance of the different processes will then determine the outcome. Experimental models, such as those described above (section 2.4 and 3.4), and computational models, such as those available for studying the effects of EP reduction on auditory nerve fiber responses (Saremi and Stenfelt, 2013) and auditory nerve fiber or synapse loss (Marmel et al., 2015) will be important to disentangle

the possible outcomes. 4.2. Compound auditory-nerve responses 4.2.1. Thresholds in quiet-aged gerbils are probably raised due to strial dysfunction ARHL is classically defined by elevated hearing thresholds, as measured by ABR or CAP (Henry et al., 1980; Mills et al., 1990). In quiet-aged gerbils, strial dysfunction alone can explain loss of thresholds (Fig. 3A). Correspondingly, in a study of human temporal bones, it was shown that the mean area of the stria vascularis correlated positively with hearing thresholds (Pauler et al., 1988); more strial degeneration correlated with larger threshold shifts. Furthermore, in cats with furosemide-induced EP reductions, CAP thresholds to clicks were elevated in a highly correlated fashion (Sewell, 1984c). The most direct evidence of age-related threshold shifts being related to EP reduction comes from a study in gerbils showing that injecting current into scala media of a gerbil cochlea with ARHL improved its thresholds by about 20 dB (Schmiedt, 1993) (Fig. 3B). Across frequency, it was found that the EP is more clearly correlated with high-frequency (16 kHz) than with lowfrequency (2 kHz) threshold shifts, both in the aged cochlea and in cochleae that received chronic furosemide infusions (Schmiedt et al., 2002a) (Fig. 3A). This results in a flat threshold shift at low frequencies and an ~8 dB/octave roll-off at higher frequencies. Based on this model, Dubno et al. (2013) classified audiograms from humans with age-related threshold shifts as likely deriving from strial dysfunction. Indeed, this correlated with a less-than-average history of noise exposure and being of older age (Dubno et al., 2013), consistent with the notion that strial dysfunction is not related to noise exposure and is evident also in quiet-aged individuals. Synaptopathy, on the other hand, does not tightly correlate with ABR or CAP threshold shifts. On the contrary, it has been shown that following noise- and ouabain-induced synaptopathy, ABR or CAP thresholds can be completely normal, despite a loss of synapses of up to 60% (Bourien et al., 2014; Kujawa and Liberman, 2009) e a condition that is now commonly referred to as hidden hearing loss (Schaette and McAlpine, 2011). Similarly, with aging, synaptopathy or SGN loss can occur in the absence of thresholds shifts, but threshold shifts were observed earlier, after a relatively small loss of synapses or SGNs of 20% or 10e20%, respectively (Makary et al., 2011; Sergeyenko et al., 2013). Since these results were obtained as a function of age, however, other factors than synaptopathy could also be responsible for the threshold shift. In quiet-aged gerbils, in which both behavioral threshold shifts and synaptopathy were evaluated, behavioral threshold shift did not correlate with the frequency-specific synapse loss along the cochlea (Gleich et al., 2016). This suggests that a different mechanism, such as strial dysfunction, caused the observed age-related threshold elevation. Rather than a loss of thresholds, it was suggested that synaptopathy results in problems with supra-threshold processing, presenting as poor speech discrimination or problems with listening in noise (Kujawa and Liberman, 2015; Lopez-Poveda, 2014; Pauler et al., 1986), although conclusive results on this issue are still pending (Plack et al., 2014; Johannesen et al., 2019). In summary, there is strong evidence that strial dysfunction results in CAP or ABR threshold elevations, whereas synaptopathy does not correlate with these measures. Since ARHL is classically defined by the loss of thresholds, synaptopathy has barely been explored as a cause of age-related hearing deficits and the exploration for evidence of hidden hearing loss has only just begun. The gerbil as an animal model in which synaptopathy can be induced in a graded fashion, and that responds well to behavioral training, is well suited to approach this experimentally.

