The Interactive Fly

Zygotically transcribed genes

Olfaction in Drosophila

Olfactory receptors Olfactory neurons of the antenna and maxillary palp Olfactory glomeruli in the antennal lobe Mushroom body and olfactory learning

  • Odor coding in the Drosophila antenna
  • What the fly's nose tells the fly's brain
  • An olfactory sensory map in the fly brain
  • A regulatory code for neuron-specific odor receptor expression
  • Combinatorial rules of precursor specification underlying olfactory neuron diversity
  • Measuring activity in olfactory receptor neurons in Drosophila: Focus on spike amplitude
  • Sex- and tissue-specific profiles of chemosensory gene expression in a herbivorous gall-inducing fly (Diptera: Cecidomyiidae)
  • Olfactory control of blood progenitor maintenance

  • Olfactory wiring, circuitry, and coding
  • Multiple sites of adaptation lead to contrast encoding in the Drosophila olfactory system
  • Precise and fuzzy coding by olfactory sensory neurons
  • Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna
  • Converging circuits mediate temperature and shock aversive olfactory conditioning in Drosophila
  • Wiring variations that enable and constrain neural computation in a sensory microcircuit
  • Non-synaptic inhibition between grouped neurons in an olfactory circuit
  • A conserved dedicated olfactory circuit for detecting harmful microbes in Drosophila
  • Current source density mapping of antennal sensory selectivity reveals conserved olfactory systems between tephritids and Drosophila
  • Differential contributions of olfactory receptor neurons in a Drosophila olfactory circuit
  • Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity
  • A presynaptic gain control mechanism fine-tunes olfactory behavior
  • Olfactory receptor neurons use gain control and complementary kinetics to encode intermittent odorant stimuli

    Functional analysis of specific receptor-encoding genes
  • Olfactory proxy detection of dietary antioxidants in Drosophila
  • Food odors trigger males to deposit a pheromone that guides aggregation and female oviposition decisions
  • Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila
  • Or47b plays a role in Drosophila males' preference for younger mates
  • Farnesol-detecting olfactory neurons in Drosophila

  • Olfactory receptors and behavior
  • Dynamical feature extraction at the sensory periphery guides chemotaxis
  • Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search
  • Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila
  • Drosophila avoids parasitoids by sensing their semiochemicals via a dedicated olfactory circuit
  • Receptors for mate recognition in Drosophila
  • Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration
  • Metabolite exchange between microbiome members produces compounds that influence Drosophila behavior.
  • Olfactory channels associated with the maxillary palp mediate short- and long-range attraction
  • Odor-evoked inhibition of olfactory sensory neurons drives olfactory perception in Drosophila
  • Artificial selection for odor-guided behavior in Drosophila reveals changes in food consumption

    Non-receptor proteins affecting olfaction
  • pdm3 acts in odor receptor expression and axon targeting of olfactory neurons
  • A CD36 ectodomain mediates insect pheromone detection via a putative tunnelling mechanism
  • Central peptidergic modulation of peripheral olfactory responses
  • Hedgehog signaling regulates the ciliary transport of odorant receptors in Drosophila
  • A role for a phospholipid intermediate in insect olfactory transduction
  • Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila

    Characterization of Drosophila odorant receptors

    Animals can detect a vast array of odors with remarkable sensitivity and discrimination. Olfactory information is first received by olfactory receptor neurons (ORNs), which transmit signals into the CNS where they are processed; this ultimately leads to behavioral responses. Electrophysiological and anatomical studies suggest that there are on the order of 36 classes of ORNs in the adult fly (30 on the antenna and 6 on the maxillary palp), each class with a distinct odor sensitivity. Classes of ORNs found in the antenna are arrayed in zones, while the classes of ORNs found in the maxillary palp are distributed in a less ordered fashion. ORNs in both the maxillary palp and the antenna extend their axons to the antennal lobe of the brain, where first-order processing of olfactory information occurs. The lobe contains 40 olfactory glomeruli, spheroidal modules where ORN axons converge and where their terminal branches form synapses with the dendrites of their target interneurons (Clyne, 1999a and references).

