A Physiologic Role for Serotonergic Transmission in Adult Rat Taste Buds

Of the multiple neurotransmitters and neuropeptides expressed in the mammalian taste bud, serotonin remains both the most studied and least understood. Serotonin is expressed in a subset of taste receptor cells that form synapses with afferent nerve fibers (type III cells) and was once thought to be essential to neurotransmission (now understood as purinergic). However, the discovery of the 5-HT_1A serotonin receptor in a subset of taste receptor cells paracrine to type III cell suggested a role in cell-to-cell communication during the processing of taste information. Functional data describing this role are lacking. Using anatomical and neurophysiological techniques, this study proposes a modulatory role for serotonin during the processing of taste information. Double labeling immunocytochemical and single cell RT-PCR technique experiments documented that 5-HT_1A-expressing cells co-expressed markers for type II cells, cells which express T1R or T2R receptors and release ATP. These cells did not co-express type III cells markers. Neurophysiological recordings from the chorda tympani nerve, which innervates anterior taste buds, were performed prior to and during intravenous injection of a 5-HT_1A receptor antagonist. These experiments revealed that serotonin facilitates processing of taste information for tastants representing sweet, sour, salty, and bitter taste qualities. On the other hand, injection of ondansetron, a 5-HT₃ receptor antagonist, was without effect. Collectively, these data support the hypothesis that serotonin is a crucial element in a finely-tuned feedback loop involving the 5-HT_1A receptor, ATP, and purinoceptors. It is hypothesized that serotonin facilitates gustatory signals by regulating the release of ATP through ATP-release channels possibly through phosphatidylinositol 4,5-bisphosphate resynthesis. By doing so, 5-HT_1A activation prevents desensitization of post-synaptic purinergic receptors expressed on afferent nerve fibers and enhances the afferent signal. Serotonin may thus play a major modulatory role within peripheral taste in shaping the afferent taste signals prior to their transmission across gustatory nerves.     

Tachykinins stimulate a subset of mouse taste cells

The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+)-imaging on isolated taste cells, it was observed that SP induces Ca(2+) -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+)-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+)-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+)-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+) responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.     

Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25

Background  

Taste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC) signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca²⁺ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP) from two taste-specific promoters to examine Ca²⁺ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca²⁺ currents were assessed with Ca²⁺ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells.     

β-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of β-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, β-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of β-catenin in these Shh⁺ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where β-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells.     

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.     

Expression of gustducin overlaps with that of type III IP3 receptor in taste buds of the rat soft palate

Type III IP3 receptor (IP3R3) is one of the common critical calcium-signaling molecules for sweet, umami, and bitter signal transduction in taste cells, and the total IP3R3-expressing cell population represents all cells mediating these taste modalities in the taste buds. Although gustducin, a taste cell-specific G-protein, is also involved in sweet, umami, and bitter signal transduction, the expression of gustducin is restricted to different subsets of IP3R3-expressing cells by location in the tongue. Based on the expression patterns of gustducin and taste receptors in the tongue, the function of gustducin has been implicated primarily in bitter taste in the circumvallate (CV) papillae and in sweet taste in the fungiform (FF) papillae. However, in the soft palate (SP), the expression pattern of gustducin remains unclear and little is known about its function. In the present paper, the expression patterns of gustducin and IP3R3 in taste buds of the SP and tongue papillae in the rat were examined by double-color whole-mount immunohistochemistry. Gustducin was expressed in almost all (96.7%) IP3R3-expressing cells in taste buds of the SP, whereas gustducin-positive cells were 42.4% and 60.1% of IP3R3-expressing cells in FF and CV, respectively. Our data suggest that gustducin is involved in signal transduction of all the tastes of sweet, umami, and bitter in the SP, in contrast to its limited function in the tongue.     

Abnormal taste perception in mice lacking the type 3 inositol 1,4,5-trisphosphate receptor

Inositol 1,4,5-trisphosphate receptor (IP3R) is one of the important calcium channels expressed in the endoplasmic reticulum and has been shown to play crucial roles in various physiological phenomena. Type 3 IP3R is expressed in taste cells, but the physiological relevance of this receptor in taste perception in vivo is still unknown. Here, we show that mice lacking IP3R3 show abnormal behavioral and electrophysiological responses to sweet, umami, and bitter substances that trigger G-protein-coupled receptor activation. In contrast, responses to salty and acid tastes are largely normal in the mutant mice. We conclude that IP3R3 is a principal mediator of sweet, bitter, and umami taste perception and would be a missing molecule linking phospholipase C beta2 to TRPM5 activation.     

