Qualitative and quantitative differences between taste buds of the rat and mouse

BACKGROUND: Numerous electrophysiological, ultrastructural, and immunocytochemical studies on rodent taste buds have been carried out on rat taste buds. In recent years, however, the mouse has become the species of choice for molecular and other studies on sensory transduction in taste buds. Do rat and mouse taste buds have the same cell types, sensory transduction markers and synaptic proteins? In the present study we have used antisera directed against PLCβ2, α-gustducin, serotonin (5-HT), PGP 9.5 and synaptobrevin-2 to determine the percentages of taste cells expressing these markers in taste buds in both rodent species. We also determined the numbers of taste cells in the taste buds as well as taste bud volume. RESULTS: There are significant differences (p < 0.05) between mouse and rat taste buds in the percentages of taste cells displaying immunoreactivity for all five markers. Rat taste buds display significantly more immunoreactivity than mice for PLCβ2 (31.8% vs 19.6%), α-gustducin (18% vs 14.6%), and synaptobrevin-2 (31.2% vs 26.3%). Mice, however, have more cells that display immunoreactivity to 5-HT (15.9% vs 13.7%) and PGP 9.5 (14.3% vs 9.4%). Mouse taste buds contain an average of 85.8 taste cells vs 68.4 taste cells in rat taste buds. The average volume of a mouse taste bud (42,000 μm(3)) is smaller than a rat taste bud (64,200 μm(3)). The numerical density of taste cells in mouse circumvallate taste buds (2.1 cells/1000 μm(3)) is significantly higher than that in the rat (1.2 cells/1000 μm(3)). CONCLUSION: These results suggest that rats and mice differ significantly in the percentages of taste cells expressing signaling molecules. We speculate that these observed dissimilarities may reflect differences in their gustatory processing

Reconsidering Association Testing Methods Using Single-Variant Test Statistics as Alternatives to Pooling Tests for Sequence Data with Rare Variants

Association tests that pool minor alleles into a measure of burden at a locus have been proposed for case-control studies using sequence data containing rare variants. However, such pooling tests are not robust to the inclusion of neutral and protective variants, which can mask the association signal from risk variants. Early studies proposing pooling tests dismissed methods for locus-wide inference using nonnegative single-variant test statistics based on unrealistic comparisons. However, such methods are robust to the inclusion of neutral and protective variants and therefore may be more useful than previously appreciated. In fact, some recently proposed methods derived within different frameworks are equivalent to performing inference on weighted sums of squared single-variant score statistics. In this study, we compared two existing methods for locus-wide inference using nonnegative single-variant test statistics to two widely cited pooling tests under more realistic conditions. We established analytic results for a simple model with one rare risk and one rare neutral variant, which demonstrated that pooling tests were less powerful than even Bonferroni-corrected single-variant tests in most realistic situations. We also performed simulations using variants with realistic minor allele frequency and linkage disequilibrium spectra, disease models with multiple rare risk variants and extensive neutral variation, and varying rates of missing genotypes. In all scenarios considered, existing methods using nonnegative single-variant test statistics had power comparable to or greater than two widely cited pooling tests. Moreover, in disease models with only rare risk variants, an existing method based on the maximum single-variant Cochran-Armitage trend chi-square statistic in the locus had power comparable to or greater than another existing method closely related to some recently proposed methods. We conclude that efficient locus-wide inference using single-variant test statistics should be reconsidered as a useful framework for devising powerful association tests in sequence data with rare variants

Ultrastructure of mouse vallate taste buds: III. Patterns of synaptic connectivity

We have used serial high voltage electron micrographs and computergenerated, three‐dimensional reconstructions to study morphological relationships and patterns of synaptic connectivity in taste buds from the circumvallate papillae of the mouse. The intragemmal arborizations of 40 sensory nerve fibers were examined from 7 taste buds that were sectioned serially. We identified the synaptic connections from taste cells onto the reconstructed nerve fibers and classified the presynaptic taste cells based on previously established ultrastructural criteria. From these data we were able to extract the following information for the reconstructed nerve fibers: (1) the morphology of intragemmal nerve fibers and their arborizations within the taste bud, (2) the total number of synaptic connections from taste bud cells onto the nerve fibers, and (3) the taste cell types associated with each of the synapses. Fifty‐six synapses were studied. Synapses were often found to be located at either the branch points or terminations of nerve fiber processes. The maximum number of taste cells observed to synapse onto a single nerve fiber was 5. Several nerve fibers had no apparent synapses. Dark cells (type I), intermediate cells, and light cells (type II) all formed synaptic connections with sensory nerve fibers. In no cases did dark cells and light cells synapse onto the same sensory nerve fiber. Our observation that any given nerve fiber receives its synaptic input from morphologically similar taste cells provides evidence for specificity in taste bud synaptic connections. We speculate that the observed pattern of synaptic connections is related to taste bud function. Since all of the synapses onto a given nerve fiber are from morphologically similar taste cells, we postulate that there is a correlation between taste cell morphology and sensory responsiveness. Intracellular electrophysiological studies on taste cells, in which responses to focally applied chemical stimuli are followed by characterization of the ultrastructural features of the same taste cells, will prove or disprove this hypothesis