    What is the molecular basis for the distinct odor sensitivities of the different classes of ORNs? One possibility is that each class of ORN expresses a unique odorant receptor, as has been proposed for vertebrate olfactory systems (Buck, 1996). In mammals a combinatorial code is used to discriminate between odors. In mammals, any one odorant receptor recognizes multiple odorants and one odorant is recognized by multiple ORs, but different odorants are recognized by different combinations of ORs (Malnic, 1999). Another set of receptors in mammals, associated with ORNs of the vomeronasal organ, distinguishes pheromones (Matsunami, 1997). A second possibility is that each class of ORN might express a unique combination of a large set of receptors, as found in chemosensory cells of the nematode, C. elegans (Troemel, 1995). ORNs have been found to express up to four ORs each in C. elegans. Both models call for a family of receptor genes, and several lines of evidence suggest that for insects such a family would belong to the superfamily of seven-transmembrane G protein-coupled receptors (GPCRs). A genomics approach was taken to identify the odorant receptors in Drosophila. This approach, involving a search through known genome sequences for GPCRs, is arguably the best example of the power of genomics when applied to the discovery of a family of proteins. Sixteen genes have been discovered using computer programs that identify diagnostic features of the protein structure of the seven-transmembrane GPCR superfamily (Clyne, 1999a). A second studied discovered eleven GPCR proteins coding for ORNs (Vosshall, 1999). Members of this new family are highly divergent from previously defined genes. Nearly all of the genes are found to be expressed in one or both of the olfactory organs, and for a number of genes this expression is restricted to a subset of ORNs. Expression of different genes is initiated at different times during the development of the adult antenna (Clyne, 1999a), and expression of a subset of these candidate receptor genes depends on the POU-domain transcription factor, Acj6, also known as Ipou (Clyne, 1999b).

    A two-part strategy was adopted to identify odorant receptor genes from the genomic database. Initially, a computer algorithm was designed to search the Drosophila genomic sequence for open reading frames (ORFs) from candidate odorant receptor genes. Reverse transcription polymerase chain reaction (RT-PCR) was then used to see if transcripts from any of these ORFs were expressed in olfactory organs. For the computational screens, the genomic sequence data obtained by FTP from the Berkeley Drosophila Genome Project was used. ORFs of 300 bases or longer in all six frames were identified. Next, a program written to identify GPCRs statistically by their physicochemical profile was used to screen for candidate ORFs. The number of possible candidates was reduced by comparing them to Drosophila codon usage tables. Further analysis revealed that 8 of the 34 candidate ORFs correspond to genes of known function, as for example, a cyclic nucleotide-gated channel. RT-PCR with primers designed from two of the final candidates yielded amplification products from antennal cDNA. These two genes are located within 500 bp of each other at cytological position 22A (see Odorant receptor 22a and Odorant receptor 22b), and their predicted proteins are 75% identical at the amino acid level (Clyne, 1999a).

    To determine if these two candidates were part of a larger family of genes encoding seven transmembrane domain proteins, their sequences were used in BLAST searches of the Drosophila genome database to identify related genes. Homologs of the two candidates were found, and in turn, their sequences were used for further database searches. In total, 16 genes have been identified from the 16% genomic sequence currently available. This family of genes has been named DOR (for Drosophila olfactory receptor), and each individual gene is named based on its cytogenetic location in the genome. Thus, the two genes identified initially are Odorant receptor 22a and Odorant receptor 22b, which are abbreviated here as 22A.1 and 22A.2 (The final digit in this nomenclature is used to distinguish the genes at a site and does not refer to the cytogenetic band number). Of the 16 family members, 13 have been found to be expressed in either the antenna or the maxillary palp, or in both, based upon RT-PCR analysis and in situ hybridizations to RNA in tissue sections (Clyne, 1999a).

    The DOR genes have no significant similarities to any known genes, and do not appear in any of the Drosophila EST databases. However, hydropathy plots of the predicted proteins show that each has approximately seven peaks that could represent transmembrane domains. The lengths of the 16 proteins are between 369 and 403 amino acids, similar to the lengths of most previously described families of GPCRs. In addition, the spacing of the putative transmembrane domains gives rise to predicted intracellular and extracellular loops similar in size to those in many families of GPCRs. Amino acid sequence identity among the DOR genes ranges from 10%-75%, with many genes showing a relatively low level of identity to each other (20%). Two pairs of clustered genes, 22A.1/22A.2 and 33B.1/33B.2 show the highest identity, with 75% and 57% identities, respectively. However, not all clustered genes show high degrees of similarity. 33B.3, for example, is only 28% identical to both 33B.1 and 33B.2, and 46F.1 and 46F.2 are only 29% identical. In addition to exhibiting sequence identity, many of the genes contain introns in corresponding locations, consistent with their constituting a family derived from a common ancestral gene. There are 67 residues that are conserved among at least 50% of the genes, and most of these are in the C-terminal halves of the proteins. Among the conserved residues are a serine and a threonine in the intracellular C-terminal tail, residues frequently conserved in this region of GPCRs. The most divergent region in the sequences is a stretch of 30 amino acids representing part of the first extracellular loop and nearly all of transmembrane domain 3. The divergence in this region also occurs in the most conserved pairs of genes: 22A.1 and 22A.2 are 75% identical overall but only 50% identical in this region, and 33B.1 and 33B.2 are 57% identical overall but only 33% identical in this region. Transmembrane domains 3, 4, and 5 are exceptionally divergent in rat odorant receptors (Clyne, 1999a) and have been proposed to play a role in odorant binding (Buck, 1991). Some of the genes are clustered in the genome, while others are apparently isolated. Within a cluster, the average intergenic distance is on the order of 500 bp. Clustered DOR genes do not necessarily have introns in corresponding locations (e.g., 46F.1 and 46F.2), but all clustered genes have their transcriptional orientations in the same direction (Clyne, 1999a).

    To determine whether any of the DOR genes have closely related homologs, coding regions from nine of the genes were used to probe Southern blots of Drosophila genomic DNA at high or reduced stringency. Each probe appeared to detect only its own sequence at high stringency, while at lower stringency most genes detected one or two novel bands. As expected, because of the overall low level of similarity, none of these extra bands corresponded to any of the other known DOR genes. These data indicate that some of these genes have one or two closely related homologs but that none belongs to a large subfamily of highly related genes (Clyne, 1999a).

    Although, at present, the clusters of DOR genes identified in Drosophila contain smaller numbers of genes (three or fewer) than in other organisms (Troemel, 1995; Sullivan, 1996; Barth, 1996), a number of interesting features of the clustered genes are already apparent. As found in other organisms (Barth, 1996), Drosophila odorant receptor genes within a cluster are not necessarily coordinately regulated, such that genes within a cluster are expressed in different subsets of cells, and even in different olfactory organs (e.g., 46F.1 is expressed in the maxillary palp, whereas 46F.2 is expressed in the antenna). So far, all genes identified within a cluster, however, are transcribed in the same orientation. Genes within a cluster sometimes do, but sometimes do not, share intron positions, suggesting that introns may have become lost following gene duplication; a phylogenetic study revealed extensive gene duplication and intron loss among the chemoreceptor genes of C. elegans (Robertson, 1998).

    Functional analysis of odorant receptors

    A functional analysis was undertaken of the odorant receptor repertoire in the Drosophila antenna. Each receptor was expressed in a mutant olfactory receptor neuron (ORN) used as a 'decoder,' and the odor response spectrum conferred by the receptor was determined in vivo by electrophysiological recordings. The spectra of these receptors were then matched to those of defined ORNs to establish a receptor-to-neuron map. In addition to the odor response spectrum, the receptors dictate the signaling mode, i.e., excitation or inhibition, and the response dynamics of the neuron. An individual receptor can mediate both excitatory and inhibitory responses to different odorants in the same cell, suggesting a model of odorant receptor transduction. Receptors vary widely in their breadth of tuning, and odorants vary widely in the number of receptors they activate. Together, these properties provide a molecular basis for odor coding by the receptor repertoire of an olfactory organ (Hallem, 2004).

    The odorant receptor (Or) genes in Drosophila are a highly diverse family of 60 genes. Two of them, Or22a and Or22b, have been characterized in detail and were shown to be coexpressed specifically in the ab3A antennal neuron. A deletion mutant called Δhalo that lacks these receptor genes suffers loss of odorant response in the ab3A neuron. This mutant ab3A neuron has been used to characterize other odorant receptors introducing these receptors into the mutant neuron and recording the electrophysiological response to odorants (Hallem, 2004).

    For the ORNs to which a receptor has been mapped, a single odorant receptor appears sufficient to account for the complete odor response spectrum of the ORN. The simplest interpretation of these data is that these Drosophila ORNs express a single functional odorant receptor. Thus, this study provides functional data consistent with the 'one receptor-one ORN' model proposed for mammalian ORNs on the basis of molecular expression studies. Moreover, the ability of these odorant receptors to confer the odor response spectrum of a 'donor' ORN upon the recipient Δab3A neuron indicates that the receptors are the primary determinants of the odor response spectrum. These receptors do not appear to require neuron-specific or sensillum-specific perireceptor molecules in order to confer the odor response spectrum (Hallem, 2004).

