Inner hair cells are not required for survival of spiral ganglion neurons in the adult cochlea

Abstract

Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To reexamine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 d) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that (1) IHCs are not necessary for neuronal survival, (2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and (3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.

Figure 1.

Three months after IHC loss, there is no significant loss of cochlear neurons in the Slc19a2-null mice on low thiamine. A, Schematic cross-section through the cochlear duct illustrating the cell bodies of SGNs, their peripheral axons and terminals. The red outline schematizes the section plane through the osseous spiral lamina (OSL) tangential to the cochlear spiral in which SGN peripheral axons are quantified (E, F). B, Photomicrographs of the midbasal turn of the cochlear duct (∼32 kHz region) from a Slc19a2-null mouse on high thiamine (top row—IHCs present) and a knock-out mouse on low thiamine (bottom row—IHCs absent). In the high-power views at the right, the blue arrowhead points to the IHC nucleus; the white arrowhead points to the IHC hair bundle. Scale bars apply to both micrographs in each column. C, Inner and outer hair cells were counted throughout the upper basal turn in 5–7 ears from each of 4 experimental groups: wild types on high-thiamine, wild types on low thiamine, knock-outs on high thiamine and knock-outs on low thiamine. Means and SEs are shown. D, Photomicrographs of the midbasal turn (∼32 kHz region) show that loss of IHCs in the knock-out mouse on low thiamine (bottom) does not obviously affect SGN density, when compared with the wild-type (top), in which the IHCs are present. Scale bar applies to both panels. E, Peripheral axons were counted in the upper basal turn in 5–7 ears from each of the 4 experimental groups. Means and SEs are shown. F, Photomicrographs of the upper basal turn (∼22 kHz region) show SGN peripheral axons as they appear in cross-sections through the osseous spiral lamina from knock-out mice on high- vs low-thiamine diets.

Figure 2.

Three months after IHC loss, supporting cells, as well as afferent and efferent nerve terminals in the IHC area remain intact. A, Schematic of the IHC area illustrating SGN peripheral terminals, LOC efferent terminals and the two cells strongly expressing GLAST protein (inner border and inner phalangeal cells). B, Confocal micrographs of immunostained sections through the organ of Corti and osseous spiral lamina from the midbasal turn (∼32 kHz region). Three rows of OHCs are indicated by the white arrowheads in each panel. The DAPI-stained IHC nucleus (top only) is indicated by the red arrowhead. Dashed circles outline the neuropil in the IHC region where the unmyelinated SGN terminals are visible in both panels. The staining key and scale bar apply to both panels. C, Semiquantitative evaluation of LOC innervation density in confocal images such as those in D, acquired from ears in the four experimental groups. Means and SEs are shown. D, Maximum projections from confocal z-stacks of the VAT-stained LOC terminals in the IHC area. The approximate size of an IHC is indicated by the dashed circle in top panel. Scale applies to both panels. E, Maximum projections from confocal z-stacks through whole-mounts of the organ of Corti in the IHC area immunostained with an anti-GLAST antibody. The spaces where the IHCs used to be are visible in the knock-out ear maintained on low thiamine (dashed circle indicates one space). Key and scale bar applies to both panels.