Molecular composition and function of the spiral

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Molecular composition and function of the spiral ganglion neuron peripheral synapse in mice Reijntjes, Daniël Onne Jilt

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10.33612/diss.93524048

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Reijntjes, D. O. J. (2019). Molecular composition and function of the spiral ganglion neuron peripheral synapse in mice. University of Groningen. https://doi.org/10.33612/diss.93524048

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Molecular composition and function of the spiral ganglion neuron peripheral synapse in mice

Daniël Onne Jilt Reijntjes

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This research was supported by the Heinsius-Houbolt fund and the foundation for the hearing impaired child.

© D.O.J. Reijntjes, Groningen 2019. All rights reserved. No part of this thesis may be reproduced without prior permission of the author and the publishers holding copy- rights of the published articles.

Cover design: D.O.J. Reijntjes Layout: D.O.J. Reijntjes

Printed by: Gildprint - www.gildeprint.nl

ISBN printed version: 978-94-034-1702-8 ISBN electronic version: 978-94-034-1701-1

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Molecular composition and function of the spiral

ganglion neuron peripheral synapse in mice

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the

Rector Magnificus prof. C. Wijmenga and in accordance with

the decision by the College of Deans.

This thesis will be defended in public on Monday 9 September 2019 at 11.00 hours

by

Daniël Onne Jilt Reijntjes

born on the 10^th of June 1990 in Groningen

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Prof. P. van Dijk

Co-supervisor

Dr. S. J. Pyott

Assessment Committee

Prof. J. C. Billeter Prof. J. M. J. Kremer Prof. K. P. Steel

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Chapter 1 Introduction

Daniël Reijntjes

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Preface

Acquired hearing loss is becoming increasingly prevalent, has large effects on quality of life, and is currently untreatable. Acquired hearing loss arises with age and after noise exposure. Thus, with a global ageing population and an increasing exposure to loud sounds in developed and developing countries, age and exposure related hearing loss are becoming more prevalent (Stevens et al., 2011). Acquired hearing loss often co-occurs with tinnitus (ringing in the ear), hyperacusis (extreme sensitivity to sound), and vertigo (difficulty balancing, Knipper et al., 2013; Niu et al., 2016). Although hearing loss is usually not life-threatening, pathologies related to inner ear dysfunction are the third leading cause of disability globally (WHO, 2008).

The impact of hearing loss on physical wellbeing is more severe than diabetes and hy- pertension. Moreover, the impact of hearing loss on mental health is ranked second only after digestive disorders (Bainbridge and Wallhagen, 2014). Treatment options for acquired hearing loss and related pathologies are few and mostly limited to pre- scribing the use of auditory prosthetics (hearing aids, cochlear implants, and more recently, auditory brainstem implants). Although these prosthetics can offer some relief, in many cases they fail in restoring full hearing functionality (Shannon, 2012) and do not stop the progression of the pathology. Because of the increasing preva- lence, large impact on quality of life, and limited treatment options, it is important to investigate how to prevent or reverse acquired hearing loss and expand current treatment options.

Recent evidence suggests an early phase in the development of acquired hearing loss that shows a promising time window for therapeutic intervention (Liberman, 2017). During this phase of the development of acquired hearing loss, the peripheral synapses between the sensory cells and the peripheral auditory neurons are lost, pos- sibly as a first step towards overt hearing loss. Currently, we understand very little about what goes on in these peripheral synapses, and which mechanisms lead to loss of these peripheral synapses. Therefore, it is important to investigate the proteins and ion channels that contribute to the function of these peripheral synapses.

In this introduction, I will briefly explain how the auditory system extracts information from pressure waves and how fundamental properties of this system determine the critical role of the peripheral synapses between the sensory cells and the auditory neurons and how these synapses can be lost. Finally, I will introduce the chapters of my scientific work and how they contribute to our understanding of these peripheral synapses and their role in the development of acquired hearing loss.

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1.1. The perception of sound 3

1.1. The perception of sound

Audition is a complex process translating pressure changes in the external environment into information in the brain where these pressure changes are perceived as sound. Sound waves through air consist of two parts: an area of compression and an area of rarefaction (decompression). Sound waves arise when pressure is exerted onto molecules in a medium, such as air. The molecules do not progress along with the sound wave but transmit this pressure from molecule to molecule in a wave of compression and decompression. Consider a group of molecules that is subject to pressure. This group of molecules will be pushed toward a neighbouring group of molecules creating an area where molecules are compressed. In turn, the molecules in the compressed area will be pushed to molecules in a further adjacent group and so forth. Meanwhile, the area that the molecules in the first group vacated is now decompressed, and thus becomes subject to negative pressure. This negative pressure pulls the molecules from the first group back into their original position and similarly for molecules in consecutive groups. Thus, the molecules in air are not displaced but the sound wave propagates through the air (Figure 1.1A). The sound wave across these molecules can be depicted by a sine wave where the areas of compression are the peaks and the areas of decompression are the troughs (Figure 1.1B). In simplified form, sound waves can thus be described by a sine function with a specific amplitude and frequency, where the amplitude is the difference in pressure relative to the atmospheric pressure, and the frequency is related to the duration between subsequent compression peaks.