Ultrastructure of mouse vallate taste buds: II. Cell types and cell lineage

The lifespan of cells in the mouse taste bud was examined with high‐voltage electron microscopic (HVEM) autoradiography (ARG) after giving a single injection of 3H‐thymidine. Animals were killed at 1 hour, 6 hours, 12 hours, 24 hours, and then daily up through 10 days postinjection. Lingual tissues were prepared for HVEM ARG so that we could identify and characterize labeled cells. Four categories of taste cells were identified: basal, dark, intermediate, and light cells. Basal cells were polygonal cells located near the basolateral sides of the taste buds and were characterized primarily by the presence of filaments attached to the nuclear envelope. Dark and light cells had the typical features described by previous authors. Intermediate cells had features in between those of dark and light cells. Over 90% of the cells labeled in the first 2 days following injection of 3H‐thymidine were basal cells. Labeled dark cells appeared 6 hours after injection, reached their peak incidence at the fourth day postinjection, and then gradually decreased. Labeled intermediate cells were identified after the appearance of dark cells (12 hours) and reached a peak incidence at the fifth day after injection of 3H‐thymidine. Lastly, labeled light cells were first observed on the fourth day postinjection and continued to increase until the tenth day, when they constituted 45% of the labeled cells. These data support the hypothesis that there is one cell line in the mouse vallate taste bud that undergoes morphological changes in its lifespan

Immunocytochemical analysis of P2X2 in rat circumvallate taste buds

Abstract Background Our laboratory has shown that classical synapses and synaptic proteins are associated with Type III cells. Yet it is generally accepted that Type II cells transduce bitter, sweet and umami stimuli. No classical synapses, however, have been found associated with Type II cells. Recent studies indicate that the ionotropic purinergic receptors P2X2/P2X3 are present in rodent taste buds. Taste nerve processes express the ionotropic purinergic receptors (P2X2/P2X3). P2X2/P2X3Dbl−/− mice are not responsive to sweet, umami and bitter stimuli, and it has been proposed that ATP acts as a neurotransmitter in taste buds. The goal of the present study is to learn more about the nature of purinergic contacts in rat circumvallate taste buds by examining immunoreactivity to antisera directed against the purinergic receptor P2X2. Results P2X2-like immunoreactivity is present in intragemmal nerve processes in rat circumvallate taste buds. Intense immunoreactivity can also be seen in the subgemmal nerve plexuses located below the basal lamina. The P2X2 immunoreactive nerve processes also display syntaxin-1-LIR. The immunoreactive nerves are in close contact with the IP3R3-LIR Type II cells and syntaxin-1-LIR and/or 5-HT-LIR Type III cells. Taste cell synapses are observed only from Type III taste cells onto P2X2-LIR nerve processes. Unusually large, “atypical” mitochondria in the Type II taste cells are found only at close appositions with P2X2-LIR nerve processes. P2X2 immunogold particles are concentrated at the membranes of nerve processes at close appositions with taste cells. Conclusions Based on our immunofluorescence and immunoelectron microscopical studies we believe that both perigemmal and most all intragemmal nerve processes display P2X2-LIR. Moreover, colloidal gold immunoelectron microscopy indicates that P2X2-LIR in nerve processes is concentrated at sites of close apposition with Type II cells. This supports the hypothesis that ATP may be a key neurotransmitter in taste transduction and that Type II cells release ATP, activating P2X2 receptors in nerve processes.</p

Knocking Out P2X Receptors Reduces Transmitter Secretion in Taste Buds

In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors [P2X2 and P2X3 double knock-out (DKO) mice]. The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca 2+ in taste Receptor (Type II) cells from DKO mice, as from wild-type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we used reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion