Smelling on the fly: sensory cues and strategies for olfactory navigation in Drosophila

Navigating toward (or away from) a remote odor source is a challenging problem that requires integrating olfactory information with visual and mechanosensory cues. Drosophila melanogaster is a useful organism for studying the neural mechanisms of these navigation behaviors. There are a wealth of genetic tools in this organism, as well as a history of inventive behavioral experiments. There is also a large and growing literature in Drosophila on the neural coding of olfactory, visual, and mechanosensory stimuli. Here we review recent progress in understanding how these stimulus modalities are encoded in the Drosophila nervous system. We also discuss what strategies a fly might use to navigate in a natural olfactory landscape while making use of all these sources of sensory information. We emphasize that Drosophila are likely to switch between multiple strategies for olfactory navigation, depending on the availability of various sensory cues. Finally, we highlight future research directions that will be important in understanding the neural circuits that underlie these behaviors.     

Olfaction and olfactory learning in Drosophila: recent progress

The olfactory system of Drosophila resembles that of vertebrates in its overall anatomical organization, but is considerably reduced in terms of cell number, making it an ideal model system to investigate odor processing in a brain [Vosshall LB, Stocker RF: Molecular architecture of smell and taste in Drosophila. Annu Rev Neurosci 2007, 30:505-533]. Recent studies have greatly increased our knowledge about odor representation at different levels of integration, from olfactory receptors to 'higher brain centers'. In addition, Drosophila represents a favourite model system to study the neuronal basis of olfactory learning and memory, and considerable progress during the last years has been made in localizing the structures mediating olfactory learning and memory [Davis RL: Olfactory memory formation in Drosophila: from molecular to systems neuroscience. Annu Rev Neurosci 2005, 28:275-302; Gerber B, Tanimoto H, Heisenberg M: An engram found? Evaluating the evidence from fruit flies. Curr Opin Neurobiol 2004, 14:737-744; Keene AC, Waddell S: Drosophila olfactory memory: single genes to complex neural circuits. Nat Rev Neurosci 2007, 8:341-354]. This review summarizes recent progress in analyzing olfactory processing and olfactory learning in Drosophila.     

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal´s dynamic behavioral reaction.     

Innate attractiveness and associative learnability of odors can be dissociated in larval Drosophila

We investigate olfactory associative learning in larval Drosophila. A reciprocal training design is used, such that one group of animals receives a reward in the presence of odor X but not in the presence of odor Y (Train: X+ // Y), whereas another group is trained reciprocally (Train: X // Y+). After training, differences in odor preference between these reciprocally trained groups in a choice test (Test: X - Y) reflect associative learning. The current study, after showing which odor pairs can be used for such learning experiments, 1) introduces a one-odor version of such reciprocal paradigm that allows estimating the learnability of single odors. Regarding this reciprocal one-odor paradigm, we show that 2) paired presentations of an odor with a reward increase odor preference above baseline, whereas unpaired presentations of odor and reward decrease odor preference below baseline; this suggests that odors can become predictive either of reward or of reward absence. Furthermore, we show that 3) innate attractiveness and associative learnability can be dissociated. These data deepen our understanding of odor-reward learning in larval Drosophila on the behavioral level, and thus foster its neurogenetic analysis.     

Olfactory physiology in the Drosophila antenna and maxillary palp: acj6 distinguishes two classes of odorant pathways.

This article provides characterization of the electrical response to odorants in the Drosophila antenna and provides physiological evidence that a second organ, the maxillary palp, also has olfactory function in Drosophila. The acj6 mutation, previously isolated by virtue of defective olfactory behavior, affects olfactory physiology in the maxillary palp as well as in the antenna. Interestingly, abnormal chemosensory jump 6 (acj6) reduces response in the maxillary palp to all odorants tested except benzaldehyde (odor of almond), as if response to benzaldehyde is mediated through a different type of odorant pathway from the other odorants. In other experiments, different parts of the antenna are shown to differ with respect to odorant sensitivity. Evidence is also provided that antennal response to odorants varies with age, and that odorants differ in their age dependence.     