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

4.2.2. Supra-threshold responses are affected by synaptopathy and potentially also by strial dysfunction Amplitudes of the CAP and the ABR wave I, which both represent the compound activity of the auditory nerve, are typically reduced with increasing age in both humans and animals (Hellstrom and Schmiedt, 1990; Konrad-Martin et al., 2012; Sergeyenko et al., 2013). This amplitude reduction is largely uncorrelated with hearing threshold and occurs even in the absence of € hrle et al., threshold elevations (Konrad-Martin et al., 2012; Mo 2016). When thresholds are elevated, amplitudes should be compared at similar sensation levels or by using the slope of the input-output function (I/O-function), which plots amplitude as a function of sound level, to assess age-related effects on amplitude. Gerbils with age-related threshold shifts had a shallower I/O function slope (Hellstrom and Schmiedt, 1990). Noise- and ouabain-induced synaptopathy is tightly linked to reduced CAP or ABR wave I amplitude (Bourien et al., 2014; Kujawa and Liberman, 2009). Consequently, ABR wave I amplitude has been proposed as a good metric for neural degeneration (Kujawa and Liberman, 2009; Yuan et al., 2014). However, furosemideinduced EP reduction also results in a reduced CAP amplitude (Evans and Klinke, 1982), although similar sensation levels or I/O function slopes were not assessed in that study. Furthermore, although not quantified, current injections into scala media of aged animals e mimicking a normal EP e also seem to reverse CAP I/O function slope reductions (Schmiedt, 1993) (Fig. 3B). These intriguing findings call for further studies investigating the effects of strial dysfunction on response amplitudes. However, furosemide-induced changes are always accompanied by threshold losses, suggesting that, at least in the absence of threshold changes, any amplitude reduction in ABR wave I or CAP may be specifically attributable to synaptopathy. When confounding threshold losses are apparent, as they typically are in aging individuals, both synaptopathy and strial dysfunction could potentially cause amplitude reductions. Recent results from aging mice suggest a significant contribution of synaptopathy to the decline of supra-threshold envelope following responses in the ABR (Parthasarathy and Kujawa, 2018), but this study did not address potential effects of strial dysfunction. Nevertheless, animal studies such as these are important steps towards identifying supra-threshold hearing deficits and their underlying causes. As with the single-unit responses discussed above (section 4.1.2), many questions remain and require appropriate experimental or computational models. Compared to the amplitude of CAP and ABR wave I, their latency has received much less attention. There are two studies showing longer response latencies with increasing age (Konrad-Martin et al., € hrle et al., 2016). We are not aware of any studies on the 2012; Mo effects of experimentally induced EP reduction or synaptopathy on CAP or ABR wave I latency. However, computational modeling predicts that the N1 latency of the CAP would remain unaltered by ouabain-induced synaptopathy that first targets the low-SR fibers, whereas it would increase upon a base-to-apex synaptopathy that is not specific to any particular fiber type (Bourien et al., 2014). 5. Conclusion The quiet-aged gerbil suffers from a mixed strial and neural presbyacusis (Table 1), which is likely comparable to ARHL in humans that were not exposed to excessive noise throughout their lifespan (Schuknecht and Gacek, 1993). The correlation between EP reduction and altered morphology of afferent fibers in quiet-aged gerbils (Suryadevara et al., 2001) provided the first tentative evidence that strial and neural degeneration have a common underlying process or are interacting with each other. Specific interactions have not yet been demonstrated, but could include

121

