Identification of New Agonists and Antagonists of the Insect Odorant Receptor Co-Receptor Subunit

BACKGROUND: Insects detect attractive and aversive chemicals using several families of chemosensory receptors, including the OR family of olfactory receptors, making these receptors appealing targets for the control of insects. Insect ORs are odorant-gated ion channels, comprised of at least one common subunit (the odorant receptor co-receptor subunit, Orco) and at least one variable odorant specificity subunit. Each of the many ORs of an insect species is activated or inhibited by an unique set of odorants that interact with the variable odorant specificity subunits, making the development of OR directed insect control agents complex and laborious. However, several N-,2-substituted triazolothioacetamide compounds (VUAA1, VU0450667 and VU0183254) were recently shown to act directly on the highly conserved Orco subunit, suggesting that broadly active compounds can be developed. We have explored the chemical space around the VUAA1 structure in order to identify new Orco ligands. PRINCIPAL FINDINGS: We screened ORs from several insect species, using heterologous expression in Xenopus oocytes and an electrophysiological assay, with a panel of 22 compounds structurally related to VUAA1. By varying the nitrogen position in the pyridine ring and altering the moieties decorating the phenyl ring, we identified two new agonists and a series of competitive antagonists. Screening smaller compounds, similar to portions of the VUAA1 structure, also yielded competitive antagonists. Importantly, we show that Orco antagonists inhibit odorant activation of ORs from several insect species. Detailed examination of one antagonist demonstrated inhibition to be through a non-competitive mechanism. CONCLUSIONS: A similar pattern of agonist and antagonist sensitivity displayed by Orco subunits from different species suggests a highly conserved binding site structure. The susceptibility to inhibition of odorant activation by Orco antagonism is conserved across disparate insect species, suggesing the ligand binding site on Orco as a promising target for the development of novel, broadly active insect repellants

Bio-Benchmarking of Electronic Nose Sensors

BACKGROUND:Electronic noses, E-Noses, are instruments designed to reproduce the performance of animal noses or antennae but generally they cannot match the discriminating power of the biological original and have, therefore, been of limited utility. The manner in which odorant space is sampled is a critical factor in the performance of all noses but so far it has been described in detail only for the fly antenna. METHODOLOGY:Here we describe how a set of metal oxide (MOx) E-Nose sensors, which is the most commonly used type, samples odorant space and compare it with what is known about fly odorant receptors (ORs). PRINCIPAL FINDINGS:Compared with a fly's odorant receptors, MOx sensors from an electronic nose are on average more narrowly tuned but much more highly correlated with each other. A set of insect ORs can therefore sample broader regions of odorant space independently and redundantly than an equivalent number of MOx sensors. The comparison also highlights some important questions about the molecular nature of fly ORs. CONCLUSIONS:The comparative approach generates practical learnings that may be taken up by solid-state physicists or engineers in designing new solid-state electronic nose sensors. It also potentially deepens our understanding of the performance of the biological system

Gain control network conditions in early sensory coding

Gain control is essential for the proper function of any sensory system. However, the precise mechanisms for achieving effective gain control in the brain are unknown. Based on our understanding of the existence and strength of connections in the insect olfactory system, we analyze the conditions that lead to controlled gain in a randomly connected network of excitatory and inhibitory neurons. We consider two scenarios for the variation of input into the system. In the first case, the intensity of the sensory input controls the input currents to a fixed proportion of neurons of the excitatory and inhibitory populations. In the second case, increasing intensity of the sensory stimulus will both, recruit an increasing number of neurons that receive input and change the input current that they receive. Using a mean field approximation for the network activity we derive relationships between the parameters of the network that ensure that the overall level of activity of the excitatory population remains unchanged for increasing intensity of the external stimulation. We find that, first, the main parameters that regulate network gain are the probabilities of connections from the inhibitory population to the excitatory population and of the connections within the inhibitory population. Second, we show that strict gain control is not achievable in a random network in the second case, when the input recruits an increasing number of neurons. Finally, we confirm that the gain control conditions derived from the mean field approximation are valid in simulations of firing rate models and Hodgkin-Huxley conductance based models

The context of chemical communication driving a mutualism

Recent work suggests that Drosophila and Saccharomyces yeasts may establish a mutualistic association, and that this is driven by chemical communication. While individual volatiles have been implicated in the attraction of D. melanogaster, the semiochemicals affecting the behavior of the sibling species D. simulans are less well characterised. Here, we comprehensively scrutinize a broad range of volatiles produced by attractive and repulsive yeasts to experimentally evaluate the chemical nature of communication between these species. When grown in liquid or on agar-solidified grape juice, attraction to S. cerevisiae was primarily driven by 3-methylbutyl acetate (isoamyl acetate) and repulsion by acetic acid, a known attractant to D. melanogaster (also known as vinegar fly). Using T-maze choice tests and synthetic compounds we show that these responses were strongly influenced by compound concentration. Moreover, the behavioral response is further impacted by the chemical context of the environment. Thus, chemical communication between yeasts and flies is complex, and is not simply driven by the presence of single volatiles, but modulated by compound interactions. The ecological context of chemical communication needs to be taken into consideration when testing for ecologically realistic responses