The human auditory system is capable of detecting sound waves with frequencies between 20 and 20000 Hz (Micheyl et al., 2006). The intensity of sound waves can be described in sound pressure level (SPL) and expressed in decibels (dB). The mean human hearing threshold at 1 kHz, which corresponds to 0,000002 Pascal or 20 µPa was used to define 0 dB-SPL in 1933 (Fletcher and Munson, 1933), although some people are sensitive to softer sound intensities. The upper limit of human hearing is hard to quantify; instead, the auditory pain threshold is often considered to be the upper limit. The auditory pain threshold has been reported to be around a 130 dB- SPL (Silverman, 1947), which equals about 60 Pa. Expressed in sound intensity, an increase from 0 to a 130 dB-SPL equals an incredible increase of 10000000000000 (10¹³) fold, in sound energy.

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Figure 1.1.Pressure/sound wave propagation through air can be described by sine waves. Pressure waves through air are transmitted from molecule to molecule and thus propagate without propagation of the molecules. The detection and interpretation of these waves is called the sensation of sound and therefore these waves can also be described as sound waves. A) Molecules in air at rest. B) Molecules in air form wave patterns in response to pressure waves generated by the speaker. The range of displacement of an individual molecule is indicated by the *. C) Description of the wave pattern of the molecules by a sine wave with a given frequency and amplitude.

Impressive as this range of sound wave detection may be, sound generally does not consist of single sine waves but of complex sounds containing a multitude of sine waves. The real challenge of the auditory system is therefore to act as a frequency and intensity analyzer to break down complex sounds into understandable information to the brain. Furthermore, the encoding of sound has to happen with a high temporal fidelity in order to detect timing differences and for localizing sounds. To meet these requirements, the auditory system has developed complex anatomical adaptations.

1.2. Anatomy of the auditory system

The auditory system can be subdivided into a peripheral (from the ear to the brainstem) and a central component (from brainstem to cortex). The peripheral part can be further subdivided into the outer ear, the middle ear, and the inner ear (Figure 1.2). The outer ear consists of the pinna, the ear canal, and ends with the tympanic membrane (eardrum).

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1.2. Anatomy of the auditory system 5

Figure 1.2.Schematized anatomy of the peripheral auditory system. The peripheral auditory system consists of the outer, middle, and inner ear and is connected to the central auditory system by the auditory and vestibular nerves that project to the brainstem. The outer ear consists of the pinna, the ear canal, and ends at the tectorial membrane. The middle ear consists of a cavity between the tectorial membrane and the cochlea and houses the ossicles. The inner ear consists of the vestibule and the cochlea from which the auditory and vestibular nerves project to regions in the brainstem

The middle ear consists of a cavity lodged between the tympanic membrane and the bony exterior of the inner ear, which is part of the temporal bone of the skull. In the middle ear, the tympanic membrane is associated with the three ossicles (middle ear bones). The third ossicle, the stapes, pushes into the oval window, one of two membranes that cover two holes in the exterior of the cochlea. The inner ear consists of the cochlea, the auditory nerve, the vestibular organ, and the vestibular nerve, of which the latter two will not be further discussed. The auditory nerve extends from inside the cochlea to the brainstem where the peripheral auditory system joins the central auditory system.

1.2.1. The peripheral auditory system

The outer and middle ear direct sound waves and provide impedance (resistance) balancing necessary to transmit the airborne sound waves to the cochlea. Sound waves are collected from the environment by the pinna and are then propagated through the ear canal toward the tympanic membrane. Both the pinna and the ear canal play a role in providing spatial and temporal cues for the brain (Kahana and Nelson, 2006).

When the sound waves reach the tympanic membrane, the tympanic membrane is displaced by the pressure of the sound waves. To increase the sensitivity of the auditory system, the tympanic membrane then displaces the middle ear bones, which act as a lever to increase the pressure onto the oval window in the cochlea. This increase

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in pressure is required to overcome the difference in impedance between the tympanic membrane and the oval window that arises because of fluid inside the cochlea. The acoustic impedance of air is about 400 (Pa·s/m), whereas the acoustic impedance of the cochlear fluids is about 15·10⁶ (Pa·s/m). Therefore, the ratio of energy transfer from the tympanic membrane to the oval window would be only about 0,1% (Mason, 2016). The pressure gain provided by the ossicles enables sound waves to be transmitted via the oval window to the cochlea.

The cochlea (cochlea = snail) is an inflexible fluid filled chamber that houses the flexible basilar membrane that oscillates in response to sound in a frequency dependent manner. The cochlea spirals inwards and upwards creating about 2,75 turns in humans. It consists of three chambers filled with fluids that are separated from each other by two membranes, the basilar membrane and Reissner’s membrane (Fig- ure 1.3A). The two exterior fluid chambers, the scalae (scala = staircase) vestibuli and tympani, are connected at the very tip of this cochlear shell at the so called he- licotrema (helix = coil, and trema = hole) and thus share the same fluid called the perilymph. At the base of both chambers lie two membranes that cover holes in the exterior of the cochlea. The scala vestibuli ends at the oval window that associates with the ossicles and the scala tympani ends at the round window. The interior chamber, the scala media, is separated from the other two scalae by the basilar membrane and Reissner’s membrane, isolating the fluid, called endolymph, within this chamber. Thus, uncoiled, the cochlea can therefore be described as a U-shaped tube with the scala media separating both legs of the U-shape (Figure 1.3B). As sound waves reach the tympanic membrane, the ossicles exert force onto the oval window that must be followed by a corresponding motion at the round window because the cochlea is enclosed by inflexible bone and filled with incompressible fluid. Both the basilar membrane and Reissner’s membrane are non-rigid components of this otherwise rigid system and will therefore bend in response to pressure exerted at the oval window. Thus, pressure exerted at the oval window causes a fluid displacement within the cochlea. The displaced fluid generates pressure differences within the fluid that cause a passive displacement of the basilar membrane near the oval window. The displacement of the basilar membrane generates a travelling wave across this membrane that discharges energy by oscillating the basilar membrane at specific regions depending on the frequency of the sound waves. This frequency dependent oscillation results from mechanical differences in breadth and stiffness of the basilar membrane from base to apex (Figure 1.3B). This frequency specific oscillation forms the basis of the frequency selectivity of the auditory system where specific regions of the basilar membrane react to sound waves with specific frequencies.