trophic support for the auditory nerve fibers and synapses provided by the cochlear lateral wall that decreases with age-related strial dysfunction. Furthermore, intriguing studies in mice suggest that the surviving afferent and efferent synapses in an aging cochlea are both morphologically changed and can be relocated (Lauer et al., 2012; Stamataki et al., 2006). We propose that this might be another, distinct type of presbyacusis (Table 1): synaptopathic presbyacusis. Age-related strial dysfunction (summarized in section 2) is already well characterized in the quiet-aged gerbil. The gerbil also promises to be an insightful animal model for investigating the consequences of the more recently recognized synaptopathy with aging (summarized in section 3). Synaptopathy can be induced in a graded fashion in the gerbil and its consequences probed both electrophysiologically, including at the level of single auditory nerve fibers, and behaviorally. The latter is particularly important in relating experimental findings to human diagnostics. Finally, the recent sequencing of the gerbil genome (Zorio et al., 2018; Cheng et al., 2019) opens exciting new possibilities of taking gerbil research to genetic and molecular levels. Acknowledgements This work was supported by the DFG Cluster of Excellence EXC 1077/1 “Hearing4all”. We thank STELS-OL (Scientific and Technical English Language Services, Oldenburg, Germany) for English language editing. The authors declare no competing financial interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.heares.2019.02.015. References Adams, J.C., Schulte, B.A., 1997. Histopathologic observations of the aging gerbil cochlea. Hear. Res. 104, 101e111. Altschuler, R.A., Dolan, D.F., Halsey, K., Kanicki, A., Deng, N., Martin, C., Eberle, J., Kohrman, D.C., Miller, R.A., Schacht, J., 2015. Age-related changes in auditory nerve-inner hair cell connections, hair cell numbers, auditory brain stem response and gap detection in UM-HET4 mice. Neuroscience 292, 22e33. Bielefeld, E.C., Coling, D., Chen, G.D., Li, M., Tanaka, C., Hu, B.H., Henderson, D., 2008. Age-related hearing loss in the Fischer 344/NHsd rat substrain. Hear. Res. 241, 26e33. Bourien, J., Tang, Y., Batrel, C., Huet, A., Lenoir, M., Ladrech, S., Desmadryl, G., Nouvian, R., Puel, J.L., Wang, J., 2014. Contribution of auditory nerve fibers to compound action potential of the auditory nerve. J. Neurophysiol. 112, 1025e1039. Buckiova, D., Popelar, J., Syka, J., 2006. Collagen changes in the cochlea of aged Fischer 344 rats. Exp. Gerontol. 41, 296e302. Buckiova, D., Popelar, J., Syka, J., 2007. Aging cochleas in the F344 rat: morphological and functional changes. Exp. Gerontol. 42, 629e638. Cable, J., Jackson, I.J., Steel, K.P., 1993. Light (Blt), a mutation that causes melanocyte death, affects stria vascularis function in the mouse inner ear. Pigm. Cell Res. 6, 215e225. Cai, R., Montgomery, S.C., Graves, K.A., Caspary, D.M., Cox, B.C., 2018. The FBN rat model of aging: investigation of ABR waveforms and ribbon synapse changes. Neurobiol. Aging 62, 53e63. Carney, L.H., 2018. Supra-threshold hearing and fluctuation profiles: implications for sensorineural and hidden hearing loss. J. Assoc. Res. Otolaryngol. 19, 331e352. Caspary, D.M., Llano, D.A., 2018. Aging processes in the subcortical auditory system. In: Kandler, K. (Ed.), The Oxford Handbook of the Auditory Brainstem. Cheal, M.L., 1986. The gerbil: a unique model for research on aging. Exp. Aging Res. 12, 3e21. Cheng, S., Fu, Y., Zhang, Y., Xian, W., Wang, H., Grothe, B., Lu, X., Xu, X., Klug, A., McCullagh, E.A., 2019. De novo assembly of the Mongolian gerbil genome and transcriptome. BioRxiv. https://doi.org/10.1101/522516. Dazert, S., Feldman, M.L., Keithley, E.M., 1996. Cochlear spiral ganglion cell degeneration in wild-caught mice as a function of age. Hear. Res. 100, 101e106. Diaz, R.C., Vazquez, A.E., Dou, H., Wei, D., Cardell, E.L., Lingrel, J., Shull, G.E., Doyle, K.J., Yamoah, E.N., 2007. Conservation of hearing by simultaneous mutation of Na,K-ATPase and NKCC1. J. Assoc. Res. Otolaryngol. 8, 422e434. Dubno, J.R., Eckert, M.A., Lee, F.S., Matthews, L.J., Schmiedt, R.A., 2013. Classifying

