Natural variation at the Drosophila melanogaster Or22 odorant receptor locus is associated with changes in olfactory behaviour

In insects, many critical olfactory behaviours are mediated by the large odorant receptor (Or) gene family, which determines the response properties of different classes of olfactory receptor neurons (ORNs). While ORN responses are generally conserved within and between Drosophila species, variant alleles of the D. melanogaster Or22 locus have previously been shown to alter the response profile of an ORN class called ab3A. These alleles show potential clinal variation, suggesting that selection is acting at this locus. Here, we investigated if the changes seen in ab3A responses lead to changes in olfactory-related behaviours. We show that variation at the Or22 locus and in the ab3A neurons are not fully compensated for by other ORNs and lead to overall changes in antennal odorant detection. We further show that this correlates with differences in odorant preference behaviour and with differences in oviposition site preference, with flies that have the chimaeric short allele strongly preferring to oviposit on banana. These findings indicate that variation at the Or22 locus leads to changes in olfactory-driven behaviours, and add support to the idea that the ab3A neurons are of especial importance to the ecology of Drosophila flies.     

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P₂/IP₃ and PI(3,4,5)P₃, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.     

Caspase Inhibition in Select Olfactory Neurons Restores Innate Attraction Behavior in Aged Drosophila

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.     

Molecular, anatomical, and functional organization of the Drosophila olfactory system

BACKGROUND: Olfactory receptor neurons (ORNs) convey chemical information into the brain, producing internal representations of odors detected in the periphery. A comprehensive understanding of the molecular and neural mechanisms of odor detection and processing requires complete maps of odorant receptor (Or) expression and ORN connectivity, preferably at single-cell resolution. RESULTS: We have constructed near-complete maps of Or expression and ORN targeting in the Drosophila olfactory system. These maps confirm the general validity of the "one neuron--one receptor" and "one glomerulus--one receptor" principles and reveal several additional features of olfactory organization. ORNs in distinct sensilla types project to distinct regions of the antennal lobe, but neighbor relations are not preserved. ORNs grouped in the same sensilla do not express similar receptors, but similar receptors tend to map to closely appositioned glomeruli in the antennal lobe. This organization may serve to ensure that odor representations are dispersed in the periphery but clustered centrally. Integrated with electrophysiological data, these maps also predict glomerular representations of specific odorants. Representations of aliphatic and aromatic compounds are spatially segregated, with those of aliphatic compounds arranged topographically according to carbon chain length. CONCLUSIONS: These Or expression and ORN connectivity maps provide further insight into the molecular, anatomical, and functional organization of the Drosophila olfactory system. Our maps also provide an essential resource for investigating how internal odor representations are generated and how they are further processed and transmitted to higher brain centers.     

Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction

Fruit flies are attracted by a diversity of odors that signal the presence of food, potential mates, or attractive egg-laying sites. Most Drosophila olfactory neurons express two types of odorant receptor genes: Or83b, a broadly expressed receptor of unknown function, and one or more members of a family of 61 selectively expressed receptors. While the conventional odorant receptors are highly divergent, Or83b is remarkably conserved between insect species. Two models could account for Or83b function: it could interact with specific odor stimuli independent of conventional odorant receptors, or it could act in concert with these receptors to mediate responses to all odors. Our results support the second model. Dendritic localization of conventional odorant receptors is abolished in Or83b mutants. Consistent with this cellular defect, the Or83b mutation disrupts behavioral and electrophysiological responses to many odorants. Or83b therefore encodes an atypical odorant receptor that plays an essential general role in olfaction.     

Renewing olfactory receptor neurons in goldfish do not require contact with the olfactory bulb to develop normal chemical responsiveness

This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (>0.1 mmol · l⁻¹) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering. Accepted: 14 June 1997     

A computational study of odorant transport and deposition in the canine nasal cavity: implications for olfaction

Olfaction begins when an animal draws odorant-laden air into its nasal cavity by sniffing, thus transporting odorant molecules from the external environment to olfactory receptor neurons (ORNs) in the sensory region of the nose. In the dog and other macrosmatic mammals, ORNs are relegated to a recess in the rear of the nasal cavity that is comprised of a labyrinth of scroll-like airways. Evidence from recent studies suggests that nasal airflow patterns enhance olfactory sensitivity by efficiently delivering odorant molecules to the olfactory recess. Here, we simulate odorant transport and deposition during steady inspiration in an anatomically correct reconstructed model of the canine nasal cavity. Our simulations show that highly soluble odorants are deposited in the front of the olfactory recess along the dorsal meatus and nasal septum, whereas moderately soluble and insoluble odorants are more uniformly deposited throughout the entire olfactory recess. These results demonstrate that odorant deposition patterns correspond with the anatomical organization of ORNs in the olfactory recess. Specifically, ORNs that are sensitive to a particular class of odorants are located in regions where that class of odorants is deposited. The correlation of odorant deposition patterns with the anatomical organization of ORNs may partially explain macrosmia in the dog and other keen-scented species.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

Odorant Receptor Polymorphisms and Natural Variation in Olfactory Behavior in Drosophila melanogaster

Animals perceive and discriminate among a vast array of sensory cues in their environment. Both genetic and environmental factors contribute to individual variation in behavioral responses to these cues. Here, we asked to what extent sequence variants in six Drosophila melanogaster odorant receptor (Or) genes are associated with variation in behavioral responses to benzaldehyde by sequencing alleles from a natural population. Sequence analyses showed signatures of deviations from neutrality for Or42b and Or85f, and linkage disequilibrium analyses showed a history of extensive recombination between polymorphic markers for all six Or genes. We identified polymorphisms in Or10a, Or43a, and Or67b that were significantly associated with variation in response to benzaldehyde. To verify these associations, we repeated the analyses with an independent set of behavioral measurements of responses to a structurally similar odorant, acetophenone. Association profiles for both odorants were similar with many polymorphisms and haplotypes associated with variation in responsiveness to both odorants. Some polymorphisms, however, were associated with one, but not the other odorant. We also observed a correspondence between behavioral response to benzaldehyde and differences in Or10a and Or43a expression. These results illustrate that sequence variants that arise during the evolution of odorant receptor genes can contribute to individual variation in olfactory behavior and give rise to subtle shifts in olfactory perception.RESEARCHERS in many scientific fields have long appreciated that different animal species perceive the world differently. In fact, these differences are so striking that new disciplines have arisen to study the adaptations of sense organs to the environment (e.g., Ali 1978; Lythgoe 1979; Dusenbery 1992). Differences in sensory perception exist not only between species, but also between populations of a single species and between individuals within a population. What is the underlying genetic architecture for individual variation in sensory perception?Olfaction provides an excellent model for examining the underlying genetic mechanisms that result in variation in behavior. In both vertebrates and invertebrates, odorants are detected by families of odorant receptors expressed in populations of olfactory receptor neurons (ORNs), whose activation elicits a distinct spatial pattern of glomerular activity in the brain (Buck and Axel 1991; Vassar et al. 1994; Mombaerts et al. 1996; Laissue et al. 1999; Gao et al. 2000; Vosshall et al. 2000; Bhalerao et al. 2003; Wang et al. 2003). This combinatorial code allows for discrimination of a diverse repertoire of odorants.Drosophila melanogaster has a relatively simple olfactory system with only 60 odorant receptor (Or) genes (Vosshall and Stocker 2007) compared to ∼1000 in the mouse (Zhang and Firestein 2002; Zhang et al. 2004). The 60 genes are located throughout the genome, and 2 of these genes are alternatively spliced for a total of 62 identified proteins (Clyne et al. 1999; Gao and Chess 1999; Vosshall et al. 1999; Robertson et al. 2003). Furthermore, clusters of Ors throughout the genome suggest several recent gene duplication events (Robertson et al. 2003).The response spectra of individual ORNs have been extensively characterized using extracellular electrophysiological recordings from single sensilla on the antennae and maxillary palps. Recordings from basiconic sensilla on the antenna identified classes of neurons with distinct olfactory response profiles organized as two to four neurons in each sensillum with specific neuronal combinations occurring in distinct spatial regions of the antenna (de Bruyne et al. 1999, 2001).The majority of ORNs express a unique odorant receptor in addition to the highly conserved coreceptor, Or83b (Jones et al. 2005). Studies of a null mutant of Or83b implicated this receptor in positioning odorant receptor proteins in the sensory dendrites (Larsson et al. 2004; Benton et al. 2006). Odorant receptors in Drosophila have an atypical membrane topology with a cytoplasmic N terminus and an extracellular C terminus (Benton et al. 2006). Specific domains in the third cytoplasmic loops of two odorant receptors, Or22a and Or43a, have been implicated to interact with the third loop of Or83b (Benton et al. 2006). Drosophila odorant receptors act as ligand-gated nonselective cation channels formed by a dimeric complex between a unique Or and the Or83b coreceptor (Sato et al. 2008; Wicher et al. 2008).Several studies have examined ligand specificities of individual odorant receptor proteins and demonstrated that they respond to diverse and overlapping suites of ligands. Response profiles for many receptors have been characterized using the Gal4/UAS system to drive expression of individual odorant receptors in a mutant ORN lacking expression of its endogeneous receptor, followed by electrophysiological recording (Dobritsa et al. 2003; Hallem et al. 2004; Hallem and Carlson 2006). In addition, misexpression studies of Or43a resulted in a reduction of behavioral avoidance responses to benzaldehyde (Stortkuhl et al. 2005). This result combined with electrophysiological recordings from ORNs and heterologous expression in Xenopus oocytes further functionally characterized the odorant response profiles of this receptor (Wetzel et al. 2001) and identified several Or43a ligands, such as fruit- derived odorants benzaldehyde, cyclohexanone, cyclohexanol, and benzyl alcohol (Stortkuhl and Kettler 2001; Hallem et al. 2004).Despite advances in our understanding of odor coding, the molecular mechanisms responsible for variation in olfactory perception remain poorly understood. D. melanogaster is especially amenable to conducting such studies given its quantitatively simple olfactory system and since large numbers of genetically identical individuals can be reared in a common environment and these individuals can be subjected to simple, rapid, and highly reproducible quantitative behavioral assays Anholt and Mackay 2004). Here, we examine how molecular variation in odorant receptors contributes to variation in olfactory behavior in inbred lines derived from a natural population of D. melanogaster. We focused our analyses on six odorant receptors, Or7a, Or10a, Or42b, Or43a, Or67b, and Or85f, which have been shown by electrophysiology (Stortkuhl and Kettler 2001; Hallem et al. 2004; Stortkuhl et al. 2005; Hallem and Carlson 2006), through heterologous expression systems (Wetzel et al. 2001), or by calcium imaging studies (Wang et al. 2003) to respond to benzaldehyde. Significant variation in behavioral responses to benzaldehyde has been observed previously in this population and was normally distributed as is typical for a quantitative trait influenced by multiple genes (Wang et al. 2007). Here, we report associations between olfactory behavior and sequence variants in three Or genes. To validate the reliability of these associations we measured responses to a structurally similar odorant, acetophenone, in the same population, and showed that the associations with variation in responses to both odorants are largely similar with occasional molecular polymorphisms associated with variation in response to only one, but not the other odorant. These observations illustrate how sequence variants that arise during the evolution of Or genes can contribute to individual variation in olfactory behavior, how polymorphisms can give rise to subtle shifts in olfactory perception, and how naturally arising mutations within a population can combine to generate broad individual variation in sensory perception.     

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.     

Genetic Control of Wiring Specificity in the Fly Olfactory System

Precise connections established between pre- and postsynaptic partners during development are essential for the proper function of the nervous system. The olfactory system detects a wide variety of odorants and processes the information in a precisely connected neural circuit. A common feature of the olfactory systems from insects to mammals is that the olfactory receptor neurons (ORNs) expressing the same odorant receptor make one-to-one connections with a single class of second-order olfactory projection neurons (PNs). This represents one of the most striking examples of targeting specificity in developmental neurobiology. Recent studies have uncovered central roles of transmembrane and secreted proteins in organizing this one-to-one connection specificity in the olfactory system. Here, we review recent advances in the understanding of how this wiring specificity is genetically controlled and focus on the mechanisms by which transmembrane and secreted proteins regulate different stages of the Drosophila olfactory circuit assembly in a coordinated manner. We also discuss how combinatorial coding, redundancy, and error-correcting ability could contribute to constructing a complex neural circuit in general.     

Comparative genomic analysis of the odorant-binding protein family in 12 <Emphasis Type="Italic">Drosophila</Emphasis> genomes: purifying selection and birth-and-death evolution

Background  

Chemoreception is a widespread mechanism that is involved in critical biologic processes, including individual and social behavior. The insect peripheral olfactory system comprises three major multigene families: the olfactory receptor (Or), the gustatory receptor (Gr), and the odorant-binding protein (OBP) families. Members of the latter family establish the first contact with the odorants, and thus constitute the first step in the chemosensory transduction pathway.     