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Figure 1.3. The cochlea is a spiralling structure that houses the basilar membrane which responds in a frequency dependent manner to sound waves. A) The cochlea houses three fluid filled chambers that spiral around a central column of bone called the modiolus. The scala media is separated from the scala tympani and the scala vestibule by two membranes, the basilar membrane and Reissner’s membrane thus isolating the fluid in the scala media. B) The basilar membrane dissipates energy by oscillating at a specific region depending on the frequency of stimulation. This frequency dependent stimulation results from differences in the width and the stiffness of the basilar membrane. C) This frequency dependent activation can be illustrated by a tonotopic map that depicts the region where the basilar membrane oscillates most during stimulation with a given frequency.

This frequency dependent activation of specific locations is called tonotopy (tono

= frequency, topos = place) and can be depicted with a tonotopic map that is maintained from the cochlea all the way to the auditory cortex in the brain (Figure 1.3C).

These frequency dependent oscillations of the basilar membrane are transduced into electrical signals by the combined workings of the stria vascularis and the organ of Corti.

The scala media houses two structures that are vital for audition. The first, the organ of Corti, is situated on top of the basilar membrane along the length of the cochlea (Figure 1.4). The organ of Corti houses the 4 rows of sensory hair cells that are responsible for the transmission of all acoustic information to the brain. These sensory hair cells release the neurotransmitter glutamate in response to basilar membrane oscillations. To function, these sensory cells require the influx of K⁺ ions from the endolymph. The stria vascularis, which is located along the lateral wall of the scala media, is responsible for generating a potential gradient called the endocochlear potential that drives the influx of K⁺into the HCs (Figure 1.4).

The stria vascularis establishes large electrical and concentration gradients between the endolymph and the perilymph resulting in the endocochlear potential. The stria vascularis generates the endocochlear potential by utilizing a series of pumps to maintain high K⁺(≈150 mM), low Ca²⁺(≈0.05 mM), and low Na⁺(≈1 mM) concentrations in the scala media.

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Figure 1.4.The scala media houses the stria vascularis and the organ of Corti which are indispensible for normal hearing. The scala media is located between the basilar membrane and Reissner’s membrane and contains the endolymph fluid. The endolymph is maintained at a high K⁺concentration by the workings of the stria vascularis that sits along the lateral wall of the scala media. The organ of Corti houses the sensory hair cells that respond to oscillations of the basilar membrane and require the high K⁺concentration within the endolymph to function. The sensory hair cells are connected to the peripheral processes, or dendrites, of the spiral ganglion neurons (SGNs), whose cell bodies lie within the modiolus. The central processes of the SGNs, or axons, form the auditory nerve and project toward the brainstem. Adapted from a figure created by Nick M. A. Schubert published in: Reijntjes, D.O.J., et al., 2019. Sci. Rep. 9, 2573 with permission. https://creativecommons.org/licenses/by/4.0/

These concentrations are in contrast with concentrations in the perilymph in the scala vestibuli and tympani which are maintained at high Na⁺(≈150 mM), low K⁺ (≈5 mM), and low Ca²+(≈2 mM, Bosher and Warren, 1968; Gagov et al., 2018). The large [K⁺] in the endolymph helps set a positive electrical potential in the scala media of ≈80 mV called the endocochlear potential. The endocochlear potential allows the rapid influx of K⁺ions into the sensory cells when mechanically gated ion channels in the sensory cells open and thus underlines the importance of the stria vascularis.

1.2.2. The sensory hair cells

The four rows of hair cells (Figure 1.4 & Figure 1.5A) can be divided into three rows of outer hair cells (OHCs, about 12000 in humans) and one row of inner hair cells (IHCs, about 4000 in humans, Rask-Andersen et al., 2017; Wright et al., 1987).These conical cells are similar in morphology and physiology, with distinct differences per-

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taining to their specific functions. OHCs form the basis of the cochlear amplifier and the IHCs are the true sensory cells. Both types of hair cell are highly polarized with specialized structures at the apex that regulate cell depolarization and structures at the base of the cell that release neurotransmitter in response to cellular depolarization. Both cell types are rooted at their base in the basilar membrane and have stereocilia at their apex. The stereocilia of the OHCs are connected at their tips to the tectorial membrane, whereas the IHC stereocilia are unattached. When the basilar membrane oscillates in response to sound waves encountering the tympanic membrane, the stereocilia are deflected as a result of these oscillations. These deflections occur within the µs range in frogs and turtles. In mammals deflections occur even faster but have not accurately been determined thus far (Qiu and Müller, 2018).