Integrating the molecular and cellular basis of odor coding in the Drosophila antenna

We investigate how the molecular and cellular maps of the Drosophila olfactory system are integrated. A correspondence is established between individual odor receptors, neurons, and odors. We describe the expression of the Or22a and Or22b receptor genes, show localization to dendritic membranes, and find sexual dimorphism. Or22a maps to the ab3A neuron, which responds to ethyl butyrate. Analysis of a deletion mutant lacking Or22a, along with transgenic rescue experiments, confirms the mapping and demonstrates that an Or gene is required for olfactory function in vivo. Ectopic expression of Or47a in a mutant cell identifies the neuron from which it derives and its odor ligands. Ectopic expression in a wild-type cell shows that two receptors can function in a single cell. The ab3A neuron does not depend on normal odor receptor gene expression to navigate to its target in the CNS.     

The odor coding system of Drosophila

Our understanding of the molecular and cellular organization of the Drosophila melanogaster olfactory system has increased dramatically in recent years. A large family of approximately 60 odorant receptors has been identified, and many of these receptors have been functionally characterized. The odor responses of olfactory receptor neurons have been characterized, and much has been learned about how odors are represented in olfactory centers in the brain. The circuitry of the olfactory system has been studied in detail, and the developmental mechanisms that specify the wiring and functional diversity of olfactory neurons are becoming increasingly well understood. Thus, functional, anatomical and developmental studies are rapidly being integrated to form a unified picture of odor coding in this model olfactory system.     

Odorant-specific requirements for arrestin function in Drosophila olfaction

The ability to modulate olfactory sensitivity is necessary to detect chemical gradients and discriminate among a multitude of odor stimuli. Desensitization of odorant receptors has been postulated to occur when arrestins prevent the activation of downstream second messengers. A paucity of in vivo data on olfactory desensitization prompts use of Drosophila melanogaster genetics to investigate arrestins' role in regulating olfactory signaling pathways. Physiological analysis of peripheral olfactory sensitivity reveals decreased responsiveness to a host of chemically distinct odorants in flies deficient for arrestin1 (arr1), arrestin2 (arr2), or both. These phenotypes are manifest in odorant- and dose- dependent fashions. Additionally, mutants display altered adaptive properties under a prolonged exposure paradigm. Behaviorally, arr1 mutants are impaired in olfactory-based orientation towards attractive odor sources. As the olfactory deficits vary according to chemical identity and concentration, they indicate that a spectrum of arrestin activity is essential for odor processing depending upon the particular olfactory pathway involved. Arrestin mutant phenotypes are hypothesized to be a consequence of down-regulation of olfactory signaling to avoid cellular excitotoxicity. Importantly, phenotypic rescue of olfactory defects in arr1(1) mutants is achieved through transgenic expression of wild-type arr1. Taken together, these data clearly indicate that arrestins are required in a stimulus-specific manner for wild type olfactory function and add another level of complexity to peripheral odor coding mechanisms that ultimately impact olfactory behavior.     

The molecular basis of odor coding in the Drosophila larva

We have analyzed the molecular basis of odor coding in the Drosophila larva. A subset of Or genes is found to be expressed in larval olfactory receptor neurons (ORNs). Using an in vivo expression system and electrophysiology, we demonstrate that these genes encode functional odor receptors and determine their response spectra with 27 odors. The receptors vary in their breadth of tuning, exhibit both excitation and inhibition, and show different onset and termination kinetics. An individual receptor appears to transmit signals via a single ORN to a single glomerulus in the larval antennal lobe. We provide a spatial map of odor information in the larval brain and find that ORNs with related functional specificity map to related spatial positions. The results show how one family of receptors underlies odor coding in two markedly different olfactory systems; they also provide a molecular mechanism to explain longstanding observations of larval odor discrimination.     