A presynaptic gain control mechanism fine-tunes olfactory behavior

Early sensory processing can play a critical role in sensing environmental cues. We have investigated the physiological and behavioral function of gain control at the first synapse of olfactory processing in Drosophila. Olfactory receptor neurons (ORNs) express the GABA(B) receptor (GABA(B)R), and its expression expands the dynamic range of ORN synaptic transmission that is preserved in projection neuron responses. Strikingly, each ORN channel has a unique baseline level of GABA(B)R expression. ORNs that sense the aversive odorant CO(2) do not express GABA(B)Rs and do not have significant presynaptic inhibition. In contrast, pheromone-sensing ORNs express a high level of GABA(B)Rs and exhibit strong presynaptic inhibition. Furthermore, pheromone-dependent mate localization is impaired in flies that lack GABA(B)Rs in specific ORNs. These findings indicate that different olfactory receptor channels employ heterogeneous presynaptic gain control as a mechanism to allow an animal's innate behavioral responses to match its ecological needs.     

Requirement for Drosophila SNMP1 for Rapid Activation and Termination of Pheromone-Induced Activity

Pheromones are used for conspecific communication by many animals. In Drosophila, the volatile male-specific pheromone 11-cis vaccenyl acetate (cVA) supplies an important signal for gender recognition. Sensing of cVA by the olfactory system depends on multiple components, including an olfactory receptor (OR67d), the co-receptor ORCO, and an odorant binding protein (LUSH). In addition, a CD36 related protein, sensory neuron membrane protein 1 (SNMP1) is also involved in cVA detection. Loss of SNMP1 has been reported to eliminate cVA responsiveness, and to greatly increase spontaneous activity of OR67d-expressing olfactory receptor neurons (ORNs). Here, we found the snmp1¹ mutation did not abolish cVA responsiveness or cause high spontaneous activity. The cVA responses in snmp1 mutants displayed a delayed onset, and took longer to reach peak activity than wild-type. Most strikingly, loss of SNMP1 caused a dramatic delay in signal termination. The profound impairment in signal inactivation accounted for the previously reported “spontaneous activity,” which represented continuous activation following transient exposure to environmental cVA. We introduced the silk moth receptor (BmOR1) in OR67d ORNs of snmp1¹ flies and found that the ORNs showed slow activation and deactivation kinetics in response to the BmOR1 ligand (bombykol). We expressed the bombykol receptor complex in Xenopus oocytes in the presence or absence of the silk moth SNMP1 (BmSNMP) and found that addition of BmSNMP accelerated receptor activation and deactivation. Our results thus clarify SNMP1 as an important player required for the rapid kinetics of the pheromone response in insects.     

Measuring activity in olfactory receptor neurons in Drosophila: Focus on spike amplitude

Olfactory responses at the receptor level have been thoroughly described in Drosophila melanogaster by electrophysiological methods. Single sensilla recordings (SSRs) measure neuronal activity in intact individuals in response to odors. For sensilla that contain more than one olfactory receptor neuron (ORN), their different spontaneous spike amplitudes can distinguish each signal under resting conditions. However, activity is mainly described by spike frequency.Some reports on ORN response dynamics studied two components in the olfactory responses of ORNs: a fast component that is reflected by the spike frequency and a slow component that is observed in the LFP (local field potential, the single sensillum counterpart of the electroantennogram, EAG). However, no apparent correlation was found between the two elements.In this report, we show that odorant stimulation produces two different effects in the fast component, affecting spike frequency and spike amplitude. Spike amplitude clearly diminishes at the beginning of a response, but it recovers more slowly than spike frequency after stimulus cessation, suggesting that ORNs return to resting conditions long after they recover a normal spontaneous spike frequency. Moreover, spike amplitude recovery follows the same kinetics as the slow voltage component measured by the LFP, suggesting that both measures are connected.These results were obtained in ab2 and ab3 sensilla in response to two odors at different concentrations. Both spike amplitude and LFP kinetics depend on odorant, concentration and neuron, suggesting that like the EAG they may reflect olfactory information.     

Afferent induction of olfactory glomeruli requires N-cadherin

Drosophila olfactory receptor neurons (ORNs) elaborate a precise internal representation of the external olfactory world in the antennal lobe (AL), a structure analagous to the vertebrate olfactory bulb. ORNs expressing the same odorant receptor innervate common targets in a highly organized neuropilar structure inside the AL, the glomerulus. During normal development, ORNs target to specific regions of the AL and segregate into subclass-specific aggregates called protoglomeruli prior to extensive intermingling with target dendrites to form mature glomeruli. Using a panel of ORN subclass-specific markers, we demonstrate that in the adult AL, N-cadherin (N-cad) mutant ORN terminals remain segregated from dendrites of target neurons. N-cad plays a crucial role in protoglomerulus formation but is largely dispensible for targeting to the appropriate region of the AL. We propose that N-cad, a homophilic cell adhesion molecule, acts in a permissive fashion to promote subclass-specific sorting of ORN axon terminals into protoglomeruli.