The hair cell stereocilia number between 20 and 300 per hair cell, vary in size (for mammals generally between 2-8 µm high, and 0,2 µm in diameter), and are orientated in 3 rows in a v/w-shaped configuration with the tallest sterocilia at the outside and with progressively shorter stereocilia towards the center (Ciuman, 2011; Hudspeth, 1989). The stereocilia maintain this particular configuration through protein links that connect the stereocilia (Figure 1.5B). The lateral links connect the stereocilia to adjacent stereocilia and provide structural strength. The tip links connect the tips of the stereocilia together (Ciuman, 2011; Osborne and Comis, 1990). The stereocilia can be deflected either towards the tallest stereocilia or in the opposite direction. De- flections towards the tallest stereocilia will create tension by pulling on the tip links, whereas deflections in the opposite direction reduce the tension on these links. Spe- cialized ion channels called mechanotransduction channels (MET channels) sit at the base of these tip links (Beurg et al., 2009, Figure 1.5B). These MET channels are a special type of ion channel that are opened by mechanical force. In the sensory cells, the generated tension on the tip links and release of this tension provide the mechanical force that opens and closes the MET channels (Hudspeth, 2014). Upon opening of these channels, the endocochlear potential drives large amounts of K⁺ through the MET channels into the hair cells (Figure 1.5C). The influx of mostly K⁺ ions depo- larizes IHCs from around -40mV and the OHCs from around -70mV to about 0 mV (Dallos, 1986).

Depolarization of the HCs starts a cascade of events that results in the release of neurotransmitter from the base of the HCs. HC depolarization opens various ion channels that are sensitive to this voltage change in the membrane. At the base of the HCs, membrane depolarization opens voltage gated Ca²⁺ channels (CaV) that allow the influx of Ca²⁺. Subsequently, voltage gated potassium channels (KV) open to restore the homeostatic membrane potential of the HCs (Pangršič et al., 2018).

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Figure 1.5.The stereocilia are aligned in a V shape on top of the sensory hair cells and contain specific mechanotransduction channels that open in response to force. A) The stereocilia are aligned in V shapes on top of the four rows of sensory hair cells. B) The stereocilia are connected through lateral links on their flanks, and at their tips with tip links. These tip links connect to the mechanotransduction channels (MET). C) The MET channels open and close in response to mechanical force generated by deflections of the stereocilia. Figure 1.5A is taken from Schwander et. al., 2010 with permission.

The CaVs are localized to between 5-30 active zones that cluster molecular machinery for the release of synaptic vesicles. Specialized structures called synaptic ribbons dock synaptic vesicles and facilitate the rapid fusion (up to 70 Hz) of these vesicles to the HC membrane upon influx of Ca²⁺ through the CaV channels (Meyer et al., 2009; Pangršič et al., 2012, 2010; Wichmann and Moser, 2015). Fusion of the vesicles to the HC membrane releases the neurotransmitter glutamate from the vesicles into the synaptic cleft between the HC active zone and the synaptic terminals of the spiral ganglion neurons (Figure 6A-B).

Although both HC types are capable of glutamate release, they have distinct functions. The cell membrane of OHCs, but not IHCs, is filled with the protein prestin that has piezoelectric properties and contributes to a process called electromotility (Hudspeth, 2008). Prestin molecules undergo a conformational change in response to membrane potential changes of the OHCs. The prestin molecules shorten when the OHCs depolarize and elongate when the OHCs repolarize. Since prestin is spread throughout most of the OHC membrane, this conformational change results in short- ening and elongation of the OHC in response to changes in membrane potential (Yu et al., 2006). Furthermore, because the OHCs are fixed to the basilar and tectorial membranes, the conformational change of the prestin molecules pulls these structures together, intensifying the basilar membrane oscillations (Ashmore et al., 2010). A further pull on the basilar membrane comes from deflection of the stereocilia embed- ded in the tectorial membrane which further shortens the OHCs (Hudspeth, 2014).

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Figure 1.6. The inner hair cells (IHCs) associate with the peripheral terminals of 5-30 spiral ganglion neurons (SGNs) through bouton synapses and release the neurotransmitter glutamate to induce SGN ac- tivation. A) The IHCs contain presynaptic active zones that cluster several structures required for the release of glutamate. Among these structures are the synaptic ribbon and voltage gated calcium channels (CaV) that are required for the fusion of synaptic vesicles to the cell membrane to release the glutamate contained inside these vesicles. B) Ca²⁺flows into the IHC in response to IHC depolarization. The influx of Ca²⁺initiates the fusion of vesicles to the IHC membrane and the release of glutamate into the synaptic cleft. The released glutamate travels across the synaptic cleft to open glutamate receptors (GluRs) on the postsynaptic SGN peripheral terminal. The opened GluRs allow the influx of Na⁺into the postsynaptic terminal.

The combination of these two processes is described as “the outer hair cell active process” and serves to increase frequency specific amplification of the basilar membrane oscillation (Hudspeth, 2014).

The contribution to frequency dependent amplification is thought to be the most important function of the OHCs. Despite the presence of CaVs, ribbons, and connections to afferent neuron terminals, only maximal activation of the OHCs is sufficient to generate measurable postsynaptic currents in the type II SGNs (Lehar et al., 2012).

Therefore, the OHC afferent pathway is thought to play a role either in nociception (pain/harm perception, Flores et al., 2015; Lehar et al., 2012; Liu et al., 2015) or serve as a feedback regulator (Froud et al., 2015). This distinction in function between the IHCs as the true sensory cells, and the OHCs as the source of the cochlear amplifier are further exemplified by their innervation with spiral ganglion neurons.