Predicting olfactory receptor neuron responses from odorant structure

Background Olfactory receptors work at the interface between the chemical world of volatile molecules and the perception of scent in the brain. Their main purpose is to translate chemical space into information that can be processed by neural circuits. Assuming that these receptors have evolved to cope with this task, the analysis of their coding strategy promises to yield valuable insight in how to encode chemical information in an efficient way. Results We mimicked olfactory coding by modeling responses of primary olfactory neurons to small molecules using a large set of physicochemical molecular descriptors and artificial neural networks. We then tested these models by recording in vivo receptor neuron responses to a new set of odorants and successfully predicted the responses of five out of seven receptor neurons. Correlation coefficients ranged from 0.66 to 0.85, demonstrating the applicability of our approach for the analysis of olfactory receptor activation data. The molecular descriptors that are best-suited for response prediction vary for different receptor neurons, implying that each receptor neuron detects a different aspect of chemical space. Finally, we demonstrate that receptor responses themselves can be used as descriptors in a predictive model of neuron activation. Conclusions The chemical meaning of molecular descriptors helps understand structure-response relationships for olfactory receptors and their 'receptive fields'. Moreover, it is possible to predict receptor neuron activation from chemical structure using machine-learning techniques, although this is still complicated by a lack of training data

A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons

Monitoring neuronal responses to defined sensory stimuli is a powerful and widely used approach for understanding sensory coding in the nervous system. However, providing precise, stereotypic and reproducible cues while concomitantly recording neuronal activity remains technically challenging. Here we describe the fabrication and use of a microfluidics system that allows precise temporally restricted stimulation of Drosophila chemosensory neurons with an array of different chemical cues. The system can easily be combined with genetically encoded calcium sensors, and it can measure neuronal activity at single-cell resolution in larval sense organs and in the proboscis or leg of the adult fly. We describe the design of the master mold, the production of the microfluidic chip and live imaging using the calcium sensor GCaMP, expressed in distinct types of Drosophila chemosensory neurons. Fabrication of the master mold and microfluidic chips requires basic skills in photolithography and takes ~2 weeks; the same devices can be used repeatedly over several months. Flies can be prepared for measurements in minutes and imaged for up to 1 h

A Combined Perceptual, Physico-Chemical, and Imaging Approach to ‘Odour-Distances’ Suggests a Categorizing Function of the Drosophila Antennal Lobe

How do physico-chemical stimulus features, perception, and physiology relate? Given the multi-layered and parallel architecture of brains, the question specifically is where physiological activity patterns correspond to stimulus features and/or perception. Perceived distances between six odour pairs are defined behaviourally from four independent odour recognition tasks. We find that, in register with the physico-chemical distances of these odours, perceived distances for 3-octanol and n-amylacetate are consistently smallest in all four tasks, while the other five odour pairs are about equally distinct. Optical imaging in the antennal lobe, using a calcium sensor transgenically expressed in only first-order sensory or only second-order olfactory projection neurons, reveals that 3-octanol and n-amylacetate are distinctly represented in sensory neurons, but appear merged in projection neurons. These results may suggest that within-antennal lobe processing funnels sensory signals into behaviourally meaningful categories, in register with the physico-chemical relatedness of the odours

Towards plant-odor-related olfactory neuroethology in Drosophila

Drosophila melanogaster is today one of the three foremost models in olfactory research, paralleled only by the mouse and the nematode. In the last years, immense progress has been achieved by combining neurogenetic tools with neurophysiology, anatomy, chemistry, and behavioral assays. One of the most important tasks for a fruit fly is to find a substrate for eating and laying eggs. To perform this task the fly is dependent on olfactory cues emitted by suitable substrates as e.g. decaying fruit. In addition, in this area, considerable progress has been made during the last years, and more and more natural and behaviorally active ligands have been identified. The future challenge is to tie the progress in different fields together to give us a better understanding of how a fly really behaves. Not in a test tube, but in nature. Here, we review our present state of knowledge regarding Drosophila plant-odor-related olfactory neuroethology to provide a basis for new progress

Mechanisms of Maximum Information Preservation in the Drosophila Antennal Lobe

We examined the presence of maximum information preservation, which may be a fundamental principle of information transmission in all sensory modalities, in the Drosophila antennal lobe using an experimentally grounded network model and physiological data. Recent studies have shown a nonlinear firing rate transformation between olfactory receptor neurons (ORNs) and second-order projection neurons (PNs). As a result, PNs can use their dynamic range more uniformly than ORNs in response to a diverse set of odors. Although this firing rate transformation is thought to assist the decoder in discriminating between odors, there are no comprehensive, quantitatively supported studies examining this notion. Therefore, we quantitatively investigated the efficiency of this firing rate transformation from the viewpoint of information preservation by computing the mutual information between odor stimuli and PN responses in our network model. In the Drosophila olfactory system, all ORNs and PNs are divided into unique functional processing units called glomeruli. The nonlinear transformation between ORNs and PNs is formed by intraglomerular transformation and interglomerular interaction through local neurons (LNs). By exploring possible nonlinear transformations produced by these two factors in our network model, we found that mutual information is maximized when a weak ORN input is preferentially amplified within a glomerulus and the net LN input to each glomerulus is inhibitory. It is noteworthy that this is the very combination observed experimentally. Furthermore, the shape of the resultant nonlinear transformation is similar to that observed experimentally. These results imply that information related to odor stimuli is almost maximally preserved in the Drosophila olfactory circuit. We also discuss how intraglomerular transformation and interglomerular inhibition combine to maximize mutual information

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties