Thomas E. Finger

ATP Signaling Is Crucial for Communication from Taste Buds to Gustatory Nerves

Taste receptor cells detect chemicals in the oral cavity and transmit this information to taste nerves, but the neurotransmitter(s) have not been identified. We report that adenosine 5′-triphosphate (ATP) is the key neurotransmitter in this system. Genetic elimination of ionotropic purinergic receptors (P2X2 and P2X3) eliminates taste responses in the taste nerves, although the nerves remain responsive to touch, temperature, and menthol. Similarly, P2X-knockout mice show greatly reduced behavioral responses to sweeteners, glutamate, and bitter substances. Finally, stimulation of taste buds in vitro evokes release of ATP. Thus, ATP fulfils the criteria for a neurotransmitter linking taste buds to the nervous system.

Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration

Inhalation of irritating substances leads to activation of the trigeminal nerve, triggering protective reflexes that include apnea or sneezing. Receptors for trigeminal irritants are generally assumed to be located exclusively on free nerve endings within the nasal epithelium, requiring that trigeminal irritants diffuse through the junctional barrier at the epithelial surface to activate receptors. We find, in both rats and mice, an extensive population of chemosensory cells that reach the surface of the nasal epithelium and form synaptic contacts with trigeminal afferent nerve fibers. These chemosensory cells express T2R “bitter-taste” receptors and α-gustducin, a G protein involved in chemosensory transduction. Functional studies indicate that bitter substances applied to the nasal epithelium activate the trigeminal nerve and evoke changes in respiratory rate. By extending to the surface of the nasal epithelium, these chemosensory cells serve to expand the repertoire of compounds that can activate trigeminal protective reflexes. The trigeminal chemoreceptor cells are likely to be remnants of the phylogenetically ancient population of solitary chemoreceptor cells found in the epithelium of all anamniote aquatic vertebrates.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca2+. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

Type III cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin.

Taste buds contain a variety of morphological and histochemical types of elongate cells. Serotonin, neuron‐specific enolase (NSE), ubiquitin carboxyl terminal hydrolase (PGP 9.5), and neural cell adhesion molecule (N‐CAM) all have been described as being present in the morphologically defined Type III taste cells in rats. In order to determine whether these substances coexist in a single cell, we undertook immunohistochemical and ultrastructural analysis of taste buds in rats. Double‐label studies show that PGP 9.5 and NSE always colocalize. In contrast, PGP 9.5 and serotonin seldom colocalize. Further, whereas the serotonin‐immunoreactive cells are always slender and elongate, the PGP 9.5/NSE population comprise two morphological types — one slender, the other broader and pyriform. Although gustducin‐immunoreactive taste cells appear similar in overall shape to the pyriform PGP 9.5/NSE population, gustducin never colocalizes with PGP 9.5 or NSE. The serotonin‐immunoreactive taste cells have an invaginated nucleus, synaptic contacts with nerve fibers, and taper apically to a single, large microvillus. These are all characteristics of Type III taste cells described previously in rabbits (Murray [ 1973 ] Ultrastructure of Sensory Organs I. Amsterdam: North Holland. p 1–81). PGP 9.5‐immunoreactive taste cells exhibit two morphological varieties. One type is similar to the serotonin‐immunoreactive population, containing an invaginated nucleus, synapses with nerve fibers, and a single large microvillus. The other type of PGP 9.5‐immunoreactive taste cell has a large round nucleus and the apical end of the cell tapers to a tuft of short microvilli, which are characteristics of Type II taste cells. Thus, in rats, some Type III cells accumulate serotonin but do not express PGP 9.5, whereas others express PGP 9.5 but do not accumulate amines. Similarly, Type II taste cells come in at least two varieties: those immunoreactive for gustducin and those immunoreactive for PGP 9.5. J. Comp. Neurol. 440:97–108, 2001.

Correlation between Olfactory Receptor Cell Type and Function in the Channel Catfish

The olfactory epithelium of fish contains three intermingled types of olfactory receptor neurons (ORNs): ciliated, microvillous, and crypt. The present experiments were undertaken to test whether the different types of ORNs respond to different classes of odorants via different families of receptor molecules and G-proteins corresponding to the morphology of the ORN. In catfish, ciliated ORNs express OR-type receptors and Gαolf. Microvillous ORNs are heterogeneous, with many expressing Gαq/11, whereas crypt ORNs express Gαo. Retrograde tracing experiments show that ciliated ORNs project predominantly to regions of the olfactory bulb (OB) that respond to bile salts (medial) and amino acids (ventral) (Nikonov and Caprio, 2001). In contrast, microvillous ORNs project almost entirely to the dorsal surface of the OB, where responses to nucleotides (posterior OB) and amino acids (anterior OB) predominate. These anatomical findings are consistent with our pharmacological results showing that forskolin (which interferes with Gαolf/cAMP signaling) blocks responses to bile salts and markedly reduces responses to amino acids. Conversely, U-73122 and U-73343 (which interfere with Gαq/11/phospholipase C signaling) diminish amino acid responses but leave bile salt and nucleotide responses essentially unchanged. In summary, our results indicate that bile salt odorants are detected predominantly by ciliated ORNs relying on the Gαolf/cAMP transduction cascade. Nucleotides are detected by microvillous ORNs using neither Gαolf/cAMP nor Gαq/11/PLC cascades. Finally, amino acid odorants activate both ciliated and microvillous ORNs but via different transduction pathways in the two types of cells.

Nucleoside triphosphate diphosphohydrolase-2 is the ecto-ATPase of type I cells in taste buds.

