Longitudinal imaging of the taste bud in vivo with two-photon laser scanning microscopy

Taste bud cells in the tongue transduce taste information from chemicals in food and transmit this information to gustatory neurons in the geniculate ganglion that innervate taste buds. The peripheral taste system is a dynamic environment where taste bud cells are continuously replaced, but further understanding of this phenomenon has been limited by the inability to directly observe this process. To overcome this challenge, we combined chronic in vivo two-photon laser scanning microscopy with genetic labeling of gustatory neurons and taste buds to observe how cells within the taste bud change over time. This method expands the investigative possibilities beyond those offered by fixed-tissue methods. This method permits direct observation of taste bud cell entry, cell differentiation, cell loss, and arbor plasticity. We demonstrate that a few stains/dyes can be used to observe nuclei and organelles in the taste bud in vivo. We also describe a workflow for reconstructing composite z-stacks with grayscale data of both cells and arbors using ImageJ, Neurolucida 360, and Neurolucida Explorer software. Together, the methodology and software options for analyses presented here provide a novel approach for longitudinally observing taste bud cells and arbors in the taste bud in vivo.