Cytokeratin 14 is expressed in immature cells in rat taste buds

We analyzed the differentiation of taste bud cells, by precisely describing expression profiles of cytokeratins (CKs) 8 and 14 in relation to those of marker molecules including label of 5-bromo-2′-deoxy uridine (BrdU) injected. In rat circumvallate papillae, cell division was observed at the basal layer of the epithelium expressing CK14 and located outside taste buds. The progenitor cells began to migrate toward the apical surface and maintained CK14 expression at 1 day after BrdU injection (day 1). On the other hand, a minor population of newly divided cells was infrequently incorporated into taste buds and also maintained CK14 expression at day 1. In taste buds, the conversion of CK subtypes occurred from CK14 to cytokeratin 8 (CK8) at day 2–3, showing the differentiation from immature cells expressing CK14 into mature or maturing cells expressing CK8. Functionally matured cells such as taste receptor cells expressing inositol triphospate receptor type 3 (IP₃R3) never expressed CK14, suggesting that CK14 would be expressed only in immature cells. On the other hand, a small but distinct population of BrdU-positive cells still showed CK14 immunoreactivity in taste buds even at day 12, which might correspond to the cells that remain undifferentiated for a long period within taste buds.     

Expression of Genes Encoding Multi-Transmembrane Proteins in Specific Primate Taste Cell Populations

Background

Using fungiform (FG) and circumvallate (CV) taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed.

Methodology/Principal Findings

Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive), sour cells (PKD2L1-positive), as well as other taste cell populations. Transmembrane protein 44 (TMEM44), a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1), a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1), a calcium-binding transmembrane protein; and anoctamin 7 (ANO7), a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B), a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release.

Conclusions/Significance

Identification of genes encoding multi-transmembrane domain proteins expressed in primate taste buds provides new insights into the processes of taste cell development, signal transduction, and information coding. Discrete taste cell populations exhibit highly specific gene expression patterns, supporting a model whereby each mature taste receptor cell is responsible for sensing, transmitting, and coding a specific taste quality.     

Faithful expression of GFP from the PLCbeta2 promoter in a functional class of taste receptor cells

Phospholipase C-type beta2 (PLCbeta2) is expressed in a subset of cells within mammalian taste buds. This enzyme is involved in the transduction of sweet, bitter, and umami stimuli and thus is believed to be a marker for gustatory sensory receptor cells. We have developed transgenic mice expressing green fluorescent protein (GFP) under the control of the PLCbeta2 promoter to enable one to identify these cells and record their physiological activity in living preparations. Expression of GFP (especially in lines with more than one copy integrated) is strong enough to be detected in intact tissue preparations using epifluorescence microscopy. By immunohistochemistry, we confirmed that the overwhelming majority of cells expressing GFP are those that endogenously express PLCbeta2. Expression of the GFP transgene in circumvallate papillae occurs at about the same time during development as endogenous PLCbeta2 expression. When loaded with a calcium-sensitive dye in situ, GFP-positive taste cells produce typical Ca2+ responses to a taste stimulus, the bitter compound cycloheximide. These PLCbeta2 promoter-GFP transgenic lines promise to be useful for studying taste transduction, sensory signal processing, and taste bud development.     

Gustatory sensory cells express a receptor responsive to protein breakdown products (GPR92)

The ingestion of dietary protein is of vital importance for the maintenance of fundamental physiological processes. The taste modality umami, with its prototype stimulus, glutamate, is considered to signal the protein content of food. Umami was thought to be mediated by the heterodimeric amino acid receptor, T1R1 + T1R3. Based on knockout studies, additional umami receptors are likely to exist. In addition to amino acids, certain peptides can also elicit and enhance umami taste suggesting that protein breakdown products may contribute to umami taste. The recently deorphanized peptone receptor, GPR92 (also named GPR93; LPAR5), is expressed in gastric enteroendocrine cells where it responds to protein hydrolysates. Therefore, it was of immediate interest to investigate if the receptor GPR92 is expressed in gustatory sensory cells. Using immunohistochemical approaches we found that a large population of cells in murine taste buds was labeled with an GPR92 antibody. A molecular phenotyping of GPR92 cells revealed that the vast majority of GPR92-immunoreactive cells express PLCβ2 and can therefore be classified as type II cells. More detailed analyses have shown that GPR92 is expressed in the majority of T1R1-positive taste cells. These results indicate that umami cells may respond not only to amino acids but also to peptides in protein hydrolysates.     

