Selective hair cell ablation and noise exposure lead to different patterns of changes in the cochlea and the cochlear nucleus

Highlights

• Auditory neurons can survive for at least two months after selective HC ablation.

• Axon caliber and SGN soma size are significantly smaller after selective HC loss, but their density is unchanged.

• Myelin surrounding auditory neurons is maintained in the absence of HCs.

• HCs are indispensable for the GluR2 expression in the peripheral synapses.

• The VGLUT-1 expression in the CN is unchanged two months after DT injection despite complete loss of HCs and deafness.

Abstract

In experimental animal models of auditory hair cell (HC) loss, insults such as noise or ototoxic drugs often lead to secondary changes or degeneration in non-sensory cells and neural components, including reduced density of spiral ganglion neurons, demyelination of auditory nerve fibers and altered cell numbers and innervation patterns in the cochlear nucleus (CN). However, it is not clear whether loss of HCs alone leads to secondary degeneration in these neural components of the auditory pathway. To elucidate this issue, we investigated changes of central components after cochlear insults specific to HCs using diphtheria toxin receptor (DTR) mice expressing DTR only in HCs and exhibiting complete HC loss when injected with diphtheria toxin (DT). We showed that DT-induced HC ablation has no significant impacts on the survival of auditory neurons, central synaptic terminals, and myelin, despite complete HC loss and profound deafness. In contrast, noise exposure induced significant changes in synapses, myelin and CN organization even without loss of inner HCs. We observed a decrease of neuronal size in the auditory pathway, including peripheral axons, spiral ganglion neurons, and CN neurons, likely due to loss of input from the cochlea. Taken together, selective HC ablation and noise exposure showed different patterns of pathology in the auditory pathway and the presence of HCs is not essential for the maintenance of central synaptic connectivity and myelination.

Introduction

Cochlear insults induced by acoustic overstimulation or ototoxic drug administration cause damage to several types of cochlear cells including hair cells (HCs), spiral ganglion neurons (SGNs) synapses between HCs and SGNs and supporting cells (SCs) as well as to more central regions of the auditory pathways such as the cochlear nucleus (CN), resulting in sensorineural hearing loss (SNHL) (Lesperance et al., 1995, Aarnisalo et al., 2000, Kujawa and Liberman, 2009, Shibata et al., 2010, Liu et al., 2013). No effective biological treatment is available for human patients with severe or profound SNHL, although some patients with severe SNHL benefit from a cochlear implant, a prosthesis that electrically stimulates the SGNs. Preservation of healthy auditory neurons and their components, including central synapses and myelin, is thus critical for clinical success of cochlear prostheses and functional HC regeneration. Yet, the detailed mechanism of degeneration in SGNs and synapses in SNHL patients remains largely unknown due to the complexity of cochlear damage and inner ear structures.

SGNs are primary auditory neurons involved in signal transmission from the peripheral auditory receptors, the HCs in the organ of Corti, to the CN. About 95% of SGNs are type I SGNs, which are myelinated and synapse with inner HCs (IHCs) via their peripheral processes (Spoendlin, 1975). All known excitatory activity in the type I SGN pathway is glutamatergic (Flores et al., 2015). At the peripheral terminal of type I SGNs, there are numerous postsynaptic puncta that express the glutamate receptor 2 (GluR2), a critical subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) type glutamate receptor (Matsubara et al., 1996, Frank et al., 2010, Arbeloa et al., 2012). At the central terminal of type I SGNs in the CN, vesicular glutamate transporter-1 (VGLUT-1) packages the glutamate into synaptic vesicles (Zhou et al., 2007, Zeng et al., 2009). These synaptic components, including GluR2 and VGLUT-1, are required for transmission of sound-induced signals from the cochlea to the CN.

Synaptic degeneration, including decreases in GluR2 and VGLUT-1, and demyelination of auditory nerve fibers (ANFs) after cochlear insults have been reported (Zhou et al., 2007, Zeng et al., 2009, Barker et al., 2012, Tagoe et al., 2014, Wan et al., 2014, Heeringa et al., 2016). These studies used either noise or ototoxic drugs to induce cochlear damage. However, both these methods can damage other cochlear tissues, including SGNs themselves, and it is not clear whether the loss of HCs alone results in the degeneration of synapses and myelination. To determine whether the loss of HCs without any other direct cochlear tissue injury is sufficient to damage synapses and myelination, we used a novel transgenic mouse model, the DTR mouse (Golub et al., 2012), in which the gene for human diphtheria toxin receptor (hDTR) was inserted under regulation of the promoter for Pou4f3, a HC-specific transcription factor (Keithley et al., 1999). In DTR mice, a single injection of diphtheria toxin (DT) kills all cochlear HCs, leaving the cochlear neural components intact and providing an appropriate model to study responses of the auditory pathway to loss of HCs (Golub et al., 2012, Kaur et al., 2015, Tong et al., 2015). Here we compared the pathology induced by selective ablation of HCs by DT to that caused by noise-induced HC cochlear trauma. Specifically, we examined non-sensory cells in the cochleae, SGNs, myelin, CN, and synapses, including the expression of GluR2 and VGLUT-1, and determined that following DT-induced HC ablation the changes in the cochlea and the CN are minimal whereas noise exposure induced significant changes. The data suggest that central synaptic connectivity and myelination can be maintained as long as 2 months following the loss of IHCs but noise exposure can change the neural substrate even without loss of IHCs.