Putative Drosophila odor receptor OR43b localizes to dendrites of olfactory neurons

To gain insight into the role of the recently identified Drosophila seven transmembrane receptor family, we analyzed the cellular and subcellular localization of a member of this family, OR43b. The OR43b receptor is expressed exclusively in a subset of olfactory neurons in the third antennal segment. Consistent with a direct role in odorant transduction, receptor protein is concentrated within the dendrites, but is also present in the axons of the olfactory neurons in which it is expressed. OR43b protein is only detectable relatively late in development suggesting it may not be required for synaptic target choice of the olfactory neurons in which it is expressed. Flies carrying deletions removing one copy of OR43b have the same number of OR43b positive cells in the antenna as flies with two copies, suggesting that simple allelic exclusion of odor receptors may not occur in Drosophila. We show the OR43b gene on the balancer chromosome SM5 is expressed at reduced levels and contains nucleotide polymorphisms predicted to alter two amino acids in the receptor, including an arginine(128) to proline substitution in the first extracellular loop. The subcellular localization of OR43b in olfactory neurons supports the idea that some of the recently identified family of seven transmembrane receptors are odor receptors, and that Drosophila and vertebrates may differ in the developmental processes used to establish the neuronal architecture of the olfactory system.     

Translation of sensory input into behavioral output via an olfactory system

We investigate the logic by which sensory input is translated into behavioral output. First we provide a functional analysis of the entire odor receptor repertoire of an olfactory system. We construct tuning curves for the 21 functional odor receptors of the Drosophila larva and show that they sharpen at lower odor doses. We construct a 21-dimensional odor space from the responses of the receptors and find that the distance between two odors correlates with the extent to which one odor masks the other. Mutational analysis shows that different receptors mediate the responses to different concentrations of an odorant. The summed response of the entire receptor repertoire correlates with the strength of the behavioral response. The activity of a small number of receptors is a surprisingly powerful predictor of behavior. Odors that inhibit more receptors are more likely to be repellents. Odor space is largely conserved between two dissimilar olfactory systems.     

A behavioral odor similarity "space" in larval Drosophila

To provide a behavior-based estimate of odor similarity in larval Drosophila, we use 4 recognition-type experiments: 1) We train larvae to associate an odor with food and then test whether they would regard another odor as the same as the trained one. 2) We train larvae to associate an odor with food and test whether they prefer the trained odor against a novel nontrained one. 3) We train larvae differentially to associate one odor with food, but not the other one, and test whether they prefer the rewarded against the nonrewarded odor. 4) In an experiment like (3), we test the larvae after a 30-min break. This yields a combined task-independent estimate of perceived difference between odor pairs. Comparing these perceived differences to published measures of physicochemical difference reveals a weak correlation. A notable exception are 3-octanol and benzaldehyde, which are distinct in published accounts of chemical similarity and in terms of their published sensory representation but nevertheless are consistently regarded as the most similar of the 10 odor pairs employed. It thus appears as if at least some aspects of olfactory perception are "computed" in postreceptor circuits on the basis of sensory signals rather than being immediately given by them.     

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.     

果蝇嗅觉分子机理研究进展

黑腹果蝇Drosophila melanogaster是生物学研究的重要模式生物,也是探索研究生物体嗅觉奥秘的理想材料。近年来,由于分子生物学技术在神经科学领域的广泛应用,黑腹果蝇嗅觉机理研究取得了许多重大突破, 对气味分子受体及其识别机理、 嗅觉神经电信号的产生和传递、嗅觉信息的加工、编码以及记忆等方面都有了深入的了解。研究表明, 果蝇约1 300个嗅神经元(olfactory receptor neurons, ORNs)共表达62种不同的气味受体蛋白（olfactory receptor proteins, ORs）, 用以检测和识别其所感受的所有化学气味分子。许多OR所识别的气味分子配体已鉴定出来,普通的气味（如水果的气味）由数种不同的OR组合来识别,而信息素（pheromone）分子则由单种特定的OR来检测。气味信息在嗅神经元内转换成神经电信号,嗅觉电信号沿嗅神经元的轴突传递到触角叶, 再经投射神经元（projection neurons, PNs）将信息送至高级中枢如蘑菇体（mushroom body, MB）和侧角（lateral horn, LH）,最终引发行为反应。在黑腹果蝇嗅觉信息传递通路中,某些蛋白如Dock,N-cadherin,Fruitless等起着重要作用,缺失这些蛋白会导致嗅觉异常。本文对这些研究进展作一综述。     

