Neuroanatomical tracing techniques in the ear: History, state of the art, and future developments

The inner ear has long been at the cutting edge of tract tracing techniques that have shaped and reshaped our understanding of the ear’s innervation patterns. This review provides a historical framework to understand the importance of these techniques for ear innervation and for development of tracing techniques in general; it is hoped that lessons learned will help to quickly adopt transformative novel techniques and their information and correct past beliefs based on technical limitations. The technical part of the review presents details of our protocol as developed over the last 30 years. We also include arguments as to why these recommendations work best to generate the desired outcome of distinct fiber and cell labeling, and generate reliable data for any investigation. We specifically focus on two tracing techniques, in part developed and/or championed for ear innervation analysis: the low molecular multicolor dextran amine tract tracing technique and the multicolor tract tracing technique with lipophilic dyes.