122

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

human audiometric phenotypes of age-related hearing loss from animal models. J. Assoc. Res. Otolaryngol. 14, 687e701. Evans, E.F., Klinke, R., 1982. The effects of intracochlear and systemic furosemide on the properties of single cochlear nerve fibres in the cat. J. Physiol. 331, 409e427. Felix, H., Johnsson, L.G., Gleeson, M., Pollak, A., 1990. Quantitative analysis of cochlear sensory cells and neuronal elements in man. Acta Otolaryngol. Suppl. 470, 71e79. Fernandez, K.A., Jeffers, P.W., Lall, K., Liberman, M.C., Kujawa, S.G., 2015. Aging after noise exposure: acceleration of cochlear synaptopathy in "recovered" ears. J. Neurosci. 35, 7509e7520. Fetoni, A.R., Picciotti, P.M., Paludetti, G., Troiani, D., 2011. Pathogenesis of presbycusis in animal models: a review. Exp. Gerontol. 46, 413e425. Forge, A., 1976. Observations on the stria vascularis of the Guinea pig cochlea and the changes resulting from the administration of the diuretic furosemide. Clin. Otolaryngol. Allied Sci. 1, 211e219. Furman, A.C., Kujawa, S.G., Liberman, M.C., 2013. Noise-induced cochlear neuropathy is selective for fibers with low spontaneous rates. J. Neurophysiol. 110, 577e586. Gates, G.A., Mills, J.H., 2005. Presbycusis. Lancet 366, 1111e1120. Gleich, O., Wilson, S., 1993. The diameters of Guinea pig auditory nerve fibres: distribution and correlation with spontaneous rate. Hear. Res. 71, 69e79. Gleich, O., Semmler, P., Strutz, J., 2016. Behavioral auditory thresholds and loss of ribbon synapses at inner hair cells in aged gerbils. Exp. Gerontol. 84, 61e70. Gratton, M.A., Schulte, B.A., 1995. Alterations in microvasculature are associated with atrophy of the stria vascularis in quiet-aged gerbils. Hear. Res. 82, 44e52. Gratton, M.A., Schmiedt, R.A., Schulte, B.A., 1996. Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis. Hear. Res. 102, 181e190. Gratton, M.A., Schulte, B.A., Smythe, N.M., 1997. Quantification of the stria vascularis and strial capillary areas in quiet-reared young and aged gerbils. Hear. Res. 114, 1e9. Guimaraes, P., Zhu, X., Cannon, T., Kim, S., Frisina, R.D., 2004. Sex differences in distortion product otoacoustic emissions as a function of age in CBA mice. Hear. Res. 192, 83e89. Hamann, I., Gleich, O., Klump, G.M., Kittel, M.C., Strutz, J., 2004. Age-dependent changes of gap detection in the Mongolian gerbil (Meriones unguiculatus). J. Assoc. Res. Otolaryngol. 5, 49e57. Han, C., Ding, D., Lopez, M.C., Manohar, S., Zhang, Y., Kim, M.J., Park, H.J., White, K., Kim, Y.H., Linser, P., Tanokura, M., Leeuwenburgh, C., Baker, H.V., Salvi, R.J., Someya, S., 2016. Effects of long-term exercise on age-related hearing loss in mice. J. Neurosci. 36, 11308e11319. € ppl, C., 2018. Auditory Nerve Fiber Spontaneous Rate Is Heeringa, A.N., Zhang, L., Ko Decreased in an Animal Model for Presbyacusis. Midwinter Meeting of the Association for Research in Otolaryngology, San Diego, CA. Hellstrom, L.I., Schmiedt, R.A., 1990. Compound action potential input/output functions in young and quiet-aged gerbils. Hear. Res. 50, 163e174. Hellstrom, L.I., Schmiedt, R.A., 1991. Rate/level functions of auditory-nerve fibers in young and quiet-aged gerbils. Hear. Res. 53, 217e222. Henry, K.R., McGinn, M.D., Chole, R.A., 1980. Age-related auditory loss in the Mongolian gerbil. Arch. Oto-Rhino-Laryngol. 228, 233e238. Hequembourg, S., Liberman, M.C., 2001. Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice. J. Assoc. Res. Otolaryngol. 2, 118e129. Hibino, H., Nin, F., Tsuzuki, C., Kurachi, Y., 2010. How is the highly positive endocochlear potential formed? The specific architecture of the stria vascularis and the roles of the ion-transport apparatus. Pflügers Archiv. 459, 521e533. Humes, L.E., Dubno, J.R., 2010. Factors affecting speech understanding in older adults. In: Gordon-Salant, S., Frisina, R.D., Popper, A.N., Fay, R.R. (Eds.), The Aging Auditory System. Springer, pp. 211e258. Ikeda, K., Oshima, T., Hidaka, H., Takasaka, T., 1997. Molecular and clinical implications of loop diuretic ototoxicity. Hear. Res. 107, 1e8. Ishiyama, G., Tokita, J., Lopez, I., Tang, Y., Ishiyama, A., 2007. Unbiased stereological estimation of the spiral ligament and stria vascularis volumes in aging and Meniere's disease using archival human temporal bones. J. Assoc. Res. Otolaryngol. 8, 8e17. Jiang, X.W., Li, X.R., Zhang, Y.P., 2015. Changes of ribbon synapses number of cochlear hair cells in C57BL/6J mice with age. Int. J. Clin. Exp. Med. 8, 19058e19064. Johannesen, P.T., Buzo, B.C., Lopez-Poveda, E.A., 2019. Evidence for age-related cochlear synaptopathy in humans unconnected to speech-in-noise intelligibility deficits. Hear. Res. 374, 35e48. Johnsson, L.G., Hawkins Jr., J.E., 1972a. Vascular changes in the human inner ear associated with aging. Ann. Otol. Rhinol. Laryngol. 81, 364e376. Johnsson, L.G., Hawkins Jr., J.E., 1972b. Strial atrophy in clinical and experimental deafness. Laryngoscope 82, 1105e1125. Johnsson, L.G., Hawkins Jr., J.E., 1972c. Sensory and neural degeneration with aging, as seen in microdissections of the human inner ear. Ann. Otol. Rhinol. Laryngol. 81, 179e193. Keithley, E.M., Ryan, A.F., Woolf, N.K., 1989. Spiral ganglion cell density in young and old gerbils. Hear. Res. 38, 125e133. Konrad-Martin, D., Dille, M.F., McMillan, G., Griest, S., McDermott, D., Fausti, S.A., Austin, D.F., 2012. Age-related changes in the auditory brainstem response. J. Am. Acad. Audiol. 23, 18e35 quiz 74-5. Kujawa, S.G., Liberman, M.C., 2009. Adding insult to injury: cochlear nerve degeneration after "temporary" noise-induced hearing loss. J. Neurosci. 29,

