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ABSTRACT: The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant. Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.
Journal of Visualized Experiments 01/2010;
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ABSTRACT: There is broad consensus that olfactory signaling in vertebrates and the nematode C. elegans uses canonical G-protein-coupled receptor transduction pathways. In contrast, mechanisms of insect olfactory signal transduction remain deeply controversial. Genetic disruption of G proteins and chemosensory ion channels in mice and worms leads to profound impairment in olfaction, while similar mutations in the fly show more subtle phenotypes. The literature contains contradictory claims that insect olfaction uses cAMP, cGMP, or IP3 as second messengers; that insect odorant receptors couple to G(alpha)s or G(alpha)q pathways; and that insect odorant receptors are G-protein-coupled receptors or odor-gated ion channels. Here we consider all the evidence and offer a consensus model for a noncanonical mechanism of olfactory signal transduction in insects.
Current opinion in neurobiology 09/2009; 19(3):284-92. · 7.21 Impact Factor
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ABSTRACT: In animals, the sense of smell is often used as a powerful way to attract potential mates, to find food and to explore the environment. Different animals evolved different systems to detect volatile odorants, tuned to the specific needs of each species. Vertebrates and nematodes have been used extensively as models to study the mechanisms of olfaction: the molecular players are olfactory receptors (ORs) expressed in olfactory sensory neurons (OSNs) where they bind to volatile chemicals, acting as the first relay of olfactory processing. These receptors belong to the G protein-coupled receptor (GPCR) superfamily; binding to odorants induces the production and amplification of second messengers, which lead to the depolarization of the neuron. The anatomical features of the insect olfactory circuit are similar to those of mammals, and until recently it was thought that this similarity extended to the ORs, which were originally annotated as GPCRs. Surprisingly, recent evidence shows that insect ORs can act like ligand-gated ion channels, either completely or partially bypassing the amplification steps connected to the activation of G proteins. Although the involvement of G proteins in insect olfactory signal transduction is still under question, this new discovery raises fascinating new questions regarding the function of the sense of smell in insects, its evolution and potential benefits compared with its mammalian counterpart.
Journal of Experimental Biology 08/2009; 212(Pt 13):1973-9. · 3.00 Impact Factor
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ABSTRACT: In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. Here we provide evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes showed extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment.
Nature 05/2008; 452(7190):1002-6. · 36.28 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 11/2005; 50(12):1563-70.
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ABSTRACT: We describe two male-specific olfactory receptors (ORs) in the silk moth, Bombyx mori, that are mutually exclusively expressed in a pair of adjacent pheromone-sensitive neurons of male antennae: One is specifically tuned to bombykol, the sex pheromone, and the other to bombykal, its oxidized form. Both pheromone ORs are coexpressed with an OR from the highly conserved insect OR subfamily. This coexpression promotes the functional expression of pheromone receptors and confers ligand-stimulated nonselective cation channel activity. The same effects were also observed for general ORs. Both odorant and pheromone signaling pathways are mediated by means of a common mechanism in insects.
Science 04/2005; 307(5715):1638-42. · 31.20 Impact Factor
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ABSTRACT: Sex pheromones released by female moths are detected with high specificity and sensitivity in the olfactory sensilla of antennae of conspecific males. Bombykol in the silkmoth Bombyx mori was the first sex pheromone to be identified. Here we identify a male-specific G protein-coupled olfactory receptor gene, B. mori olfactory receptor 1 (BmOR-1), that appears to encode a bombykol receptor. The BmOR-1 gene is located on the Z sex chromosome, has an eight-exon/seven-intron structure, and exhibits male-specific expression in the pheromone receptor neurons of male moth antenna during late pupal and adult stages. Bombykol stimulation of Xenopus laevis oocytes expressing BmOR-1 and BmGalphaq elicited robust dose-dependent inward currents on two-electrode voltage clamp recordings, demonstrating that the binding of bombykol to BmOR-1 leads to the activation of a BmGalphaq-mediated signaling cascade. Antennae of female moths infected with BmOR-1-recombinant baculovirus showed electrophysiological responses to bombykol but not to bombykal. These results provide evidence that BmOR-1 is a G protein-coupled sex pheromone receptor that recognizes bombykol.
Proceedings of the National Academy of Sciences 12/2004; 101(47):16653-8. · 9.68 Impact Factor
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ABSTRACT: Odorant responsiveness of a mouse olfactory receptor, mOR-EG, was investigated in various heterologous cells using a variety of detection methods. Odorant-induced Ca(2+) response was observed in HEK293 cells that coexpressed mOR-EG and the promiscuous G protein, G alpha 15. Without G alpha 15, a robust increase in cAMP level was observed upon odorant-stimulation in various mammalian cells. A luciferase reporter gene assay using zif268 promoter was adopted to amplify the cAMP signals. In Xenopus laevis oocytes, odorant-stimulated currents were recorded when mOR-EG cRNA was co-injected with either G alpha 15 or cAMP-dependent channel. These results suggest that odorant responsiveness can be monitored via a signaling pathway mediated by endogenous G alphas or transfected G alpha 15 in heterologous cell systems. Various functional assays for a heterologously expressed olfactory receptor reported in this study, are potentially useful for high-throughput ligand screening and functional analyses of hundreds of olfactory receptors.
Biochemical and Biophysical Research Communications 07/2003; 305(4):964-9. · 2.48 Impact Factor
