Ontogeny of the Solitary Chemosensory Cells in the Zebrafish, Danio rerio

Secondary epidermal solitary chemosensory cells (SCCs) are widespreadamong the primary aquatic vertebrates. They resemble taste budsensory cells in fine structure and may be innervated from facialor spinal nerves. According to previous studies, SCCs may constitutea water sampling system in the contexts of predator avoidance,habitat recognition and, in some cases, finding food. By quantitativescanning (SEM) and transmission electron microscopy (TEM) in60 specimens (57 SEM, 3 TEM) of 16 developmental stages, frompre-hatchlings to adults, we describe the ontogenetic developmentof SCC densities and shapes of sensory apices in the zebrafish,Danio rerio. This is put into perspective with the ontogenyof external taste buds. Just prior to hatching, 3 days afterfertilization (3d AF), sensory apices of SCCs penetrate betweenthe squamous epidermal cells, whereas taste bud pores only appearat the onset of exogenous feeding (5d AF). SCC densities increasesharply from hatching shortly after metamorphosis (25d AF) upto 6 x 10³ per mm² on the head and remain relatively constantin density thereafter. Conservatively estimated, there may be     

Chemoreception Regulates Chemical Access to Mouse Vomeronasal Organ: Role of Solitary Chemosensory Cells

Controlling stimulus access to sensory organs allows animals to optimize sensory reception and prevent damage. The vomeronasal organ (VNO) detects pheromones and other semiochemicals to regulate innate social and sexual behaviors. This semiochemical detection generally requires the VNO to draw in chemical fluids, such as bodily secretions, which are complex in composition and can be contaminated. Little is known about whether and how chemical constituents are monitored to regulate the fluid access to the VNO. Using transgenic mice and immunolabeling, we found that solitary chemosensory cells (SCCs) reside densely at the entrance duct of the VNO. In this region, most of the intraepithelial trigeminal fibers innervate the SCCs, indicating that SCCs relay sensory information onto the trigeminal fibers. These SCCs express transient receptor potential channel M5 (TRPM5) and the phospholipase C (PLC) β2 signaling pathway. Additionally, the SCCs express choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) for synthesizing and packaging acetylcholine, a potential transmitter. In intracellular Ca²⁺ imaging, the SCCs responded to various chemical stimuli including high concentrations of odorants and bitter compounds. The responses were suppressed significantly by a PLC inhibitor, suggesting involvement of the PLC pathway. Further, we developed a quantitative dye assay to show that the amount of stimulus fluid that entered the VNOs of behaving mice is inversely correlated to the concentration of odorous and bitter substances in the fluid. Genetic knockout and pharmacological inhibition of TRPM5 resulted in larger amounts of bitter compounds entering the VNOs. Our data uncovered that chemoreception of fluid constituents regulates chemical access to the VNO and plays an important role in limiting the access of non-specific irritating and harmful substances. Our results also provide new insight into the emerging role of SCCs in chemoreception and regulation of physiological actions.     

Expression of catfish amino acid taste receptors inXenopus oocytes

We demonstrate that poly (A⁺)RNA isolated from catfish barbels directs the expression of functional amino acid taste receptors in theXenopus oocyte. The activity of these receptors is monitored in ovo by the two electrode voltage clamp technique. Specific conductance changes recorded in response to amino acid stimulation are analogous to those recorded electrophysiologically from intact catfish barbels. These responses exhibit specificity, reproducibility, rapid onset and termination, and desensitization to repetitive stimulation. A functional assay system that encompasses the full complement of transduction events from the ligand-receptor interaction to subsequent conductance changes is necessary to identify molecular components responsible for these events. Our results demonstrate that theXenopus oocyte can be used to characterize and identify clones coding for amino acid taste receptors analogous to its use in studying receptor molecules for other neuroactive compounds.Special issue is dedicated to Dr. Sidney Udenfriend.     

Expression of genes coding melatonin and serotonin receptors in rodent skin

Targeted search for expression of melatonin and serotonin receptors genes in the skin of C57BL/6J mice showed expression of MT1B (but not MT1A). Mouse skin and hamster melanomas also expressed 5HT2B and 5HT7. We identified two novel isoforms of MT1A and 5HT7.     

Specific receptors for prostaglandins in airways

The relative bronchomotor activities of prostaglandins (PG) E1, E2, F2 alpha, F2 beta and I2 and of three synthetic E prostaglandin analogues (TR4161, TR4367 and TR4752) were determined on a large number of isolated preparations of guinea-pig trachea and human bronchial muscle. Each prostaglandin was capable of eliciting both contraction and relaxation, the relative incidence of these responses partly depending on concentration. TR4161 was a virtually pure relaxant; TR4367 was virtually devoid of bronchomotor activity; and TR4752 was a potent relaxant, devoid of contractant activity. The results also provided distinct rank orders of approximate potency for contraction and relaxation. Tachyphylaxis to the relaxant activities of PGE1 and TR4752 confirmed the underlying contractant activity of the two natural E prostaglandins. Antagonism with a high dose of indomethacin of the contractant actions of PGE1, PGE2 and PGF2 alpha confirmed the presence of relaxant activities in each. Inhaled aerosols of the same natural and synthetic prostaglandins were evaluated for irritant activity on the airways, using the cough response of the restrained conscious cat. All of them, except TR4161, elicited severe coughing. The rank order of potencies for irritancy differed from those for tracheobronchial contractant and relaxant activities. These findings suggest that the three responses studied arise from the activation of three distinct PG receptors in the airways. We propose the terms chi (contractant), psi (relaxant) and omega (irritant) for these putative receptors for prostaglandins or possibly other prostanoids.     

Specific receptors for prostaglandins in airways

The relative bronchomotor activities of prostaglandins (PG) E₁, E₂, F_2α, F_2β and I₂ and of three synthetic E prostaglandin analogues (TR4161, TR4367 and TR4752) were determined on a large number of isolated preparations of guinea-pig trachea and human bronchial muscle. Each prostaglandin was capable of eliciting both contraction and relaxation, the relative incidence of these responses partly depending on concentration. TR4161 was a virtually pure relaxant; TR4367 was virtually devoid of bronchomotor activity; and TR4752 was a potent relaxant, devoid of contractant activity. The results also provided distinct rank orders of approximate potency for contraction and relaxation. Tachyphylaxis to the relaxant activities of PGE₁ and TR4752 confirmed the underlying contractant activity of the two natural E prostaglandins. Antagonism with a high dose of indomethacin of the contractant actions of PGE₁, PGE₂ and PGF_2α confirmed the presence of relaxant activities in each.Inhaled aerosols of the same natural and synthetic prostaglandins were evaluated for irritant activity on the airways, using the cough response of the restrained conscious cat. All of them, except TR4161, elicited severe coughing. The rank order of potencies for irritancy differed from those for tracheobronchial contractant and relaxant activities.These findings suggest that the three responses studied arise from the activation of three distinct PG receptors in the airways. We propose the terms χ (contractant), ψ (relaxant) and ω (irritant) for these putative receptors for prostaglandins or possibly other prostanoids.     

Chemosensory control of pollen ingestion in the hoverfly Eristalis tenax by labellar taste hairs

The labellar gustatory system of the dronefly Eristalis tenax L. (Syrphidae; Diptera) that enables the fly to discriminate between pollen and nectar is investigated, and the triggering of pollen ingestion is examined. In behavioural preference tests, exhaustively extracted pollen of the sunflower Helianthus annuus is consumed in smaller amounts than untreated pollen, indicating that water-soluble substances are important for acceptance. Dry pollen is preferred over moist pollen in which the grains stick together, suggesting that mechanical properties of the pollen also play a role in its sensory assessment. Electrophysiological studies of the labellar taste hairs reveal that aqueous extracts of pollen (2% w/v) stimulate the salt receptor cell, but not the sugar receptor cell. The response of the water receptor cell remains the same as to pure water (or standard electrolyte, 10 mmol · l⁻¹ KCl). Of the 20 amino acids tested, the salt cell is sensitive only to proline in a submillimolar range. Behavioural experiments support the electrophysiological findings. When KCl is applied at concentrations eliciting salt-cell spike frequencies equal to those produced by pollen extract (which is often accepted), the water receptor cell is inhibited and a pronounced rejection behaviour occurs. This rejection of concentrated salt solution in Eristalis is therefore mainly mediated by the inhibition of the water cell. Accepted: 27 November 1999     

Dormancy of Solitary Metastatic Cells

After arriving in a secondary site metastatic cells either begin proliferating, undergo apoptosis or remain as solitary dormant cells. The process of metastasis, although dangerous, is extremely inefficient with the majority of the cells undergoing apoptosis and thus becoming clinically irrelevant. Of the cells that begin proliferating, the few that make it past the micrometastasis stage may be of immediate clinical relevance. Dormant cells, while not of immediate clinical concern, are believed to be at least in part responsible for cancer recurrence that can occur decades after apparently successful initial treatment. Dormant solitary cells are different from “dormant” micrometastases, in which active proliferation is balanced by apoptosis. The mechanisms of cell cycle regulation and the function of the molecules regulating this process are well understood. However, there is relatively little known about the mechanisms controlling cell cycle regulation and dormancy of solitary metastatic cells. There are several inherent difficulties impeding the study of solitary cells. This review paper will examine the models used in the study of dormant solitary metastatic cells, methods of imaging and studying these cells, the molecular mechanisms believed to be responsible for solitary cell dormancy, and finally the unique treatment challenges posed by these cells.     