14077e14085. Kujawa, S.G., Liberman, M.C., 2015. Synaptopathy in the noise-exposed and aging cochlea: primary neural degeneration in acquired sensorineural hearing loss. Hear. Res. 330, 191e199. Lang, H., Schulte, B.A., Schmiedt, R.A., 2005. Ouabain induces apoptotic cell death in type I spiral ganglion neurons, but not type II neurons. J. Assoc. Res. Otolaryngol. 6, 63e74. Lang, H., Jyothi, V., Smythe, N.M., Dubno, J.R., Schulte, B.A., Schmiedt, R.A., 2010. Chronic reduction of endocochlear potential reduces auditory nerve activity: further confirmation of an animal model of metabolic presbyacusis. J. Assoc. Res. Otolaryngol. 11, 419e434. Lauer, A.M., Fuchs, P.A., Ryugo, D.K., Francis, H.W., 2012. Efferent synapses return to inner hair cells in the aging cochlea. Neurobiol. Aging 33, 2892e2902. Lee, F.S., Matthews, L.J., Dubno, J.R., Mills, J.H., 2005. Longitudinal study of pure-tone thresholds in older persons. Ear Hear. 26, 1e11. Liberman, L.D., Wang, H., Liberman, M.C., 2011. Opposing gradients of ribbon size and AMPA receptor expression underlie sensitivity differences among cochlearnerve/hair-cell synapses. J. Neurosci. 31, 801e808. Liberman, M.C., 1982. Single-neuron labeling in the cat auditory nerve. Science 216, 1239e1241. Liberman, M.C., 1990. Effects of chronic cochlear de-efferentation on auditory-nerve response. Hear. Res. 49, 209e223. Liberman, M.C., 2017. Noise-induced and age-related hearing loss: new perspectives and potential therapies. F1000Res 6, 927. Liberman, M.C., Oliver, M.E., 1984. Morphometry of intracellularly labeled neurons of the auditory nerve: correlations with functional properties. J. Comp. Neurol. 223, 163e176. Liberman, M.C., Kujawa, S.G., 2017. Cochlear synaptopathy in acquired sensorineural hearing loss: manifestations and mechanisms. Hear. Res. 349, 138e147. Liberman, M.C., Liberman, L.D., Maison, S.F., 2014. Efferent feedback slows cochlear aging. J. Neurosci. 34, 4599e4607. Liberman, M.C., Liberman, L.D., Maison, S.F., 2015. Chronic conductive hearing loss leads to cochlear degeneration. PLoS One 10, e0142341. Lin, F.R., Thorpe, R., Gordon-Salant, S., Ferrucci, L., 2011. Hearing loss prevalence and risk factors among older adults in the United States. J. Gerontol. A Biol. Sci. Med. Sci. 66, 582e590. Lopez-Poveda, E.A., 2014. Why do I hear but not understand? Stochastic undersampling as a model of degraded neural encoding of speech. Front. Neurosci. 8, 348. Makary, C.A., Shin, J., Kujawa, S.G., Liberman, M.C., Merchant, S.N., 2011. Age-related primary cochlear neuronal degeneration in human temporal bones. J. Assoc. Res. Otolaryngol. 12, 711e717. Mannstrom, P., Ulfhake, B., Kirkegaard, M., Ulfendahl, M., 2013. Dietary restriction reduces age-related degeneration of stria vascularis in the inner ear of the rat. Exp. Gerontol. 48, 1173e1179. Marmel, F., Rodriguez-Mendoza, M.A., Lopez-Poveda, E.A., 2015. Stochastic undersampling steepens auditory threshold/duration functions: implications for understanding auditory deafferentation and aging. Front. Aging Neurosci. 7. Merchan-Perez, A., Liberman, M.C., 1996. Ultrastructural differences among afferent synapses on cochlear hair cells: correlations with spontaneous discharge rate. J. Comp. Neurol. 371, 208e221. Meyer, A.C., Frank, T., Khimich, D., Hoch, G., Riedel, D., Chapochnikov, N.M., Yarin, Y.M., Harke, B., Hell, S.W., Egner, A., Moser, T., 2009. Tuning of synapse number, structure and function in the cochlea. Nat. Neurosci. 12, 444e453. Mills, D.M., Schmiedt, R.A., 2004. Metabolic presbycusis: differential changes in auditory brainstem and otoacoustic emission responses with chronic furosemide application in the gerbil. J. Assoc. Res. Otolaryngol. 5, 1e10. Mills, D.M., Norton, S.J., Rubel, E.W., 1993. Vulnerability and adaptation of distortion product otoacoustic emissions to endocochlear potential variation. J. Acoust. Soc. Am. 94, 2108e2122. Mills, J.H., Schmiedt, R.A., Kulish, L.F., 1990. Age-related changes in auditory potentials of Mongolian gerbil. Hear. Res. 46, 201e210. Mistrik, P., Ashmore, J., 2009. The role of potassium recirculation in cochlear amplification. Curr. Opin. Otolaryngol. Head Neck Surg. 17, 394e399. €hrle, D., Ni, K., Varakina, K., Bing, D., Lee, S.C., Zimmermann, U., Knipper, M., Mo Ruttiger, L., 2016. Loss of auditory sensitivity from inner hair cell synaptopathy can be centrally compensated in the young but not old brain. Neurobiol. Aging 44, 173e184. Moser, T., Starr, A., 2016. Auditory neuropathy–neural and synaptic mechanisms. Nat. Rev. Neurol. 12, 135e149. Mustapha, M., Fang, Q., Gong, T.W., Dolan, D.F., Raphael, Y., Camper, S.A., Duncan, R.K., 2009. Deafness and permanently reduced potassium channel gene expression and function in hypothyroid Pit1dw mutants. J. Neurosci. 29, 1212e1223. Nin, F., Hibino, H., Doi, K., Suzuki, T., Hisa, Y., Kurachi, Y., 2008. The endocochlear potential depends on two Kþ diffusion potentials and an electrical barrier in the stria vascularis of the inner ear. Proc. Natl. Acad. Sci. U. S. A. 105, 1751e1756. Nouvian, R., Beutner, D., Parsons, T.D., Moser, T., 2006. Structure and function of the hair cell ribbon synapse. J. Membr. Biol. 209, 153e165. Ohlemiller, K.K., 2006. Contributions of mouse models to understanding of age- and noise-related hearing loss. Brain Res. 1091, 89e102. Ohlemiller, K.K., 2009. Mechanisms and genes in human strial presbycusis from animal models. Brain Res. 1277, 70e83. Ohlemiller, K.K., Gagnon, P.M., 2004. Apical-to-basal gradients in age-related cochlear degeneration and their relationship to "primary" loss of cochlear