Tastants evoke cAMP signal in taste buds that is independent of calcium signaling

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca²⁺. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca²⁺. Specifically, 5 µM thapsigargin or 3 µM A-23187 (calcium ionophore), both of which increase intracellular Ca²⁺ concentration ([Ca²⁺]_i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca²⁺, suggesting that it is dependent on Ca²⁺ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase C₂ (i.e., cells that elevate [Ca²⁺]_i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca²⁺ may represent independent second messenger signals downstream of taste receptors. calcium-sensitive adenylyl cyclase; capacitative entry; cross talk; taste transduction     

Labeling afferent nerves in the tongue by peripheral transganglionic transport of HRP and WGA-HRP in the rat

Peripheral transganglionic transport of horseradish pcroxidase(HRP) and wheat germ agglutinin–horseradish peroxidaseconjugate (WGA–HRP) was used to label afferent fibersin the taste buds and lingual epithelium of the rat. Microinjectionsof the tracer were made in the brain stem central projectionarea of the afferent nerves to the tongue. Optimal labelingof nerve endings in the tongue was obtained when 2 µlof 20% HRP was injected into the brain stem and postinjectionsurvival times of 24–36 h were used. The distributionof single nerves was studied by using this tracing procedurein combination with strategic transections of the various afferentnerves supplying the tongue. Labeled nerve fibers from the combinedchorda tympani–lingual nerve were found in the epitheliumand in taste buds in the fungiform and anterior foliate papillaeof the anterior 3/4 of the tongue. Labeled nerve fibers in theepithelium of the anterior 2/3 of the tongue but none in tastebuds were found when the lingual nerve alone was studied, althoughnumerous perigeminal fibers were found. The glossopharyngealnerve was found to innervate die posterior 1/4 of the tongueepithelium including the taste buds of the circumvallate papillae.The glossopharyngeal nerve on one side was found to innervatethe taste buds on both sides of the midline. The results showthat this tracing procedure can be a useful supplement to othermethods for studying afferent nerves in the tongue.     

Are there efferent synapses in fish taste buds?

In fish, nerve fibers of taste buds are organized within the bud's nerve fiber plexus. It is located between the sensory epithelium consisting of light and dark elongated cells and the basal cells. It comprises the basal parts and processes of light and dark cells that intermingle with nerve fibers, which are the dendritic endings of the taste sensory neurons belonging to the cranial nerves VII, IX or X. Most of the synapses at the plexus are afferent; they have synaptic vesicles on the light (or dark) cells side, which is presynaptic. In contrast, the presumed efferent synapses may be rich in synaptic vesicles on the nerve fibers (presynaptic) side, whereas the cells (postsynaptic) side may contain a subsynaptic cistern; a flat compartment of the smooth endoplasmic reticulum. This structure is regarded as a prerequisite of a typical efferent synapse, as occurring in cochlear and vestibular hair cells. In fish taste buds, efferent synapses are rare and were found only in a few species that belong to different taxa. The significance of efferent synapses in fish taste buds is not well understood, because efferent connections between the gustatory nuclei of the medulla with taste buds are not yet proved.     

Expression of T1Rs and gustducin in palatal taste buds of mice

The palatal region of the oral cavity in rodents houses 100-300 taste buds and is particularly sensitive to sweet and umami compounds; yet, few studies have examined the expression patterns of transduction-related molecules in this taste field. We investigated the interrelationships between members of the T1R family and between each T1R and gustducin in palatal taste buds. Similar to lingual taste buds, T1R1 and T1R2 are generally expressed in separate palatal taste cells. In contrast to lingual taste buds, however, T1R2 and T1R3-positive palatal taste cells almost always coexpress gustducin, suggesting that sweet taste transduction in the palate is almost entirely dependent on gustducin. T1R1-positive palate taste cells coexpress gustducin about half the time, suggesting that other G proteins may contribute to the transduction of umami stimuli in this taste field.     

Genetic tracing of the neural pathway for bitter taste in t2r5-WGA transgenic mice

To visualize the neural pathways originating from bitter taste receptor cells (TRCs), we generated transgenic mice expressing the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse T2R5 gene promoter/enhancer (t2r5-WGA mice). WGA mRNA was specifically expressed in bitter TRCs. The WGA protein was detected in bitter TRCs and nerve processes in taste buds, but not in sweet, umami, or sour TRCs. The WGA protein was transferred to a subset of sensory neurons in the geniculate and nodose/petrosal ganglia. These results suggest that bitter TRCs, which are devoid of synaptic structures, are innervated by gustatory neurons and that bitter sensory information is directly transmitted to specific gustatory neurons. The t2r5-WGA mice provide a useful tool for identifying gustatory relay neurons in the peripheral sensory ganglia responsible for aversive sensations.