    Eleven of the receptors expressed conferred odor response spectra that did not match those of identified ORNs. Not all antennal ORNs have been characterized, e.g., only a limited survey of ORNs in trichoid sensilla is currently available. It seems likely that most of these 11 unmapped receptors derive from ORNs that have not yet been defined. At the same time, a number of defined ORN classes, such as ab1A, have not been matched to a receptor. One possibility is that these ORNs express Or genes whose expression was not initially detected in the antenna and that have not been tested in this study. Alternatively, some could express gustatory receptor (Gr) genes, many of which are expressed in taste organs, where some have been functionally implicated in taste or pheromone perception but at least three of which are expressed in the antenna. Another formal possibility is that some of the unmapped receptors in fact derive from unmatched ORNs but act in pairs in these ORNs or in conjunction with perireceptor molecules that are not available to receptors expressed in ab3A. Several Or genes did not confer detectable odorant sensitivity upon the Δab3A neuron. These receptors could be nonfunctional in vivo, or they could respond specifically to a ligand not present in the odorant panel, such as a pheromone. Finally, two of the receptors analyzed, Or43a and Or43b, have also been functionally characterized by others using different approaches. These results are similar to those reported previously, with only a few exceptions (Hallem, 2004).

    The receptor-to-neuron map provided in this study does not reveal a simple logic relating the ORN and the receptor that it expresses. For example, adjacent ORNs do not consistently express receptors encoded by adjacent genes. Although some pairs of neurons (such as ab5A, ab5B and ab2A, ab2B) express receptors that are relatively closely related by sequence similarity, this relationship does not hold universally: the receptors of ab3A and ab3B are distantly related. Thus, these studies define a problem whose solution is likely to be complex: the evolution of the receptor-to-neuron map (Hallem, 2004).

    The odorant receptor dictates the odor response spectrum of the ORN in which it is expressed in many and perhaps all cases. The results of this study also indicate that the receptor is the primary determinant of three other ORN response properties: spontaneous firing rate, signaling mode, and response dynamics. All four of these properties are likely to play critical roles in odor coding, and some are closely related. For example, the level of spontaneous activity affects the capacity of inhibitory signaling as a mode of information transmission: a high level of spontaneous activity provides a wide operating range in which inhibition can act. A high spontaneous firing level could also affect the sensitivity of the ORN or could have effects on the state of postsynaptic neurons in the antennal lobe. Thus, these results demonstrate a critical role for the odorant receptor in multiple aspects of odor coding. The complexity of the odor code transmitted from the peripheral olfactory organs to the brain depends primarily on the functional properties of odorant receptors (Hallem, 2004).

    The signaling mode of an ORN was found to be determined by its odorant receptor. Different receptors, when expressed in the same ORN and given the same odorant stimulus, can confer responses that differ in signaling mode. A second finding is that a single receptor can mediate both excitatory and inhibitory responses (Hallem, 2004).

    A simple model could explain how the receptor determines both the signaling mode and spontaneous firing rate of the ORN. According to this model, in the absence of odorants, receptors exist in an equilibrium between an 'active' conformation that leads to activation of the G protein-mediated signal transduction cascade and an 'inactive' conformation that does not. The equilibrum constant differs for different receptors, thus explaining differences in spontaneous firing rate among ORNs. The binding of an excitatory odorant stabilizes the active conformation of the receptor, leading to an increase in firing rate. The binding of an inhibitory odorant stabilizes the inactive conformation, leading to a decrease in firing rate. A particular odorant, such as 1-hexanol, might stabilize the active conformation of some receptors, such as Or67a, but stabilize the inactive conformation of other receptors, such as Or47b. Similar models have also been proposed for other GPCRs (Hallem, 2004).

    Response termination kinetics, like spontaneous firing rate, signaling mode, and odor response spectrum, is determined by the odorant receptor. By what mechanism does termination kinetics depend on the receptor but not on the cellular context in which the receptor is expressed? One possibility is that termination kinetics depends primarily on the dissociation constant of the receptor for its odorant ligand. This possibility could explain why an individual receptor can show differences in the termination rate for two odorants, as has been observed with Or7a for E2-hexenal and benzaldehyde. It could also explain why two receptors can show differences in the termination rate for the same odorant, as observed with Or47a and Or98a for pentyl acetate (Hallem, 2004).

    Drosophila ORNs operate in different environments. They reside in different olfactory organs, in sensilla of radically different morphology, and in different molecular contexts, e.g., in proximity to different OBPs. Moreover, the receptors are themselves remarkably divergent in sequence. Given this heterogeneity, one might have expected severe limitations on the ability of receptors to function normally when expressed ectopically in different ORNs (Hallem, 2004).

