The spiral ganglion is an essential functional component of the peripheral auditory system. Most types of hearing loss are associated with spiral ganglion cell degeneration which is irreversible due to the inner ear's lack of regenerative capacity. Recent studies revealed the existence of stem cells in the postnatal spiral ganglion, which gives rise to the hope that these cells might be useful for regenerative inner ear therapies. Here, we provide an in-depth analysis of sphere-forming stem cells isolated from the spiral ganglion of postnatal mice. We show that spiral ganglion spheres have characteristics similar to neurospheres isolated from the brain. Importantly, spiral ganglion sphere cells maintain their major stem cell characteristics after repeated propagation, which enables the culture of spheres for an extended period of time. In this work, we also demonstrate that differentiated sphere-derived cell populations not only adopt the immunophenotype of mature spiral ganglion cells but also develop distinct ultrastructural features of neurons and glial cells. Thus, our work provides further evidence that self-renewing spiral ganglion stem cells might serve as a promising source for the regeneration of lost auditory neurons.

Ribbon synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) in the inner ear are damaged by noise trauma and with aging, causing 'synaptopathy 'and hearing loss. Co-cultures of neonatal denervated organs of Corti and newly introduced SGNs have been developed to find strategies for improving IHC synapse regeneration, but evidence of the physiological normality of regenerated synapses is missing. This study utilizes IHC optogenetic stimulation and SGN recordings, showing that newly formed IHC synapses are indeed functional, exhibiting glutamatergic excitatory postsynaptic currents. When older organs of Corti were plated, synaptic activity probed by deconvolution, showed more mature release properties, closer to the highly specialized mode of IHC synaptic transmission that is crucial for coding the sound signal. This newly developed functional assessment of regenerated IHC synapses provides a powerful tool for testing approaches to improve synapse regeneration.

The spiral ganglion neurons constitute the primary connection between auditory hair cells and the brain. The spiral ganglion afferent fibers and their synapse with hair cells do not regenerate to any significant degree in adult mammalian ears after damage. We have investigated gene expression changes after kainate-induced disruption of the synapses in a neonatal cochlear explant model in which peripheral fibers and the afferent synapse do regenerate. We compared gene expression early after damage, during regeneration of the fibers and synapses, and after completion of in vitro regeneration. These analyses revealed a total of 2.5% differentially regulated transcripts (588 out of 24,000) based on a threshold of p<0.005. Inflammatory response genes as well as genes involved in regeneration of neural circuits were upregulated in the spiral ganglion neurons and organ of Corti, where the hair cells reside. Prominent genes upregulated at several time points included genes with roles in neurogenesis (Elavl4 and Sox21), neural outgrowth (Ntrk3 and Ppp1r1c), axonal guidance (Rgmb and Sema7a), synaptogenesis (Nlgn2 and Psd2), and synaptic vesicular function (Syt8 and Syn1). Immunohistochemical and in situ hybridization analysis of genes that had not previously been described in the cochlea confirmed their cochlear expression. The time course of expression of these genes suggests that kainate treatment resulted in a two-phase response in spiral ganglion neurons: an acute response consistent with inflammation, followed by an upregulation of neural regeneration genes. Identification of the genes activated during regeneration of these fibers suggests candidates that could be targeted to enhance regeneration in adult ears.

HMG domain transcription factor, Sox2, is a critical gene for the development of cochlear hair cells, the receptor cells for hearing, but this has been ascribed to expansion of the progenitors that become hair cells. Here, we show that Sox2 activated Atoh1, a transcription factor important for hair cell differentiation, through an interaction with the 3' enhancer of Atoh1. Binding to consensus sequences in the Atoh1 enhancer was dependent on the level of Sox2, and the extent of enhancer binding correlated to the extent of activation. Atoh1 activation by Sox2 was required for embryonic hair cell development: deletion of Sox2 in an inducible mutant, even after progenitor cells were fully established, halted development of hair cells, and silencing also inhibited postnatal differentiation of hair cells induced by inhibition of γ-secretase. Sox2 is thus required in the cochlea to both expand the progenitor cells and initiate their differentiation to hair cells.

Sensorineural hearing loss (SNHL) caused by noise exposure and attendant loss of glutamatergic synapses between cochlear spiral ganglion neurons (SGNs) and hair cells is the most common sensory deficit worldwide. We show here that systemic administration of a bisphosphonate to mice 24 h after synaptopathic noise exposure regenerated synapses between inner hair cells and SGNs and restored cochlear function. We further demonstrate that this effect is mediated by inhibition of the mevalonate pathway. These results are highly significant because they suggest that bisphosphonates could reverse cochlear synaptopathy for the treatment of SNHL.

Auditory neuropathy is caused by the loss of afferent input to the brainstem via the components of the neural pathway comprising inner hair cells and the first order neurons of the spiral ganglion. Recent work has identified the synapse between cochlear primary afferent neurons and sensory hair cells as a particularly vulnerable component of this pathway. Loss of these synapses due to noise exposure or aging results in the pathology identified as hidden hearing loss, an initial stage of cochlear dysfunction that goes undetected in standard hearing tests. We show here that repulsive axonal guidance molecule a (RGMa) acts to prevent regrowth and synaptogenesis of peripheral auditory nerve fibers with inner hair cells. Treatment of noise-exposed animals with an anti-RGMa blocking antibody regenerated inner hair cell synapses and resulted in recovery of wave-I amplitude of the auditory brainstem response, indicating effective reversal of synaptopathy.

Ouabain application to the round window can selectively destroy type-I spiral ganglion cells, producing an animal model of auditory neuropathy. To assess the long-term effects of this deafferentation on synaptic organization in the organ of Corti and cochlear nucleus, and to ask whether surviving cochlear neurons show any post-injury plasticity in the adult, we quantified the peripheral and central synapses of type-I neurons at posttreatment times ranging from 1 to 3 months. Measures of normal DPOAEs and greatly reduced auditory brainstem responses (ABRs) confirmed the neuropathy phenotype. Counts of presynaptic ribbons and postsynaptic glutamate receptor patches in the inner hair cell area decreased with post-exposure time, as did counts of cochlear nerve terminals in the cochlear nucleus. Although these counts provided no evidence of new synapse formation via branching from surviving neurons, the regular appearance of ectopic neurons in the inner hair cell area suggested that neurite extension is not uncommon. Correlations between pathophysiology and histopathology showed that ABR thresholds are very insensitive to even massive neural degeneration, whereas the amplitude of ABR wave 1 is a better metric of synaptic degeneration.