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko neurons. J. Comp. Neurol. 479, 103e116. Ohlemiller, K.K., Dahl, A.R., Gagnon, P.M., 2010. Divergent aging characteristics in CBA/J and CBA/CaJ mouse cochleae. J. Assoc. Res. Otolaryngol. 11, 605e623. Ohlemiller, K.K., Rice, M.E., Lett, J.M., Gagnon, P.M., 2009. Absence of strial melanin coincides with age-associated marginal cell loss and endocochlear potential decline. Hear. Res. 249, 1e14. Ohlemiller, K.K., 2018. Lessons from rodent models for genetic and age-related hearing loss. In: Dent, M.L., Fay, R.R., Popper, A.N. (Eds.), Rodent Bioacoustics, vol. 67. Springer, pp. 165e190. Otte, J., Schuknecht, H.F., Kerr, A.G., 1978. Ganglion cell populations in normal and pathological human cochleae. Implications for cochlear implantation. Laryngoscope 125, 1038. Parthasarathy, A., Kujawa, S.G., 2018. Synaptopathy in the aging cochlea: characterizing early-neural deficits in auditory temporal envelope processing. J. Neurosci. 38, 7108e7119. Pauler, M., Schuknecht, H.F., Thornton, A.R., 1986. Correlative studies of cochlear neuronal loss with speech-discrimination and pure-tone thresholds. Arch. OtoRhino-Laryngol. 243, 200e206. Pauler, M., Schuknecht, H.F., White, J.A., 1988. Atrophy of the stria vascularis as a cause of sensorineural hearing loss. Laryngoscope 98, 754e759. Petitpre, C., Wu, H., Sharma, A., Tokarska, A., Fontanet, P., Wang, Y., Helmbacher, F., Yackle, K., Silberberg, G., Hadjab, S., Lallemend, F., 2018. Neuronal heterogeneity and stereotyped connectivity in the auditory afferent system. Nat. Commun. 9, 3691. Pike, D.A., Bosher, S.K., 1980. The time course of the strial changes produced by intravenous furosemide. Hear. Res. 3, 79e89. Plack, C.J., Barker, D., Prendergast, G., 2014. Perceptual consequences of "hidden" hearing loss. Trends Hear 18. Popelar, J., Groh, D., Pelanova, J., Canlon, B., Syka, J., 2006. Age-related changes in cochlear and brainstem auditory functions in Fischer 344 rats. Neurobiol. Aging 27, 490e500. Puel, J.L., 1995. Chemical synaptic transmission in the cochlea. Prog. Neurobiol. 47, 449e476. Puel, J.L., Saffiedine, S., Gervais d'Aldin, C., Eybalin, M., Pujol, R., 1995. Synaptic regeneration and functional recovery after excitotoxic injury in the Guinea pig cochlea. C R Acad Sci III 318, 67e75. Reijntjes, D.O.J., Pyott, S.J., 2016. The afferent signaling complex: regulation of type I spiral ganglion neuron responses in the auditory periphery. Hear. Res. 336, 1e16. Rickheit, G., Maier, H., Strenzke, N., Andreescu, C.E., De Zeeuw, C.I., Muenscher, A., Zdebik, A.A., Jentsch, T.J., 2008. Endocochlear potential depends on Cl- channels: mechanism underlying deafness in Bartter syndrome IV. EMBO J. 27, 2907e2917. Ruan, Q., Ma, C., Zhang, R., Yu, Z., 2014. Current status of auditory aging and antiaging research. Geriatr. Gerontol. Int. 14, 40e53. Ruel, J., Nouvian, R., Gervais d'Aldin, C., Pujol, R., Eybalin, M., Puel, J.L., 2001. Dopamine inhibition of auditory nerve activity in the adult mammalian cochlea. Eur. J. Neurosci. 14, 977e986. Ruggero, M.A., Rich, N.C., 1991. Furosemide alters organ of corti mechanics: evidence for feedback of outer hair cells upon the basilar membrane. J. Neurosci. 11, 1057e1067. Ryan, A., 1976. Hearing sensitivity of the Mongolian gerbil, Meriones unguiculatis. J. Acoust. Soc. Am. 59, 1222e1226. Rybak, L.P., Morizono, T., 1982. Effect of furosemide upon endolymph potassium concentration. Hear. Res. 7, 223e231. Rybak, L.P., Wright, L.B., Whitworth, C., 1984. Cochlear effects of locally applied inhibitors. Arch. Oto-Rhino-Laryngol. 240, 207e213. Saitoh, Y., Hosokawa, M., Shimada, A., Watanabe, Y., Yasuda, N., Murakami, Y., Takeda, T., 1995. Age-related cochlear degeneration in senescence-accelerated mouse. Neurobiol. Aging 16, 129e136. Sakaguchi, N., Crouch, J.J., Lytle, C., Schulte, B.A., 1998. Na-K-Cl cotransporter expression in the developing and senescent gerbil cochlea. Hear. Res. 118, 114e122. Salt, A.N., Konishi, T., 1979. Effects of noise on cochlear potentials and endolymph potassium concentration recorded with potassium-selective electrodes. Hear. Res. 1, 343e363. Salt, A.N., Melichar, I., Thalmann, R., 1987. Mechanisms of endocochlear potential generation by stria vascularis. Laryngoscope 97, 984e991. Santos-Sacchi, J., Wu, M., Kakehata, S., 2001. Furosemide alters nonlinear capacitance in isolated outer hair cells. Hear. Res. 159, 69e73. Saremi, A., Stenfelt, S., 2013. Effect of metabolic presbyacusis on cochlear responses: a simulation approach using a physiologically-based model. J. Acoust. Soc. Am. 134, 2833e2851. Schaette, R., McAlpine, D., 2011. Tinnitus with a normal audiogram: physiological evidence for hidden hearing loss and computational model. J. Neurosci. 31, 13452e13457. Schmiedt, R.A., 1993. Cochlear potentials in quiet-aged gerbils: does the aging cochlea need a jump start? In: Verrillo, R.T. (Ed.), Sensory Research: Multimodal Perspectives. Lawrence Erlbaum Associates, Publishers, Hillsdale, New Jersey, pp. 91e103. Schmiedt, R.A., 1996. Effects of aging on potassium homeostasis and the endocochlear potential in the gerbil cochlea. Hear. Res. 102, 125e132. Schmiedt, R.A., 2010. The physiology of cochlear presbycusis. In: Gordon-Salant, S., Frisina, R.D., Popper, A.N., Fay, R.R. (Eds.), The Aging Auditory System, vol. 34. Springer, pp. 9e38.