    Many odorant receptors were found to function normally with respect to a variety of parameters when expressed in the ab3A neuron, and at least some receptors were found to function in a number of diverse neurons. Receptors normally expressed in ORNs of trichoid and coeloconic sensilla (atXA and acXB, respectively) can function in a basiconic sensillum (ab3), despite differences in morphology and OBP content, and antennal receptors can function in the maxillary palp, a developmentally and morphologically distinct organ. Odorant receptors from the malaria vector mosquito Anopheles gambiae have been shown to function in a Drosophila ORN. While it is certainly possible that some receptors, such as those specialized for pheromone detection, might function normally only in their native contexts, the results suggest a broad compatibility between most receptors and ORNs (Hallem, 2004).

    Nearly the entire repertoire of Or receptors has been examined in a highly sensitive olfactory organ, the Drosophila antenna. This analysis has allowed a consideration of the molecular basis of odor coding across an entire olfactory organ, with respect both to the mechanisms of coding and to the functional organization of the system (Hallem, 2004).

    The odor response spectra of these receptors was examined with an odorant panel that is both chemically diverse and ecologically relevant. The odorants include acetate esters, organic acids, alcohols, an aldehyde, ketones, and a monoterpene ester. All of these odorants can be found in either bananas, apples, oranges, pineapples, or black currants. Ethyl acetate, for example, constitutes 33% of the volatiles in pineapple (Hallem, 2004).

    In their natural environment, flies encounter not only a vast array of odorants but also a vast range of odorant concentrations, ranging from low concentrations for a fly in flight to high concentrations for a fly immersed in rotting fruit. In this study, 500 ms pulses of air were directed over odorant solutions that varied in dilution from 10-8 to 10-2. Although these doses are referred to in terms of the dilutions of odorant in the solvent, i.e., '10-2,' these air pulses then undergo a large dilution in another air stream before reaching the fly. It is not known how many molecules of odorant are thereby carried from their hydrophobic solvent to the antenna at room temperature or how this exposure compares to that of a fly standing on a fermenting fruit at higher temperatures. However, virtually all of the firing rates measured in this study are below the maximum firing rates observed for Drosophila ORNs and are thus within the dynamic ranges of ORNs. Moreover, the responses observed are comparable in magnitude to those produced by exposure to natural food sources such as banana, orange, pineapple, mango, and grape, all of which was found to yield responses of ~80-270 spikes/s from ab2A and ab3A neurons. It will be of interest to extend the sampling of odor space to include not only a broader panel of odorants at a wide range of concentrations but, perhaps most important, mixtures of odorants, as flies in the wild rarely encounter a pure odorant (Hallem, 2004).

    All receptors characterized are distinct. Odor response spectra differ between receptors that are encoded by tightly linked genes, receptors that map to neighboring neurons in the same sensillum, and receptors that are more closely related in sequence. At the same time, there is overlap among response spectra. Some odorants elicited strong responses from ~1/3 of the tested receptors. Different receptors vary in their breadth of tuning with respect to the odorant panel: some respond strongly to a single odorant and others to as many as ~70% of the volatile odorants selected for inclusion in the panel (Hallem, 2004).

    The functional overlap among receptors expands the coding capacity of the system by allowing for combinatorial coding, which has been documented previously in other systems. Coding capacity is further expanded, however, by additional diversity in receptor function: receptors confer not only the odor response spectrum but also the response mode and the response dynamics upon the ORNs that express them, as well as the level of spontaneous activity. Thus, there are several degrees of freedom available to each receptor, and the response of the system is multidimensional not only by virtue of its multiplicity of receptors but also by virtue of the multiplicity of response characteristics exhibited by each receptor (Hallem, 2004).

    The olfactory system encodes not only odorant quality, i.e., the identity of an odorant stimulus, but also its intensity. Analysis of a large population of receptors revealed that different odorants are encoded differently across different concentrations. Some odorants elicit strong responses from multiple receptors even at low concentrations, whereas others do not. These results show that differential receptor activation provides a rich coding space in which to register odor intensity (Hallem, 2004).

    These results provide an underlying molecular basis for odor coding, whose cellular basis has been the focus of several recent studies. Optical imaging and electrophysiological studies showed that different odorants activate distinct but overlapping subsets of glomeruli in the antennal lobe of Drosophila and that higher odorant concentrations elicit stronger responses and activate larger numbers of glomeruli. Extensive electrophysiological recordings from projection neurons reveal that they differ in breadth of tuning, signaling mode, and response dynamics, and it will be of interest to determine how the diverse odorant receptor responses described in this study are ultimately transformed into those of the projection neurons. Of particular interest in the representation of odors in the antennal lobe is the role of local interneurons, which form widespread connections among glomeruli and which could register the simultaneous activation of receptors that recognize different features of an odor stimulus (Hallem, 2004).