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1.2.3. The spiral ganglion neurons

The spiral ganglion neurons (SGNs) form the first neural node in the auditory pathway and transmit all auditory information (frequency, intensity, temporal, and spatial) to the brain (Figure 1.7A). The SGNs are unlike most other neurons in the sense that they are bipolar neurons (Nayagam et al., 2011) with both long central and peripheral processes flanking a central cell body or soma. The SGN somas (or somata) are located in Rosenthal’s canal that spirals along the modiolus. Their peripheral processes or dendrites extend towards either the IHCs for type I SGNs or OHCs for type II SGNs. The central processes join together in the modiolus to form the auditory nerve and extend toward the cochlear nucleus in the brainstem. In humans there are about 35000 SGNs (Ishiyama et al., 2001; Schuknecht, 1978). For mammals it is estimated that 90-95% are type I SGNs and 5-10% are type II SGNs (Spoendlin, 1985). IHCs are innervated by ≈10-20 type I SGNs each, depending on their location on the tonotopic map (Meyer et al., 2009; Meyer and Moser, 2010). Moreover, each type I SGN makes only a single connection to one IHC (Liberman, 1980a). In contrast, the type II SGNs form synaptic connections with ≈3-20 OHCs, and each OHC has around 3 connections to type II SGNs (Huang et al., 2012; Lehar et al., 2012;

Rusznák and Szucs, 2009). The type I SGNs have a larger diameter than the type IIs, ≈1-2 µm versus ≈0.5-1 µm (Berglund and Ryugo, 1987) and, unlike the type II SGNs, are myelinated for most of the central and peripheral processes. In addition, the SGN soma can be myelinated in some mammals (Rattay et al., 2013). Myelin- isation allows for a greater conduction velocity through the SGNs for neurons with a diameter over 1 µm (Nave and Werner, 2014), further suggesting that the type II SGNs transmit information to the brain that is not subject to stringent temporal demands required by acoustic information.

The type I SGNs produce electrical signals in response to glutamate released from the IHCs. The final segment of the type I SGN dendrite is unmyelinated and forms a synaptic bouton connection onto an IHC opposite an active zone containing a presynaptic ribbon. The synaptic bouton contains a variety of proteins necessary for the generation of action potentials (APs). Most importantly, glutamate receptors (GluRs) are localized to these synaptic boutons and are activated by the glutamate released from the IHCs. Upon glutamate binding, GluRs, open their ion channel pores, resulting in the influx of cations including Na⁺. This influx of cations, upon sufficient depolarization, causes voltage gated Na⁺ channels to open, which results in addi- tional Na⁺ influx that triggers an AP to propagate along the SGNs (Glowatzki and Fuchs, 2002; Lehar et al., 2012; Martinez-Monedero et al., 2016). The APs generated in this way allow the brain to reconstruct information of the sound waves by integrat- ing the responses of multiple SGNs.

Individual SGNs show functional differences in their activation determined by their characteristic frequency, spontaneous activity, and dynamic range. These dif-

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ferences in activation are used by the auditory system to encode for the different information contained in sound waves (frequency, intensity, temporal, and spatial information). As basilar membrane oscillation is frequency dependent, so are IHC stimulation and thus SGN action potential generation. However, frequencies near the optimal frequency also oscillate the basilar membrane, albeit to a lesser extent.

Therefore, these near optimal frequencies can produce APs in SGNs when the sound waves have a higher intensity. The sensitivity of each SGN to sound waves of a particular frequency follows a v-shaped pattern (Figure 1.7c) surrounding the sound wave frequency to which it is most sensitive (Heil and Peterson, 2015). Depending on the stimulation frequency, SGNs increase their rate of AP firing dependent on the intensity of the sound wave in a sigmoidal fashion up to ≈400 Hz (Figure 1.7d) until the response becomes saturated (Heil and Peterson, 2015). The intensity range over which the SGNs increase their activity is called the dynamic range and differs between SGNs. Of the 5-30 SGNs contacting each inner hair cell, some have a low and others have a high activation threshold that is inversely correlated with spontaneous activity (Figure 1.7c). The SGNs can be grouped based on their spontaneous activity ranging from <1 spike/s up to 120 spikes/s (Liberman, 1978). These subgroups are specifically distributed over the inner hair cell membrane where the low spontaneously active SGNs localize to the modiolar side of the IHCs and the high spontaneously active SGNs localize to the pillar side of the IHCs (Figure 1.7b). These groups further differ in their dynamic range, where high threshold fibers in general have larger dynamic ranges (Heil and Peterson, 2015; Liberman, 2017; Winter et al., 1990). These differences in sensitivity likely serve to extend the range of intensities for which the auditory system can encode at a given frequency, where subpopulations of SGNs respond to low intensity sound waves and others start responding only at higher intensities (Liberman, 2017). The full spectrum of action potentials generated by all the SGNs combined thus allows interpretation of the incoming sound waves in the brainstem and subsequent regions of the central auditory pathway.

1.2.4. The central auditory system

The central auditory system extends from the brainstem toward the auditory cortex in the cerebrum and integrates the input from the peripheral auditory system in order to process auditory information and, if necessary, adjust the function of the peripheral auditory system. The central processes of the SGNs project from the SGN somas (or somata) to the brainstem, where they form synaptic connections to neurons in the cochlear nucleus. From here the acoustic signal is sent on to a number of other nodes in the central auditory system that interpret temporal and spatial cues and contribute to cognitive perception of sound.

