Efficient quantification of afferent cochlear ultrastructure using design-based stereology

The afferent synapse between the auditory nerve fiber and the inner hair cell (IHC) represents a critical junction for hearing. Elucidation of the structure at this site will help establish the substrate for normal sound encoding as well as pathologic processes associated with hearing dysfunction. Previous applications of unbiased (design-based) stereological principles have expanded our knowledge of neuro-morphological changes evident with the light microscope. Applying these principles at the level of the synapse is a promising morphometric approach for the efficient sampling of large reference spaces with electron microscopy. This study tests the accuracy of using ultra-thin sections at a fixed interval, known as disector pairs, to quantify afferent innervation density. We analyzed the total numbers of afferent terminals, synaptic thickenings, and synaptic bodies associated with each IHC in the C57BL/6J mouse cochlea, and confirmed the accuracy of the stereological approach in comparison to three-dimensional reconstructions of serial alternate sections. The higher sampling efficiency of the disector pair method rapidly increases precision while also reducing the largest source of variability, inter-animal differences. We conclude that ultrastructural quantification of afferent innervation can be accomplished in the cochlea using efficient design-based stereology.