123

Schmiedt, R.A., Mills, J.H., Adams, J.C., 1990. Tuning and suppression in auditory nerve fibers of aged gerbils raised in quiet or noise. Hear. Res. 45, 221e236. Schmiedt, R.A., Mills, J.H., Boettcher, F.A., 1996. Age-related loss of activity of auditory-nerve fibers. J. Neurophysiol. 76, 2799e2803. Schmiedt, R.A., Lang, H., Okamura, H.O., Schulte, B.A., 2002a. Effects of furosemide applied chronically to the round window: a model of metabolic presbyacusis. J. Neurosci. 22, 9643e9650. Schmiedt, R.A., Okamura, H.O., Lang, H., Schulte, B.A., 2002b. Ouabain application to the round window of the gerbil cochlea: a model of auditory neuropathy and apoptosis. J. Assoc. Res. Otolaryngol. 3, 223e233. Schuknecht, H.F., 1964. Further observations on the pathology of presbycusis. Arch. Otolaryngol. 80, 369e382. Schuknecht, H.F., Woellner, R.C., 1955. An experimental and clinical study of deafness from lesions of the cochlear nerve. J. Laryngol. Otol. 69, 75e97. Schuknecht, H.F., Gacek, M.R., 1993. Cochlear pathology in presbycusis. Ann. Otol. Rhinol. Laryngol. 102, 1e16. Schuknecht, H.F., Watanuki, K., Takahashi, T., Belal Jr., A.A., Kimura, R.S., Jones, D.D., Ota, C.Y., 1974. Atrophy of the stria vascularis, a common cause for hearing loss. Laryngoscope 84, 1777e1821. Schulte, B.A., Schmiedt, R.A., 1992. Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils. Hear. Res. 61, 35e46. Sergeyenko, Y., Lall, K., Liberman, M.C., Kujawa, S.G., 2013. Age-related cochlear synaptopathy: an early-onset contributor to auditory functional decline. J. Neurosci. 33, 13686e13694. Sewell, W.F., 1984a. The relation between the endocochlear potential and spontaneous activity in auditory nerve fibres of the cat. J. Physiol. 347, 685e696. Sewell, W.F., 1984b. Furosemide selectively reduces one component in rate-level functions from auditory-nerve fibers. Hear. Res. 15, 69e72. Sewell, W.F., 1984c. The effects of furosemide on the endocochlear potential and auditory-nerve fiber tuning curves in cats. Hear. Res. 14, 305e314. Shi, L.J., Chang, Y., Li, X.W., Aiken, S.J., Liu, L.J., Wang, J., 2016. Coding deficits in noise-induced hidden hearing loss may stem from incomplete repair of ribbon synapses in the cochlea. Front. Neurosci. 10, 231. Shrestha, B.R., Chia, C., Wu, L., Kujawa, S.G., Liberman, M.C., Goodrich, L.V., 2018. Sensory neuron diversity in the inner ear is shaped by activity. Cell 174, 1229e1246 e17. Spicer, S.S., Schulte, B.A., 2005. Pathologic changes of presbycusis begin in secondary processes and spread to primary processes of strial marginal cells. Hear. Res. 205, 225e240. Stamataki, S., Francis, H.W., Lehar, M., May, B.J., Ryugo, D.K., 2006. Synaptic alterations at inner hair cells precede spiral ganglion cell loss in aging C57BL/6J mice. Hear. Res. 221, 104e118. Stankovic, K., Rio, C., Xia, A., Sugawara, M., Adams, J.C., Liberman, M.C., Corfas, G., 2004. Survival of adult spiral ganglion neurons requires erbB receptor signaling in the inner ear. J. Neurosci. 24, 8651e8661. Strawbridge, W.J., Wallhagen, M.I., Shema, S.J., Kaplan, G.A., 2000. Negative consequences of hearing impairment in old age: a longitudinal analysis. Gerontol. 40, 320e326. Suga, F., Lindsay, J.R., 1976. Histopathological observations of presbycusis. Ann. Otol. Rhinol. Laryngol. 85, 169e184. Sugawara, M., Murtie, J.C., Stankovic, K.M., Liberman, M.C., Corfas, G., 2007. Dynamic patterns of neurotrophin 3 expression in the postnatal mouse inner ear. J. Comp. Neurol. 501, 30e37. Sun, S., Babola, T., Pregernig, G., So, K.S., Nguyen, M., Su, S.M., Palermo, A.T., Bergles, D.E., Burns, J.C., Müller, U., 2018. Hair cell mechanotransduction regulates spontaneous activity and spiral ganglion subtype specification in the auditory system. Cell 174, 1247e1263. Suryadevara, A.C., Schulte, B.A., Schmiedt, R.A., Slepecky, N.B., 2001. Auditory nerve fibers in young and quiet-aged gerbils: morphometric correlations with endocochlear potential. Hear. Res. 161, 45e53. Syka, J., 2010. The Fischer 344 rat as a model of presbycusis. Hear. Res. 264, 70e78. Tarnowski, B.I., Schmiedt, R.A., Hellstrom, L.I., Lee, F.S., Adams, J.C., 1991. Age-related changes in cochleas of Mongolian gerbils. Hear. Res. 54, 123e134. Thomopoulos, G.N., Spicer, S.S., Gratton, M.A., Schulte, B.A., 1997. Age-related thickening of basement membrane in stria vascularis capillaries. Hear. Res. 111, 31e41. Tsuji, J., Liberman, M.C., 1997. Intracellular labeling of auditory nerve fibers in Guinea pig: central and peripheral projections. J. Comp. Neurol. 381, 188e202. Ueberfuhr, M.A., Fehlberg, H., Goodman, S.S., Withnell, R.H., 2016. A DPOAE assessment of outer hair cell integrity in ears with age-related hearing loss. Hear. Res. 332, 137e150. Viana, L.M., O'Malley, J.T., Burgess, B.J., Jones, D.D., Oliveira, C.A., Santos, F., Merchant, S.N., Liberman, L.D., Liberman, M.C., 2015. Cochlear neuropathy in human presbycusis: confocal analysis of hidden hearing loss in post-mortem tissue. Hear. Res. 327, 78e88. Wan, G., Corfas, G., 2015. No longer falling on deaf ears: mechanisms of degeneration and regeneration of cochlear ribbon synapses. Hear. Res. 329, 1e10. Wan, G., Gomez-Casati, M.E., Gigliello, A.R., Liberman, M.C., Corfas, G., 2014. Neurotrophin-3 regulates ribbon synapse density in the cochlea and induces synapse regeneration after acoustic trauma. Elife 3. Wang, J., Puel, J.L., 2018. Toward cochlear therapies. Physiol. Rev. 98, 2477e2522. Wangemann, P., Liu, J., Marcus, D.C., 1995. Ion transport mechanisms responsible for Kþ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro. Hear. Res. 84, 19e29. Wu, P.Z., Liberman, L.D., Bennett, K., de Gruttola, V., O'Malley, J.T., Liberman, M.C.,