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Figure 1.7.Subgroups of spiral ganglion neurons (SGNs) respond differently to basilar membrane oscillations extending the range of hearing. The SGNs can be classified based on their stimulation threshold which is inversely correlated to their spontaneous activity, or spontaneous rate (SR). These subtypes of SGN have a preferential distribution across the IHC membrane. a) serial section of the organ of Corti showing SGN innervations at the basal pole of the inner hair cell. b) Subgroups of SGN that differ in spontaneous activity innervate the IHC on different sides of the basal pole. c) Activation thresholds across frequencies for two exemplar SGNs with different spontaneous activity. The SGNs have the lowest thresholds for basilar membrane oscillations at their characteristic frequency. d) The cumulative activity (spontaneous + evoked) of two exemplar SGNs in response to stimulation of different intensity. Low spontaneously active SGNs are typified by higher thresholds and larger dynamic ranges. Figures taken from Liberman 2017 with permission.

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Figure 1.8. Schematic of efferent innervation from the brainstem to the cochleae. Neurons from the cochlear nucleus project mostly to the contralateral olivary complexes. From both the medial (MOC) and lateral olivary complexes (LOC), efferent neurons project back to both the ipsilateral and contralateral cochleae. The MOC neurons end up contacting the outer hair cells directly to control the cochlear amplifier. Neurons from the LOC end up contacting the peripheral part of the type I SGN dendrites underneath the inner hair cells where they influence SGN activity. COCB = crossed olivocochlear bundle. UOCB = uncrossed olivocochlear bundle. CN = cochlear nucleus. MOC = medial olivary complex. LOC = lateral olivary complex. Figure taken from Guinan 2006 with permission.

Depending on the input from the peripheral auditory system, the central auditory system can then provide feedback on the peripheral auditory system in order to max- imize sensitivity or protect the auditory system from harm. Interneurons from the cochlear nucleus innervate neurons in the medial olivary (MOC) and lateral olivary (LOC) complexes (Figure 1.8). From these complexes ≈400 MOC neurons (in humans) project back to the cochleae to form synaptic connections directly onto the OHCs, whereas ≈1000 LOC neurons (in humans) project back to the cochleae to form synaptic connections on the peripheral segment of the dendrites of the type I SGNs (Lopez-Poveda, 2018). The MOC neurons serve to inhibit the OHCs and thus the cochlear amplifier (Glowatzki and Fuchs, 2000), thereby reducing the basilar membrane oscillations and thus SGN activity. The function of the LOC fibers is unclear, but several studies suggest that these LOC fibers can have both activating and in- hibiting effects on the type I SGNs (reviewed in Reijntjes and Pyott, 2016). Therefore, these LOC fibers may contribute to setting the excitability of the individual SGNs.

Furthermore, the LOC neurons could also play a role in protecting the SGN dendrites from acquired hearing loss.

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1.3. Acquired hearing loss

Acquired hearing loss due to age and noise exposure is likely not the result of a single damaged mechanism or pathway but the result of the accruement of damage to various parts of the auditory system over time. Classic work suggests that acquired hearing loss can be divided into four different categories: sensory, neural, metabolic, and conductive (Schuknecht, 1964). These four categories are based on patterns in the audiogram of patients suffering from hearing loss and are associated with damage to specific anatomical features of the auditory system. 1) Sensory hearing loss is marked by a steep increase in threshold for the higher frequencies combined with loss of the sensory hair cells in the high frequency part of the basilar membrane. 2) Neural hearing loss is marked by limited threshold shifts and difficulty in certain as- pects of speech perception combined with loss of the SGNs. 3) Metabolic hearing loss is indicated by an increase in threshold across all frequency ranges and associated with loss of cells in the stria vascularis. 4) Conductive hearing loss is indicated by gradual threshold shifts from low to high frequency regions combined with no obvious cellular deficits. Since this original classification, two more groups are considered, mixed and indeterminate, which together comprise >25% of patients and indicate the extent of variability and overlap in the contribution of the four described categories of acquired hearing loss (Tu and Friedman, 2018).

The development of acquired hearing loss is also dependent on genetic variation and general processes of ageing that affect all cells. Many genes are known to be involved in congenital hearing loss from young ages. In humans, over 100 genes contribute to hereditary non-syndromic hearing loss. Recent genetic screens have as- sessed the effect of genes on age-related hearing loss and identified five novel genes that are specifically related to hearing loss at mature to late life stages. As these experiments are time and cost intensive, genes associated with age- and noise-related hearing loss have been understudied, and these five genes are likely just scratching the surface of the number of genes involved in acquired hearing loss (Bowl et al., 2017;

Bowl and Brown, 2018). More general age-related processes are thought to interact with age- and noise-related hearing loss. One hypothesis for cellular ageing is the formation of reactive oxygen species (ROS) that can break down cellular structures by oxidation. ROS are formed naturally in mitochondria, the energy factories of cells (Chandrasekaran et al., 2017). With ageing, the clearance of these ROS molecules can be reduced in some patients resulting in increased cellular damage. Mitochon- dria are widely expressed in both the stria vascularis and the SGNs, and therefore, these structures may be especially influenced by ageing (Yang et al., 2015). Thus, variation in genetic background and the normal ageing process can affect the development of the type of acquired hearing loss.