    The patterns of receptor activation described in this study may not provide all the information necessary for odorant discrimination. For example, the temporal structure of olfactory information has been shown to be critical for odor coding in several systems, and there are other ways of analyzing the temporal dynamics of neuronal activity. However, all of the parameters measured in this study are likely to be essential to odor coding. Olfactory responses are based on the activities of the first-order neurons of the system, the ORNs, and the activities of these neurons are in turn based on the activities of the receptors that have been characterized in this study (Hallem, 2004).

    Odor coding depends on the existence of multiple ORN classes, each with different response characteristics. This organization depends ultimately on the regulated expression of individual receptors in defined subsets of ORNs. Little is known about the mechanisms by which ORNs select, from among a large repertoire, which genes to express. The receptor-to-neuron map established in this study provides a foundation for exploring the developmental mechanisms by which the molecular basis of odor coding in this system is established (Hallem, 2004).

    Molecular determinants of odorant receptor function in insects

    The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later computational methods can be added to further understanding of how specific odorants are detected by receptors. Using a comparative approach this study identified two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection (Ray, 2014).

    Insect olfactory receptors are heteromeric ligand-gated ion channels

    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. This study provides 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 show 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 (Sato, 2008).

    Taken together, these data provide compelling evidence that a heteromeric complex of a conventional insect OR and the highly conserved Or83b family co-receptor has the characteristics of a cation non-selective ion channel directly gated by odour or pheromone ligands. It is concluded that G-protein-mediated signalling is negligible in producing the current elicited by odour activation of insect OR heteromultimers. These findings provide insight into long-argued insect olfactory transduction mechanisms and may explain the lack of clear consensus on the role of second messengers in this process. The insect ORs share no homology with any previously described ion channel and do not contain any known ion selectivity filter motifs. Insect OR activity is not inhibited by Gd3+, a lanthanide that is a broad-spectrum ion channel inhibitor. Therefore, although the ionic permeability reported in this study for Na+, K+ and Ca2+ would be consistent with the properties of non-selective cation channels, a molecular basis for this novel ionotropic activity remains to be elucidated. The spontaneous activity of the OR complex found in this study seems to account for previous observations that olfactory sensory neurons exhibit bipolar electrical activity and become electrically negative on the deletion of Or83b in vivo. Given that there are 62 and 79 potential ligand-binding OR subunits in Drosophila and Anopheles, respectively, the insect ORs may represent the largest single family of ion-channel-like proteins in any organism. This work also raises the intriguing possibility that the insect gustatory system, which senses bitter and sweet tastants as well as carbon dioxide, shares this ionotropic coupling mechanism with the insect ORs. In fact, an ionotropic sugar-gated channel in fleshfly taste cells has previously been reported. This finding offers the caveat that other orphan receptors classified as G-protein-coupled receptors merely because of their putative seven-transmembrane topology may instead possess ligand-gated channel activities, as has been shown previously for light-activated channelrhodopsin. This work has important implications for worldwide efforts to identify specific inhibitors for the insect ORs, which may prove useful in controlling host-seeking behaviours of disease-vector insects such as mosquitoes (Sato, 2008).

    From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors1 that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila6. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. This study shows that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca2+ concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling (Wicher, 2008).

    Sex- and tissue-specific profiles of chemosensory gene expression in a herbivorous gall-inducing fly (Diptera: Cecidomyiidae)

