We thank Kavisca Kuruparanantha for her contributions to tissue staining and the imaging of circumvallate taste buds, Jennifer Xu for staining and imaging of innervation to the papilla, Kaytee Horn for animal care and genotyping, and Liqun Ma for her tissue staining of the soft-palate taste buds. This project was supported by R21 DC014857 and R01 DC007176 to R.F.K and F31 DC017660 to L.O.

NOTE: All animals were cared for in accordance with the guidelines set by the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the NIH Guide for the Care and Use of Laboratory Animals. Phox2b-Cre mice (MMRRC strain 034613-UCD, NP91Gsat/Mmcd) or TrkBCreER mice (Ntrk2tm3.1(cre/ERT2)Ddg) were bred with tdTomato reporter mice (Ai14). AdvillinCreER47 were bred with Phox2b-flpo48 and Ai65. For 5-ethyn...

The development of an approach to consistently collect and stain whole taste buds from three oral cavity taste regions (fungiform, circumvallate, and the palate) provides significant improvements for analyzing taste-transducing cells, tracking newly incorporated cells, innervation, and relationships between these structures. In addition, it facilitates the localization of a potential secondary neuron marker both within or outside of a labeled population50. This is particularly relevant given that ...

Taste buds are collections of 50-100 specialized epithelial cells that bind subsets of chemical-taste stimuli present in the oral cavity. Taste-transducing cells are generally thought to exist as types1,2,3,4,5,6,7,8,9, initially based on electron microscopy criteria that were later correlated with molecular markers. Type II cells express phospholipase C-beta 2 (PLC&#946;2)2 and transient receptor potential cation channel, subfamily M member 51 and include cells that transduce sweet, bitter, and umami1,10. Type III cells express carbonic anhydrase 4 (Car4)11 and synaptosomal-associated protein 258 and denote cells that primarily respond to sour taste11. The cells that transduce saltiness have not been as clearly delineated12,13,14, but could potentially include Type I, Type II&#160;and Type III cells15,16,17,18,19.The taste-bud environment is complex and dynamic, given that taste-transducing cells continuously turn over throughout adulthood and are replaced by basal progenitors3,20,21. These taste-transducing cells connect to pseudo-unipolar nerve fibers from the geniculate and petrosal ganglia, which pass taste information to the brainstem. These neurons have primarily been categorized based on the kind of taste information they carry22,23 because information about their morphology has been elusive until recently24. Type II cells communicate with nerve fibers via calcium homeostasis modulator protein 1 ion channels25, whereas Type III cells communicate via classical synapses8,26. Further characterization of taste bud cells-including transducing cell type lineages, factors that influence their differentiation, and the structures of connecting arbors are all areas of active investigation.
Taste-bud studies have been hindered by several technical challenges. The heterogenous and dense tissues that make up the tongue significantly reduce antibody permeability for immunohistochemistry27,28,29. These obstacles have necessitated sectioning protocols that result in the splitting of taste buds across sections so that measurements are either approximated based on representative sections or summed across sections. Previously, representative thin sections have been used to approximate both volume values and transducing-cell counts30. Thicker serial sectioning allows for the imaging of all taste-bud sections and the summing of measurements from each section31. Cutting such thick sections and selecting only whole taste buds biases sampling towards smaller taste buds32,33,34. Nerve innervation estimates from sectioned taste buds have been based on analyses of pixel numbers13,35, if quantified at all36,37,38. These measurements completely ignore the structure and number of individual nerve arbors, because arbors are split (and usually poorly labeled). Lastly, although peeling away the epithelium does permit entire taste buds to be stained39,40, it also removes taste-bud nerve fibers and could disrupt the normal relationships between cells. Therefore, investigations of the structural relationships within taste buds have been limited because of this disruption caused by staining approaches.
Whole-structure collection eliminates the need for representative sections and allows the determination of absolute-value measurements of volumes, cell counts, and structure morphologies41. This approach also increases accuracy, limits bias, and reduces technical variability. This last element is important because taste buds show considerable biological variability both within34,42 and across regions43,44, and whole taste-bud analyses allow absolute cell numbers to be compared between control and experimental conditions. Furthermore, the ability to collect intact taste buds permits the analysis of the physical relationships between different transducing cells and their associated nerve fibers. Because taste-transducing cells may communicate with each other45 and do communicate with nerve fibers46, these relationships are important for normal function. Thus, loss-of-function conditions may not be due to a loss of cells, but instead to changes in cell relationships. Provided here is a method for collecting whole taste buds to achieve the benefits of absolute measurements for refining volume analyses for both taste buds and their innervations, taste-cell counts and shapes, and for facilitating analyses of transducing-cell relationships and nerve-arbor morphologies. Two workflows are also presented downstream of this novel whole-mount method for tissue preparation: 1) for analyzing taste bud volume and total innervation and 2) for sparse-cell genetic labeling of taste neurons (with subsets of taste-transducing cells labeled) and subsequent analyses of taste-nerve arbor morphology, numbers of taste-cell types and their shapes, and the use of image analysis software to analyze the physical relationships between transducing cells and those between transducing cells and their nerve arbors. Together, these workflows provide a novel approach to tissue preparation and for the analyses of whole taste buds and the complete morphology of their innervating arbors.

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whole-mount staining, visualization, and analysis of fungiform, circumvallate, and palate taste buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

Staining of the lingual epithelium with antibodies to dsRed and keratin-8 (a general taste-bud marker) labeled both whole taste buds and all taste-bud innervation in Phox2b-Cre:tdTomato mice50,51 (Figure 3A). Imaging these taste buds from their pores to their bases gave the highest resolution x-y plane images (Figure 3A,B). The contour function of the pixel-based imaging program was used...

Watch this Scientific Journal Video about Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds at JoVE.com

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

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.

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Abstract