Interestingly, recent findings in both humans and animals suggest that loss of the

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1.3. Acquired hearing loss 17

synaptic contacts between the sensory hair cells and the spiral ganglion neurons leads to a “hidden hearing loss” that precedes classical types of hearing loss. Mouse models indicate that the synaptic connections and the peripheral process of the SGN can be lost in response to age-related and noise-induced damage without loss of the SGN somas or the IHCs (Kujawa and Liberman, 2009). In addition, post mortem studies of human temporal bones suggest that the IHC-SGN synaptic connections can be lost without loss of the sensory hair cells in humans (Viana et al., 2015). This kind of damage, called synaptopathy, precedes any observable hearing loss in audiograms as the pure tone thresholds are mostly insensitive to synapse loss. Unfortunately, it is difficult to examine this pathology in humans, as assessing this kind of damage re- quires histological examination of human temporal bones. Therefore, it is currently unclear what the extent of synaptopathy and hidden hearing loss is in the human population and thus whether synaptopathy truly is the first step on the road to acquired hearing loss.

It is currently unknown exactly how synaptopathy arises and thus how to prevent or reverse synaptopathy. The leading hypothesis for the development of synaptopathy is excitotoxicity induced by overexposure of the peripheral process of the SGN to glutamate (Ruel et al., 2007). The opening of the glutamate receptor ion channel pore results in the influx of Na⁺ ions which increases the total intracellular ion concentration. Other ion channels are primed to restore the intracellular ion concentrations to their homeostatic levels. It is thought that prolonged glutamate receptor activation results in an influx of too many Na⁺ ions for the cell to restore intracellular ion concentrations resulting in an osmotic imbalance. This imbalance attracts water through osmosis, which could eventually lead to the rupture of the terminal process of the SGNs. In support of this hypothesis, exposure of the cochlea to large doses of glutamate results in observable vacuoles (fluid filled membranes) underneath the IHCs at the location of the synaptic contacts (Ruel et al., 2007). In addition to the toxic effect of Na⁺influx, Ca²⁺influx is thought to trigger apoptotic pathways or deregulate the mitochondria but there is little direct evidence in the cochlea for this mechanism.

The specific molecular mechanisms involved in SGN excitotoxicity are unknown. A further complicating factor is that not all SGNs may be equally vulnerable to excitotoxicity but specifically the low spontaneously active SGNs may be at risk (Furman et al., 2013). The low spontaneously active SGNs are not thought to contribute to auditory thresholds but instead are thought to extend the dynamic range of hearing to distinguish suprathreshold sound stimuli. In a variety of mammals, after moderate sound exposure, auditory thresholds recover despite the observation that up to 40%

of synaptic connections can be lost. Thus, it might be that the lost synaptic contacts are mostly low spontaneously active SGNs as the auditory thresholds are unaffected (Liberman, 2017). Furthermore, in guinea pig, exposure of the SGNs to glutamate resulted in the loss of responses of the low spontaneously active SGNs specifically (Furman et al., 2013). Finally, in humans, a common clinical complaint is the fail-

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ure to recognize speech in noise and other complaints suggestive of a loss of dynamic range (Vermiglio et al., 2012). These results imply that the low spontaneously active SGNs are more vulnerable to glutamate exposure and excitotoxicity than the high spontaneously active SGNs.

1.4. Molecular composition and function of the spiral ganglion neuron peripheral synapse

Regulation of SGN function is largely dependent on a variety of ion channels and proteins in the peripheral synapse. The excitability of the SGNs is determined in the peripheral synapse where the action potential generation site is located (Hossain, 2005). Ion channels are largely responsible for determining neuronal excitability by regulating both active and passive membrane characteristics, such as the resting membrane potential, action potential thresholds, durations, firing rates and timing (Rusznák and Szucs, 2009). Furthermore, each peripheral synapse contains a specialized structure called the postsynaptic density (PSD), which serves to cluster a variety of proteins that shape postsynaptic responses of the SGNs. These proteins co- determine neuronal excitability by controlling the expression of GluRs at the synapse (Chen 2007, 2009). Finally, lateral efferent innervation from the LOC can modulate SGN function, likely by acting on ion channels and or proteins in the PSD (Reijntjes and Pyott, 2016). Therefore, the function of SGNs is tightly regulated by a variety of molecular mechanisms. Although much work has been done to discover the precise nature of these mechanisms and the ion channels and proteins that underlie them, many of these processes and their components remain undetermined. Since the physiological responses and the vulnerability to acquired hearing loss is heterogenous in SGNs, it is likely that there are differences in the mechanisms that control SGN excitability and function between SGNs. Thus, elucidation of the ion channels and proteins that determine SGN function and their relative expression between SGNs will contribute to our understanding of SGN function and pathology.

1.5. This thesis

The spiral ganglion neurons (SGNs) are the first action potential generating neurons in the auditory pathway. The type I SGNs contact the sensory inner hair cells via their peripheral dendrites and relay auditory information to the brainstem via their central axon fibers. Individual afferent fibers show differences in response properties that are essential for normal hearing. Furthermore, this heterogeneity has been proposed to underlie an increased vulnerability to noise and age-related damage of a subgroup of SGNs. The mechanisms that give rise to the heterogeneity of SGN

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1.5. This thesis 19

afferent responses and their vulnerability are very poorly understood but are likely already in place at the peripheral dendrites where synapses are formed and action potentials are generated. In order to understand what drives this heterogeneity in SGN function and vulnerability, it is paramount to understand the molecular architecture and function of the SGN peripheral terminal, which is currently poorly understood. This thesis contributes to our understanding of the molecular architecture and function of the SGN peripheral terminal by relating the contribution of specific ion channels and synaptic proteins in the peripheral dendrite to SGN function.