Molecular receptors of taste agents

All representatives of higher eukaryotes can probably differentially perceive nutrients and poisonous substances. Molecular mechanisms of transduction of taste information have been best studied for mammals and for the fruit fly Drosophila. Here, we consider receptor mechanisms and conjugated primary signal processes of stimulation of taste receptor cells by stimuli of various taste modalities.     

Channels as taste receptors in vertebrates

Taste reception is fundamental for proper selection of food and beverages. Chemicals detected as taste stimuli by vertebrates include a large variety of substances, ranging from inorganic ions (e.g., Na⁺, H⁺) to more complex molecules (e.g., sucrose, amino acids, alkaloids). Specialized epithelial cells, called taste receptor cells (TRCs), express specific membrane proteins that function as receptors for taste stimuli. Classical view of the early events in chemical detection was based on the assumption that taste substances bind to membrane receptors in TRCs without permeating the tissue. Although this model is still valid for some chemicals, such as sucrose, it does not hold for small ions, such as Na⁺, that actually diffuse inside the taste tissue through ion channels. Electrophysiological, pharmacological, biochemical, and molecular biological studies have provided evidence that indeed TRCs use ion channels to reveal the presence of certain substances in foodstuff. In this review, we focus on the functional and molecular properties of ion channels that serve as receptors in taste transduction.     

Sex-specific non-pheromonal taste receptors in Drosophila

Taste receptors have recently been reported in Drosophila [1,2], but little is known of the relation between receptor and response. Morphological studies of the distribution of chemosensory sensilla indicate that the fruit fly has two major sites of gustation: the proboscis and the legs [3]. The taste sensilla on both these sites are similar in structure and each sensillum generally houses four gustatory neurons [4]. Early anatomical observations have demonstrated a sexual dimorphism in the number of tarsal sensilla [5] and in their central projections [6]. We measured the electrophysiological responses of the prothoracic taste sensilla to non-pheromonal substances--salts, sugars and water--and found a clear sexual dimorphism. From the response profile of individual sensilla, we were able to distinguish three types of tarsal sensilla in females as against only two types in males. The female-specific type, which responded specifically to sugar, was absent in males except when male gustatory neurons were genetically feminised. The fact that tarsal gustatory hairs exhibit a sexual dimorphism that affects the perception of non-pheromonal compounds suggests that sexual identity is more complex than has previously been thought [7,8].     

Hedonic taste in Drosophila revealed by olfactory receptors expressed in taste neurons

Taste and olfaction are each tuned to a unique set of chemicals in the outside world, and their corresponding sensory spaces are mapped in different areas in the brain. This dichotomy matches categories of receptors detecting molecules either in the gaseous or in the liquid phase in terrestrial animals. However, in Drosophila olfactory and gustatory neurons express receptors which belong to the same family of 7-transmembrane domain proteins. Striking overlaps exist in their sequence structure and in their expression pattern, suggesting that there might be some functional commonalities between them. In this work, we tested the assumption that Drosophila olfactory receptor proteins are compatible with taste neurons by ectopically expressing an olfactory receptor (OR22a and OR83b) for which ligands are known. Using electrophysiological recordings, we show that the transformed taste neurons are excited by odor ligands as by their cognate tastants. The wiring of these neurons to the brain seems unchanged and no additional connections to the antennal lobe were detected. The odor ligands detected by the olfactory receptor acquire a new hedonic value, inducing appetitive or aversive behaviors depending on the categories of taste neurons in which they are expressed i.e. sugar- or bitter-sensing cells expressing either Gr5a or Gr66a receptors. Taste neurons expressing ectopic olfactory receptors can sense odors at close range either in the aerial phase or by contact, in a lipophilic phase. The responses of the transformed taste neurons to the odorant are similar to those obtained with tastants. The hedonic value attributed to tastants is directly linked to the taste neurons in which their receptors are expressed.     

Expression and purification of functional ligand-binding domains of T1R3 taste receptors

Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.     

Espin cytoskeletal proteins in the sensory cells of rodent taste buds

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 ± 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 ± 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP₃R₃)_, α-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP₃R₃, PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP₃R₃, α-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of ～3 μm-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10–12 μm into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells.