Taste Bud Connectome: Implications for Taste Information Processing.

Taste buds contain multiple cell types, two of which mediate transduction of specific taste qualities: Type III cells transduce sour while Type II cells transduce either sweet, or bitter or umami. In order to discern the degree of interaction between different cell types and specificity of connectivity with the afferent nerve fibers (NFs), we employed serial blockface scanning electron microscopy (sbfSEM) through five circumvallate mouse taste buds. Points of contact between Type II and Type III cells are rare and lack morphologically identifiable synapses, suggesting that interaction between these cell types does not occur via synapses. Of the 127 NFs that make synaptic contacts with taste cells in the sampling volume, ∼70% (n = 91) synapse with only one taste cell while 32 fibers synapse exclusively with multiple Type II cells or multiple Type III cells. Our data do not rule out multimodal fibers innervating Type II cells of separate taste qualities. Notably, four fibers (∼3%) synapse with both Type II and Type III cells, forming both mitochondrial and vesicular synapses on the different cell types. Since Type II and Type III cells transduce different taste qualities, these dual connected fibers are not consistent with a absolute labeled-line encoding system. Further, our data reveal considerable variation in both the number of synapses per cell/nerve pair and the number of innervating NFs per taste cell, both of which likely have consequences for encoding taste quality and concentration. Finally, we identify a subset of Type II cells which may represent an immature stage.SIGNIFICANCE STATEMENT Taste buds, the sensory end organs for the sense of taste, contain multiple types of sensory cells, with each responding to one of the primary tastes: salt, sweet, sour, bitter, and umami. In order to determine the degree of interaction between cell types and specificity of connectivity to afferent nerves, we employed serial blockface electron microscopy (EM) of mouse circumvallate taste buds. We find no synapses between cell types within the taste bud suggesting that any interactions are indirect. While the majority of nerve fibers (NFs) connect to a single type of taste cell, 3.1% of the fibers branch to receive input from taste cells of different specificities. Thus, taste cannot entirely be carried along NFs dedicated to single taste qualities.

Death in the taste bud: Morphological features of dying taste cells and engulfment by Type I cells.

Taste buds comprise 50-100 epithelial derived cells, which are renewed throughout the life of an organism. Immature cells enter the bud at its base, maturing into one of three distinct cell types. How taste cells die and/or exit the bud, however, remains unclear. Here we present morphological data obtained through Serial Blockface Scanning Electron Microscopy of murine circumvallate taste buds, revealing several taste cells at the end of their life (4-6 per bud). Cells we identify as dying share certain morphological features typical of apoptosis: swollen endoplasmic reticulum, large lysosomes, degrading organelles, distended outer nuclear membranes, heterochromatin reorganization, cell shrinkage, and cell and/or nuclear fragmentation. Based on these features, we divide the cells into "early" and "late" stage dying cells. Most early stage dying cells have Type II cell morphologies, while a few display Type III cell features. Many dying cells maintain contacts with nerve fibers, but those fibers often appear detached from the main trunk of an afferent nerve fiber. Dying cells, like mature Type II and Type III taste cells, are surrounded by Type I taste cells, the glial-like cells of the bud. In many instances Type I cells appear to be engulfing their dying neighbors, suggesting a novel, phagocytic role for Type I cells. Surprisingly, virtually no Type I cells, which have the shortest residence time in taste buds, display features of apoptosis. The ultimate fate of Type I cells therefore remains enigmatic.

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.

Immunocytochemical analysis of P2X2 in rat circumvallate taste buds.

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/P2X3(Dbl-/-) 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 IP(3)R3-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.

Three-dimensional reconstructions of mouse circumvallate taste buds using serial blockface scanning electron microscopy: I. Cell types and the apical region of the taste bud.

Taste buds comprise four types of taste cells: three mature, elongate types, Types I-III; and basally situated, immature postmitotic type, Type IV cells. We employed serial blockface scanning electron microscopy to delineate the characteristics and interrelationships of the taste cells in the circumvallate papillae of adult mice. Type I cells have an indented, elongate nucleus with invaginations, folded plasma membrane, and multiple apical microvilli in the taste pore. Type I microvilli may be either restricted to the bottom of the pore or extend outward reaching midway up into the taste pore. Type II cells (aka receptor cells) possess a large round or oval nucleus, a single apical microvillus extending through the taste pore, and specialized "atypical" mitochondria at functional points of contact with nerve fibers. Type III cells (aka "synaptic cells") are elongate with an indented nucleus, possess a single, apical microvillus extending through the taste pore, and are characterized by a small accumulation of synaptic vesicles at points of contact with nerve fibers. About one-quarter of Type III cells also exhibit an atypical mitochondrion near the presynaptic vesicle clusters at the synapse. Type IV cells (nonproliferative "basal cells") have a nucleus in the lower quarter of the taste bud and a foot process extending to the basement membrane often contacting nerve processes along the way. In murine circumvallate taste buds, Type I cells represent just over 50% of the population, whereas Types II, III, and IV (basal cells) represent 19, 15, and 14%, respectively.

Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex.

Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-µm) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca2+ upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles.

Immunocytochemical analysis of syntaxin-1 in rat circumvallate taste buds.

Mammalian buds contain a variety of morphological taste cell types, but the type III taste cell is the only cell type that has synapses onto nerve processes. We hypothesize that taste cell synapses utilize the SNARE protein machinery syntaxin, SNAP-25, and synaptobrevin, as is used by synapses in the central nervous system (CNS) for Ca2+-dependent exocytosis. Previous studies have shown that taste cells with synapses display SNAP-25- and synaptobrevin-2-like immunoreactivity (LIR) (Yang et al. [2000a] J Comp Neurol 424:205-215, [2004] J Comp Neurol 471:59-71). In the present study we investigated the presynaptic membrane protein, syntaxin-1, in circumvallate taste buds of the rat. Our results indicate that diffuse cytoplasmic and punctate syntaxin-1-LIR are present in different subsets of taste cells. Diffuse, cytoplasmic syntaxin-1-LIR is present in type III cells while punctate syntaxin-1-LIR is present in type II cells. The punctate syntaxin-1-LIR is believed to be associated with Golgi bodies. All of the synapses associated with syntaxin-1-LIR taste cells are from type III cells onto nerve processes. These results support the proposition that taste cell synapses use classical SNARE machinery such as syntaxin-1 for neurotransmitter release in rat circumvallate taste buds.

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 PLCbeta2, alpha-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 PLCbeta2 (31.8% vs 19.6%), alpha-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 microm3) is smaller than a rat taste bud (64,200 microm3). The numerical density of taste cells in mouse circumvallate taste buds (2.1 cells/1000 microm3) is significantly higher than that in the rat (1.2 cells/1000 microm3). 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.

Synaptobrevin-2-like immunoreactivity is associated with vesicles at synapses in rat circumvallate taste buds.

Synaptobrevin is a vesicle-associated membrane protein (VAMP) that is believed to play a critical role with presynaptic membrane proteins (SNAP-25 and syntaxin) during regulated synaptic vesicle docking and exocytosis of neurotransmitter at the central nervous system. Synaptic contacts between taste cells and nerve processes have been found to exist, but little is known about synaptic vesicle docking and neurotransmitter release at taste cell synapses. Previously we demonstrated that immunoreactivity to SNAP-25 is present in taste cells with synapses. Our present results show that synaptobrevin-2-like immunoreactivity (-LIR) is present in approximately 35% of the taste cells in rat circumvallate taste buds. Synaptobrevin-2-LIR colocalizes with SNAP-25-, serotonin-, and protein gene product 9.5-LIR. Synaptobrevin-2-LIR also colocalizes with immunoreactivity for type III inositol 1,4,5-triphosphate receptor (IP3R3), a taste-signaling molecule in taste cells. All IP3R3-LIR taste cells express synaptobrevin-2-LIR. However, approximately 27% of the synaptobrevin-2-LIR taste cells do not display IP3R3-LIR. We believe, based on ultrastructural and biochemical features, that both type II and type III taste cells display synaptobrevin-2-LIR. All of the synapses that we observed from taste cells onto nerve processes express synaptobrevin-2-LIR, as well as some taste cells without synapses. By using colloidal gold immunoelectron microscopy, we found that synaptobrevin-2-LIR is associated with synaptic vesicles at rat taste cell synapses. The results of this study suggest that soluble NSF attachment receptor (SNARE) machinery may control synaptic vesicle fusion and exocytosis at taste cell synapses.

Morphologic characterization of rat taste receptor cells that express components of the phospholipase C signaling pathway.

Rat taste buds contain three morphologically distinct cell types that are candidates for taste transduction. The physiologic roles of these cells are, however, not clear. Inositol 1,4,5-triphosphate (IP(3)) has been implicated as an important second messenger in bitter, sweet, and umami taste transductions. Previously, we identified the type III IP(3) receptor (IP(3)R3) as the dominant isoform in taste receptor cells. In addition, a recent study showed that phospholipase Cbeta(2) (PLCbeta(2)) is essential for the transduction of bitter, sweet, and umami stimuli. IP(3)R3 and PLCbeta(2) are expressed in the same subset of cells. To identify the taste cell types that express proteins involved in PLC signal transduction, we used 3,3'diaminobenzidine tetrahydrochloride immunoelectron microscopy and fluorescence microscopy to identify cells with IP(3)R3. Confocal microscopy was used to compare IP(3)R3 or PLCbeta(2) immunoreactivity with that of some known cell type markers such as serotonin, protein gene-regulated product 9.5, and neural cell adhesion molecule. Here we show that a large subset of type II cells and a small subset of type III cells display IP(3)R3 immunoreactivity within their cytoplasm. These data suggest that type II cells are the principal transducers of bitter, sweet, and umami taste transduction. However, we did not observe synapses between type II taste cells and nerve fibers. Interestingly, we observed subsurface cisternae of smooth endoplasmic reticulum at the close appositions between the plasma membrane of type II taste cells and nerve processes. We speculate that some type II cells may communicate to the nervous system via subsurface cisternae of smooth endoplasmic reticulum in lieu of conventional synapses.

Microwave processing of gustatory tissues for immunohistochemistry.

We use immunohistochemistry to study taste cell structure and function as a means to elucidate how taste receptor cells communicate with nerve fibers and adjacent taste cells. This conventional method, however, is time consuming. In the present study we used taste buds from rat circumvallate papillae to compare conventional immunohistochemical tissue processing with microwave processing for the colocalization of several biochemical pathway markers (PLCß2, syntaxin-1, IP3R3, α-gustducin) and the nuclear stain, Sytox. The results of our study indicate that in microwave versus conventional immunocytochemistry: (1) fixation quality is improved; (2) the amount of time necessary for processing tissue is decreased; (3) antigen retrieval is no longer needed; (4) image quality is superior. In sum, microwave tissue processing of gustatory tissues is faster and superior to conventional immunohistochemical tissue processing for many applications.

The candidate sour taste receptor, PKD2L1, is expressed by type III taste cells in the mouse.

The transient receptor potential channel, PKD2L1, is reported to be a candidate receptor for sour taste based on molecular biological and functional studies. Here, we investigated the expression pattern of PKD2L1-immunoreactivity (IR) in taste buds of the mouse. PKD2L1-IR is present in a few elongate cells in each taste bud as reported previously. The PKD2L1-expressing cells are different from those expressing PLCbeta2, a marker of Type II cells. Likewise PKD2L1-immunoreactive taste cells do not express ecto-ATPase which marks Type I cells. The PKD2L1-positive cells are immunoreactive for neural cell adhesion molecule, serotonin, PGP-9.5 (ubiquitin carboxy-terminal transferase), and chromogranin A, all of which are present in Type III taste cells. At the ultrastructural level, PKD2L1-immunoreactive cells form synapses onto afferent nerve fibers, another feature of Type III taste cells. These results are consistent with the idea that different taste cells in each taste bud perform distinct functions. We suggest that Type III cells are necessary for transduction and/or transmission of information about "sour", but have little or no role in transmission of taste information of other taste qualities.

A three dimensional serial reconstruction of neuronal distributions in the hypostome of a Hydra.

The numbers, types, and distributions of neurons in a hypostome of Hydra littoralis were determined from electron micrographs of serial (0.25 µm thick) sections. In 1,080 serial sections examined we found 75 sensory cells and 949 centrally located ganglion cells. More than 96% of the 1,024 neurons identified had a single cilium. Sensory cells were most numerous near the apex of the hypostome. Proceeding away from the apex, they steadily decreased in numbers; at 120 µm they were no longer observed. Ganglion cells were bimodally distributed; some were associated with sensory cells at the apex, but most were found at the sites of tentacle origin. We observed, throughout the hypostome, a total of 64 neuronal clusters (three or more contiguous neurons), with an average of five and a maximum of 11 neurons in a cluster. Clusters were distributed similarly to ganglion cells: an initial concentration of clusters near the apex; the majority at the hypostometentacle junctions. Each neuron identified was traced through succeeding sections in which it was observed. We used a three coordinate system to create a three-dimensional reconstruction of the neuronal locations in the hypostome. Although the functional significance of the neuronal distributions we observed is unknown, we suggest that neurons at the apex of the hypostome transduce sensory information involved in feeding behavior. The neuronal concentrations at sites of tentacle origin may be responsible for initiating Contraction Burst Pulses associated with rhythmic behavioral patterns of Hydra or coordinating tentacle movements involved in prey capture, ingestion or locomotion.