124

€ppl / Hearing Research 376 (2019) 111e124 A.N. Heeringa, C. Ko

2018. Primary neural degeneration in the human cochlea: evidence for hidden hearing loss in the aging ear. Neuroscience. https://doi.org/10.1016/ j.neuroscience.2018.07.053. Xiao, A.Y., Wei, L., Xia, S., Rothman, S., Yu, S.P., 2002. Ionic mechanism of ouabaininduced concurrent apoptosis and necrosis in individual cultured cortical neurons. J. Neurosci. 22, 1350e1362. Yamasoba, T., Lin, F.R., Someya, S., Kashio, A., Sakamoto, T., Kondo, K., 2013. Current concepts in age-related hearing loss: epidemiology and mechanistic pathways. Hear. Res. 303, 30e38. Yuan, Y., Shi, F., Yin, Y., Tong, M., Lang, H., Polley, D.B., Liberman, M.C., Edge, A.S., 2014. Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy. J. Assoc. Res. Otolaryngol. 15, 31e43. €ppl, C., 2016. Loss of Inner Hair Cell Afferent Synapses in Zhang, L., Stachowiak, R., Ko Ageing Gerbils. Midwinter Meeting of the Association for Research in

Otolaryngology, San Diego, CA. €ppl, C., 2017. Coding of Temporal Information in the Zhang, L., Steenken, F., Ko Auditory Nerve of Aging Gerbils. Midwinter Meeting of the Association for Research in Otolaryngology, Baltimore, MA. €ppl, C., 2018. Concurrent gradients Zhang, L., Engler, S., Koepcke, L., Steenken, F., Ko of ribbon volume and AMPA-receptor patch volume in cochlear afferent synapses on gerbil inner hair cells. Hear. Res. 364, 81e89. Zheng, X.Y., Henderson, D., McFadden, S.L., Ding, D.L., Salvi, R.J., 1999. Auditory nerve fiber responses following chronic cochlear de-efferentation. J. Comp. Neurol. 406, 72e86. Zorio, D.A.R., Monsma, S., Sanes, D.H., Golding, N.L., Rubel, E.W., Wang, Y., 2018. De novo sequencing and initial annotation of the Mongolian gerbil (Meriones unguiculates) genome. Genomics. https://doi.org/10.1016/j.ygeno.2018.03.001 (in press).