    The chemical senses of insects mediate behaviors that are closely linked to survival and reproduction. The order Diptera contains two model organisms, the vinegar fly Drosophila melanogaster and the mosquito Anopheles gambiae, whose chemosensory genes have been extensively studied. Representing a third dipteran lineage with an interesting phylogenetic position, and being ecologically distinct by feeding on plants, the Hessian fly (Mayetiola destructor Say, Diptera: Cecidomyiidae) genome sequence has recently become available. Among plant-feeding insects, the Hessian fly is unusual in 'reprogramming' the plant to create a superior food and in being the target of plant resistance genes, a feature shared by plant pathogens. Chemoreception is essential for reproductive success, including detection of sex pheromone and plant-produced chemicals by males and females, respectively. This study identified genes encoding 122 odorant receptors (OR), 28 gustatory receptors (GR), 39 ionotropic receptors (IR), 32 odorant binding proteins, and 7 sensory neuron membrane proteins in the Hessian fly genome. Illumina-sequenced transcriptome reads were mapped to the genome to explore gene expression in male and female antennae and terminal abdominal segments. The results reveal that a large number of chemosensory genes have up-regulated expression in the antennae, and the expression is in many cases sex-specific. Sex-specific expression is particularly evident among the Or genes, consistent with the sex-divergent olfactory-mediated behaviors of the adults. In addition, the large number of Ors in the genome but the reduced set of Grs and divergent Irs suggest that the short-lived adults rely more on long-range olfaction than on short-range gustation. Also, this study reports up-regulated expression of some genes from all chemosensory gene families in the terminal segments of the abdomen, which play important roles in reproduction. This study shows that a large number of the chemosensory genes in the Hessian fly genome have sex- and tissue-specific expression profiles. These findings provide the first insights into the molecular basis of chemoreception in plant-feeding flies, representing an important advance toward a more complete understanding of olfaction in Diptera and its links to ecological specialization (Andersson, 2014).

    Feeding regulates sex pheromone attraction and courtship in Drosophila females

    In Drosophila melanogaster, gender-specific behavioural responses to the male-produced sex pheromone cis-vaccenyl acetate (cVA) rely on sexually dimorphic, third-order neural circuits. This study shows that nutritional state in female flies modulates cVA perception in first-order olfactory neurons. Starvation increases, and feeding reduces attraction to food odour, in both sexes. Adding cVA to food odour, however, maintains attraction in fed females, while it has no effect in males. Upregulation of sensitivity and behavioural responsiveness to cVA in fed females is paralleled by a strong increase in receptivity to male courtship. Functional imaging of the antennal lobe (AL), the olfactory centre in the insect brain, shows that olfactory input to DA1 and VM2 glomeruli is also modulated by starvation. Knocking down insulin receptors in neurons converging onto the DA1 glomerulus suggests that insulin-signalling partly controls pheromone perception in the AL, and adjusts cVA attraction according to nutritional state and sexual receptivity in Drosophila females (Lebreton, 2015).

    Drosophila males and females meet on ripe fruit where they feed, mate and oviposit. Accordingly, they perceive food olfactory cues and pheromones as an ensemble. That environmental and social cues cannot be dissociated in natural habitats is reflected by the behavioural and chemical ecology of the fly. Grosjean (2011) established how food odours enhance the sexual behaviour of Drosophila males. Projection neurons downstream of sensory neurons dedicated to pheromone and food odours converge in the pheromone processing region of the lateral horn, to promote male courtship behaviour. This study shows that females and males use a first-order olfactory pathway for the integration of male-produced sex pheromone cVA and food signals, and that the female behavioural response to sex and food odours is modulated by its nutritional state, which also influences sexual receptivity (Lebreton, 2015).

    The male-produced sex pheromone cVA functions to increase female receptivity to male courtship. Behavioural studies of a blend of cVA and food odour vs. food odour alone show behavioural synergism and a response modulation in fed females, and demonstrate that the olfactory pathways responding to these signals are interconnected. Starved females prioritize the search for food, cVA has no effect on their upwind flight response and their odour preference in a choice test. Fed females, on the other hand, which are sexually receptive, showed a clear response to the blend of cVA and food odour. Fed males, in comparison, showed little activity in response to olfactory stimuli. Unlike females, males preferred cVA only when starved, supporting the idea that starvation increases odour sensitivity in males, disregarding the nature of the stimulus (Lebreton, 2015).

    Adult Drosophila females require nutrient intake for reproductive functions, including oogenesis. An association between nutritional state and reproductive behaviour is a well-conserved feature in many other animals and even in mammals, a decrease in sexual receptivity is accompanied by a loss of preference for social odours signals (Lebreton, 2015).

    A sexually dimorphic behavioural response to cVA, i.e. increased female receptivity to male courtship vs. male-male aggression and courtship inhibition, relies on sexually dimorphic third-order neurons. Food-related odour, by itself, enhances male courtship behaviour through activation of sexually dimorphic courtship circuitry (Lebreton, 2015).

    The modulation of cVA perception in starved vs. fed females shown in this study effects first-order olfactory neurons in the AL. cVA stimulates the DA1 glomerulus. In addition, it elicits a response in two isomorphic glomeruli, DM2 and VM2, which also respond to vinegar odour. The response pattern in VM2 to cVA, as well as the behavioral response to a blend of cVA and food odours are starvation-dependent and gender-specific. It remains to be determined how olfactory input modulation and behavioral response modulation are interconnected (Lebreton, 2015).