Genetic and functional subdivision of the Drosophila antennal lobe

Olfactory systems confer the recognition and discrimination of a large number of structurally distinct odor molecules. Recent molecular analysis of odorant receptor (OR) genes and circuits has led to a model of odor coding in which a population of olfactory sensory neurons (OSNs) expressing a single OR converges upon a unique olfactory glomerulus. Activation of the OR can thus be read out by the activation of its cognate glomerulus. Drosophila is a powerful system in which to test this model because the entire repertoire of 62 ORs can be manipulated genetically. However, a complete understanding of how fly olfactory circuits are organized is lacking. Here, we present a nearly complete map of OR projections from OSNs to the antennal lobe (AL) in the fly brain. Four populations of OSNs coexpress two ORs along with Or83b, and a fifth expresses one OR and one gustatory receptor (GR) along with Or83b. One glomerulus receives coconvergent input from two separate populations of OSNs. Three ORs label sexually dimorphic glomeruli implicated in sexual courtship and are thus candidate Drosophila pheromone receptors. This olfactory sensory map provides an experimental framework for relating ORs to glomeruli and ultimately behavior.     

Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly

Three classes of neurons form synapses in the antennal lobe of Drosophila, the insect counterpart of the vertebrate olfactory bulb: olfactory receptor neurons, projection neurons, and inhibitory local interneurons. We have targeted a genetically encoded optical reporter of synaptic transmission to each of these classes of neurons and visualized population responses to natural odors. The activation of an odor-specific ensemble of olfactory receptor neurons leads to the activation of a symmetric ensemble of projection neurons across the glomerular synaptic relay. Virtually all excited glomeruli receive inhibitory input from local interneurons. The extent, odor specificity, and partly interglomerular origin of this input suggest that inhibitory circuits assemble combinatorially during odor presentations. These circuits may serve as dynamic templates that extract higher order features from afferent activity patterns.     

Reversing Stimulus Timing in Visual Conditioning Leads to Memories with Opposite Valence in Drosophila

Animals need to associate different environmental stimuli with each other regardless of whether they temporally overlap or not. Drosophila melanogaster displays olfactory trace conditioning, where an odor is followed by electric shock reinforcement after a temporal gap, leading to conditioned odor avoidance. Reversing the stimulus timing in olfactory conditioning results in the reversal of memory valence such that an odor that follows shock is later on approached (i.e. relief conditioning). Here, we explored the effects of stimulus timing on memory in another sensory modality, using a visual conditioning paradigm. We found that flies form visual memories of opposite valence depending on stimulus timing and can associate a visual stimulus with reinforcement despite being presented with a temporal gap. These results suggest that associative memories with non-overlapping stimuli and the effect of stimulus timing on memory valence are shared across sensory modalities.     

Cortical feedback and gating in odor discrimination and generalization

A central question in neuroscience is how context changes perception. In the olfactory system, for example, experiments show that task demands can drive divergence and convergence of cortical odor responses, likely underpinning olfactory discrimination and generalization. Here, we propose a simple statistical mechanism for this effect based on unstructured feedback from the central brain to the olfactory bulb, which represents the context associated with an odor, and sufficiently selective cortical gating of sensory inputs. Strikingly, the model predicts that both convergence and divergence of cortical odor patterns should increase when odors are initially more similar, an effect reported in recent experiments. The theory in turn predicts reversals of these trends following experimental manipulations and in neurological conditions that increase cortical excitability.     

Cross-modal interactions between olfaction and touch

We report two experiments designed to investigate the nature of any cross-modal interactions between olfactory and tactile information processing. In Experiment 1, we assessed the influence of olfactory cues on the tactile perception of fabric softness using computer-controlled stimulus presentation. The results showed that participants rated fabric swatches as feeling significantly softer when presented with a lemon odor than when presented with an animal-like odor, demonstrating that olfactory cues can modulate tactile perception. In Experiment 2, we assessed whether this modulatory effect varied as a function of the particular odors being used and/or of the spatial coincidence between the olfactory and tactile stimuli. The results replicated those reported in Experiment 1 thus further supporting the claim that people's rating of tactile stimuli can be modulated by the presence of an odor. Taken together, the results of the two experiments reported here support the existence of a cross-modal interaction between olfaction and touch.