PROJECT SUMMARY The adult cochlea presents many technical hurdles to researchers studying the normal function of its constituent cell types, and how these cell types respond to injury, aging and degeneration. Because the cochlea is encased in strong bone and contains relatively small numbers of cells compared to other sensory organs such as the retina, isolating cells to characterize their structure, physiology or gene expression is challenging. Moreover, histological methods to make the cochlea more accessible, such as decalcification, are harsh treatments that can degrade mRNA or protein. In this pilot R21 proposal, we will assess the feasibility of a recently-developed technique known as MERFISH – Multiplexed Error-Robust Fluorescence In Situ Hybridization – to simultaneously detect and quantify the expression of hundreds of genes in the cochlea. We believe this technique is sensitive enough to reveal quantitative differences in gene expression along the developmental and tonotopic gradients that exist along the basal-apical axis of the cochlea. Developing a robust protocol to detect, quantify and spatially localize hundreds of genes at once has the ability to transform our understanding of the normal function of the cochlea, and how gene expression changes in the cochlea and spiral ganglion after noise exposure, hair cell loss, aging and drug administration. The goal of this proposal is to demonstrate the feasibility of MERFISH in the neonatal and adult cochlea and to develop protocols to allow MERFISH to be used by the hearing and balance community.