The presence of one or more calcium‐dependent ecto‐ATPases (enzymes that hydrolyze extracellular 5′‐triphosphates) in mammalian taste buds was first shown histochemically. Recent studies have established that dominant ecto‐ATPases consist of enzymes now called nucleoside triphosphate diphosphohydrolases (NTPDases). Massively parallel signature sequencing (MPSS) from murine taste epithelium provided molecular evidence suggesting that NTPDase2 is the most likely member present in mouse taste papillae. Immunocytochemical and enzyme histochemical staining verified the presence of NTPDase2 associated with plasma membranes in a large number of cells within all mouse taste buds. To determine which of the three taste cell types expresses this enzyme, double‐label assays were performed with antisera directed against the glial glutamate/aspartate transporter (GLAST), the transduction pathway proteins phospholipase Cβ2 (PLCβ2) or the G‐protein subunit α‐gustducin, and serotonin (5HT) as markers of type I, II, and III taste cells, respectively. Analysis of the double‐labeled sections indicates that NTPDase2 immunoreactivity is found on cell processes that often envelop other taste cells, reminiscent of type I cells. In agreement with this observation, NTPDase2 was located to the same membrane as GLAST, indicating that this enzyme is present in type I cells. The presence of ecto‐ATPase in taste buds likely reflects the importance of ATP as an intercellular signaling molecule in this system. J. Comp. Neurol. 497:1–12, 2006.

Epithelial Na+ channel subunits in rat taste cells: Localization and regulation by aldosterone

Amiloride‐sensitive Na+ channels play an important role in transducing Na+ salt taste. Previous studies revealed that in rodent taste cells, the channel shares electrophysiological and pharmacological properties with the epithelial Na+ channel, ENaC. Using subunit‐specific antibodies directed against α, β, and γ subunits of rat ENaC (rENaC), we observed cytoplasmic immunoreactivity for all three subunits in nearly all taste cells of fungiform papillae, and in about half of the taste cells in foliate and vallate papillae. The intensity of labeling in cells of vallate papillae was significantly lower than that of fungiform papillae, especially for β and γ subunits. Dual localization experiments showed that immunoreactivity for the taste cell‐specific G protein, gustducin, occurs in a subset of rENaC positive taste cells.

Differential distribution of olfactory receptor neurons in goldfish: structural and molecular correlates.

The olfactory system of many terrestrial vertebrates comprises a main olfactory organ and a vomeronasal organ each containing a morphologically distinct type of olfactory receptor neuron (ORN). The two cell types also differ in the expression of G‐proteins and odorant receptor molecules. Fish do not have a vomeronasal organ, and their olfactory neurons—three different morphological types—are contained in one epithelium. The olfactory organ of goldfish appears as a rosette, with the sensory epithelium lying along the proximal portion of each lamella, where it attaches to the midline raphe. Using immunocytochemistry, in situ hybridization, and electron microscopy, we tested whether a correlation exists between receptor cell morphology, distribution of cell type within the sensory epithelium, and expression of odorant receptors and G‐proteins. A strong correlation exists between ORN morphology, type of odorant receptor and G‐protein expressed and the distribution of sensory cells within the olfactory epithelium. The Buck and Axel type of odorant receptor and Gαolf are expressed in tall ciliated ORNs distributed homogenously across the entire sensory epithelium. In contrast, microvillous ORNs expressing V2R‐like receptors, and Gαo, Gαq, or Gαi‐3, and crypt type ORNs expressing Gαo and Gαq, are preferentially located along the dorsal margin of the epithelium and near the midline raphe. V2R‐ and OR‐type receptor molecules do not colocalize in one cell, and only crypt‐type ORNs express more than one G‐protein. J. Comp. Neurol. 477:347–359, 2004.

Expression of taste receptors in Solitary Chemosensory Cells of rodent airways

BackgroundChemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs.MethodsWe utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP).ResultsEpithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways.ConclusionsElements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.

Trigeminal collaterals in the nasal epithelium and olfactory bulb: A potential route for direct modulation of olfactory information by trigeminal stimuli

The nasal epithelium is richly invested with peptidergic (substance P and calcitonin gene‐related peptide [CGRP]) trigeminal polymodal nociceptors, which respond to numerous odorants as well as irritants. Peptidergic trigeminal sensory fibers also enter the glomerular layer of the olfactory bulb. To test whether the trigeminal fibers in the olfactory bulb are collaterals of the epithelial trigeminal fibers, we utilized dual retrograde labeling techniques in rats to identify the trigeminal ganglion cells innervating each of these territories. Nuclear Yellow was injected into the dorsal nasal epithelium, and True Blue was injected into the olfactory bulb of the same side. Following a survival period of 3–7 days, the trigeminal ganglion contained double‐labeled, small (11.8 × 8.0 μm), ellipsoid ganglion cells within the ethmoid nerve region of the ganglion. Tracer injections into the spinal trigeminal complex established that these branched trigeminal ganglion cells also extended an axon into the brainstem. These results indicate that some trigeminal ganglion cells with sensory endings in the nasal epithelium also have branches reaching directly into both the olfactory bulb and the spinal trigeminal complex. These trigeminal ganglion cells are unique among primary sensory neurons in having two branches entering the central nervous system at widely distant points. Furthermore, the collateral innervation of the epithelium and bulb may provide an avenue whereby nasal irritants could affect processing of coincident olfactory stimuli. J. Comp. Neurol. 444:221–226, 2002. Erratum: J. Comp. Neurol. 2002;448(4):423.© 2002 Wiley‐Liss, Inc.