Ultrastructure of noise-induced cochlear synaptopathy

Overexposure to noise can eliminate synaptic connections between inner hair cells (IHCs) and auditory nerve fibers (ANFs), even if hair-cell function recovers. This noise-induced synaptopathy has been extensively studied in confocal microscopy of cochleas immunostained for pre- and post-synaptic markers, however, understanding the nature and sequence of damage requires ultrastructural analysis. Here, we used focused ion beam scanning electron microscopy to serially mill, image, segment and reconstruct the unmyelinated terminals of ANFs in mice, 1 day and 1 week after synaptopathic exposure. At both survivals, ANF terminals were normal in number, but synaptic counts were reduced by half. Most non-synapsing fibers remained in close proximity to IHCs and contained healthy-looking organelles. ANFs showed a transient increase in mitochondrial content and efferent innervation at 1 day. Fibers maintaining synaptic connections showed hypertrophy of pre-synaptic ribbons at both 1 day and 1 week. Non-synaptic fibers were lower in mitochondrial content and typically on the modiolar side of the IHC, consistent with selective damage to ANFs with high-thresholds and low spontaneous rates. Even 1 week post-exposure, most unmyelinated ANF terminals remained intact despite loss of synaptic specializations. Thus, the first step in the degenerative process is not explosion and retraction of the neuron.