This whole-mount dissection and analysis workflow provides a novel approach to analyze the complete morphology of individual taste nerve arbors, transducing cell type numbers, and the physical relationships between cells. Collecting the entire lingual epithelium permits us to measure the absolute number of cells and volume of innervation within the whole taste bud. This approach is more accurate than estimates of cell structure based on sampling from sections, which reduces technical variability.
This is also the first method providing detailed analysis of the taste bud cells in relation to their innervation. This method can enhance investigations into the potential circuitry within the taste bud, as well as disease processes and chemotherapies that disrupt normal taste function. In most of these cases, it's unclear which structure or relationship is disrupted, so maintaining all of those factors intact allows for all analyses within a single preparation.
The most common mistake is trying to avoid damaging the epithelium by not dissecting close enough to the underside of the epithelium. It's imperative to remove as much underlying tissue as possible before freezing the tissue in the tissue mold. It's also important to ensure the epithelium is laying flat in the bottom of the tissue mold.
Begin by thawing and rinsing the tongue in 0.1 molar phosphate buffer. Then place 1/2 of the anterior tongue containing the fungiform papillae on a glass slide under a dissecting microscope. Use blunt-ended forceps and dissection scissors to remove the muscle and hold the tissue open as the lingual epithelium is curved, ensuring a flat orientation of tissue by keeping the blades of the coarse dissection scissors parallel to the epithelium.
Discard the ventral non-keratinized epithelium of the tongue as it contains no taste buds. Then use fine dissection scissors for closer dissection to the underside of the keratinized epithelium. Use blunt-ended forceps to lay a piece of epithelium into a tissue mold and ensure that it lays flat.
Then add a drop of optimal cutting temperature compound, or OCT, to the tissue. Place the tissue mold on a previously cooled metal base under the dissecting scope, then continue to tap the tissue lightly with the forceps until the OCT has frozen, ensuring that the tissue freezes as flat as possible. Once the OCT has frozen, quickly add additional OCT and place the mold in a beaker of cooled 2-methylbutane until frozen.
Mount the OCT mold on the cryostat and cut it into 20 micron sections. Collect each section and view it under the light microscope to assess its proximity to the base of the epithelium. After the tissue is shaved from the underside of the epithelium, thaw the epithelium and rinse it twice in 0.1 molar PB on a shaker.
Cut the hard palate anterior to the junction of the soft and hard palate, then separate the soft palate from the underlying tissue, making sure any remaining bone fragments are cut away and removing additional muscle and connective tissue. Hold the palate with blunt-ended forceps and remove the remaining glands and lose connective tissue by gently scraping them with a razor blade. Whole taste buds and all taste bud innervation in Phox2b-Cre TdTomato mice were labeled by staining the lingual epithelium with antibodies for DsRed and keratin eight.
Taste bud volume was measured and revealed no correlations between taste bud volumes and innervation volumes in either the fungiform or the circumvallate measurement regions. The administration of a low dose of tamoxifen in TrkB CreER TdTomato mice causes gene recombination and the labeling of a small number of neurons. A whole-mount taste bud stained with taste transducing cell markers carbonic anhydrase four and phospholipase C beta two is shown here.
This taste bud has two labeled terminal arbors, which are shown with the taste bud removed after reconstructing the fibers. Using cell pixel-based imaging software to determine the closest proximity between nerve fibers and taste transducing cells revealed that out of 19 taste transducing cells, the blue terminal arbor was within 200 nanometers of the light blue carbonic anhydrase four plus cell. The terminal arbor associated with the green tracing is shown in magenta and is within 200 nanometers of both the light and dark blue carbonic anhydrase four plus cells.
Whole-mount keratin eight and 5-ethynyl-2'deoxyuridine or EdU staining of fungiform taste buds revealed that EdU-labeled cells were present both within and outside of the taste buds. The magenta and purple nuclei are observed outside of the keratin eight plus border of the taste bud. The yellow, teal, and blue cells were within the taste bud.
When attempting this protocol, the tissue dissection and freezing steps are pivotal. These tissue dissection methods can be followed by staining for a variety of cells and structures to analyze the relationships both within the taste bud and outside the taste bud, but within the papilla. This is the first analysis of whole taste arbors within the taste bud, as well as their relationships with taste transducing cells.
Preserving and staining for several of these elements in a single preparation both expands the possibilities for analysis and refines the measurements that are possible.