In chapter 1 I introduce important concepts of the auditory pathway that are key in understanding the importance of the SGNs and their peripheral dendrites to normal auditory function. Furthermore, I will introduce current ideas about SGN heterogeneity, their vulnerability, and their role in acquired hearing loss as a framework to understand the contribution of the work that my colleagues and I have performed.

In chapter 2 we outline key players that contribute to shaping the functional re- sponses of SGNs in an extensive review of the complicated machinery located in and around the SGN peripheral terminal. The review focuses on the contribution of the various glutamate receptors and ion channels present, as well as glutamate reuptake by the surrounding cells and the effect of neurotransmitters released by the lateral efferent system in order to describe an afferent signalling complex that regulates SGN activity.

In chapter 3 we describe novel sodium dependent potassium channels (KN a1.1

and KN a1.2) in the type I SGNs. The properties of these ion channels make them ideal to regulate SGN activity in the SGN terminal. We further describe that loss of these ion channels in a knockout mouse model (KN a1.1and KN a1.2double knockout) severely impacts SGN function in vitro and auditory function in vivo.

In chapter 4 we investigate the contribution of KN a1.1and KN a1.2to acquired hearing loss by examining a KN a1.1and KN a1.2double knockout mouse model. We describe a set of experiments that follow SGN functionality and vulnerability after noise exposure and with increasing age.

In chapter 5 we study gradients in the size of synaptic proteins between two sub- groups of SGN synapses: those contacting the pillar and modiolar facings of the IHCs.

We find evidence to support the existence of size gradients in presynaptic proteins but find irregularities in the size gradients of postsynaptic proteins. These irregularities warrant reconsideration of previous hypotheses about the determination of SGN subgroups and the mechanisms that shape SGN vulnerability.

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Finally, in chapter 6 I will outline the scientific impact of these studies and ex- amine the implications for future research.

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Chapter 2 The afferent signaling complex:

Regulation of type I spiral ganglion neuron responses in the

auditory periphery

This chapter has been published as: Reijntjes, D.O.J., Pyott, S.J., 2016. Hearing Research 336:1-16.

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Abstract

The spiral ganglion neurons (SGNs) are the first action potential generating neurons in the auditory pathway. The type I SGNs contact the sensory inner hair cells via their peripheral dendrites and relay auditory information to the brainstem via their central axon fibers. Individual afferent fibers show differences in response properties that are essential for normal hearing. The mechanisms that give rise to the heterogeneity of afferent responses are very poorly understood but are likely already in place at the peripheral dendrites where synapses are formed and action potentials are generated. To identify these molecular mechanisms, this review synthesizes a variety of literature and comprehensively outlines the cellular and molecular components positioned to regulate SGN afferent dendrite excitability, especially following glutamate release. These components include 1) proteins of the SGN postsynapses and neighboring supporting cells that together shape glutamatergic signaling, 2) the ion channels and transporters that determine the intrinsic excitability of the SGN afferent dendrites, and 3) the neurotransmitter receptors that extrinsically modify this excitability via synaptic input from the lateral olivocochlear efferents. This cellular and molecular machinery, together with presynaptic specializations of the inner hair cells, can be collectively referred to as the type I afferent signaling complex. As this review underscores, interactions of this signaling complex determine excitability of the SGN afferent dendrites and the afferent fiber responses. Moreover, this complex establishes the environmental milieu critical for the development and maintenance of the SGN afferent dendrites and synapses. Motivated by these important functions, this review also indicates areas of future research to elucidate the contributions of the afferent signaling complex to both normal hearing and also hearing loss.

2.1. Overview

The encoding of sound stimuli imposes enormous demands on the auditory system. In meeting this challenge, neurons of the auditory system show morphological, physiological, and molecular specializations that enable fast, sustained, and tempo- rally reliable synaptic transmission over a wide dynamic range. The spiral ganglion neurons (SGNs) are the first action potential (AP) generating neurons in the auditory pathway. The central projections of these bipolar cells supply all of the auditory input from the inner hair cells (IHCs) to the central nervous system (CNS) via their axons, which are myelinated and collectively form the auditory nerve. The peripheral dendrites of the type I SGNs are unmyelinated and form synaptic contacts at the bases of these IHCs, the true sensory cells of the auditory system. Remarkably, each SGN receives input from just a single IHC via a single synapse.

While all type I SGNs exhibit extraordinary temporal precision of their sound-

Molecular composition and function of the spiral

Molecular composition and function of the spiral ganglion neuron peripheral synapse in mice

Molecular composition and function of the spiral

ganglion neuron peripheral synapse in mice

PhD thesis

Daniël Onne Jilt Reijntjes

Co-supervisor

Assessment Committee

Table of contents

Chapter 1

Introduction

Preface

1.1. The perception of sound

1.2. Anatomy of the auditory system

1.2.1. The peripheral auditory system

1.3. Acquired hearing loss

1.4. Molecular composition and function of the spiral ganglion neuron peripheral synapse

1.5. This thesis

Chapter 2

The afferent signaling complex:

Regulation of type I spiral ganglion neuron responses in the

auditory periphery

Abstract

2.1. Overview