    The global metabolic cue insulin and local signalling with short neuropeptide F (sNPF) have been shown to interact in the AL to regulate the attraction response to food cues according to nutritional state. Following feeding, insulin (via activation of InR) inhibits the expression of sNPF receptors in DM1 OSNs and therefore decreases sensitivity to food odours by reducing synaptic transmission. The results confirm that DM1, DM2 and DM4 glomeruli, which respond to starvation, are activated by vinegar odour. Disruption of insulin signalling in DA1, on the other hand, induces a loss of the preference for cVA in fed females. This suggests that insulin acts on the female olfactory system to regulate pheromone attraction (Lebreton, 2015).

    Insulin is a key regulator of insect development, metabolism and behaviour. The role of insulin in regulating Drosophila sexual behaviour remains, nonetheless, controversial. Although insulin regulates female remating, it does not affect sexual receptivity in unmated females, which was confirmed by using a temperature-sensitive mutant of InR. This suggests that the nutritional state regulates both pheromone perception and sexual receptivity in females through two distinct mechanisms. Insulin signalling is required, at least, in the DA1 glomerulus to induce pheromone attraction and in the DM1 glomerulus to reduce food attraction in fed Drosophila females. The mechanisms by which the same hormonal pathway can both up- and downregulate sensitivity to different odours are yet unknown. A combination of excitatory and inhibitory local interneurons or projection neurons, receiving differential OSN input, may underlie such a bimodal response (Lebreton, 2015).

    Another scenario pertains to the participation of sugar receptors in feeding-induced olfactory response modulation. Sugar receptors function to sense external, as well as internal sugars in the hemolymph, and very recently, it has further been shown that antennal neurons, expressing Gr64b together with Orco, coincidently project to DA1 and VM251. This finding will certainly stimulate future work on the physiological mechanisms regulating sexual behaviour as a function of nutritional state in Drosophila (Lebreton, 2015).

    Drosophila courtship is a classical paradigm for studying the neural logic of innate behaviour. Research emphasis has been placed on the male-produced sex pheromone cVA and the neural circuits encoding sex-specific behavioural responses. The DA1 glomerulus is known to contribute to cVA attraction. This study has shown that cVA activates also the sexually isomorphic DM2 and VM2 glomeruli, which respond to vinegar, and that perception of cVA and food odour interacts in these glomeruli, in a gender-specific fashion. It follows that investigations of physiological and behavioural responses to cVA should take habitat or food odours into account, since in nature, the flies perceive social and environmental signals as an ensemble (Lebreton, 2015).

    The behavioural response to olfactory stimuli is not a constant, but is modulated, following mating or feeding, to match physiological internal states. In this study, behavioural tests showed that the olfactory attraction to food odour and sex pheromone is modulated according to nutritional state and sexual receptivity (Lebreton, 2015).

    Food odors trigger males to deposit a pheromone that guides aggregation and female oviposition decisions: 9-tricosene activates antennal basiconic Or7a receptors

    Animals use olfactory cues for navigating complex environments. Food odors in particular provide crucial information regarding potential foraging sites. Many behaviors occur at food sites, yet how food odors regulate such behaviors at these sites is unclear. Using Drosophila melanogaster as an animal model, this study found that males deposit the pheromone 9-tricosene upon stimulation with the food-odor apple cider vinegar. This pheromone acts as a potent aggregation pheromone and as an oviposition guidance cue for females. Genetic, molecular, electrophysiological, and behavioral approaches were used to show that 9-tricosene activates antennal basiconic Or7a receptors, a receptor activated by many alcohols and aldehydes such as the green leaf volatile E2-hexenal. Loss of Or7a+ neurons or the Or7a receptor abolishes aggregation behavior and oviposition site-selection towards 9-tricosene and E2-hexenal. 9-Tricosene thus functions via Or7a to link food-odor perception with aggregation and egg-laying decisions (Lin, 2015).

    An antennal carboxylesterase from Drosophila melanogaster, Esterase 6, is a candidate odorant-degrading enzyme toward food odorants

    Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors. Recently work has shown that an extracellular carboxylesterase, Esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, tests were performed to see EST-6 could play a broader function in the antennae. Recombinant EST-6 was found to be able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate showed that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, a role as an ODE is hypothesized for this enzyme toward food volatiles.

    Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila

    Ionotropic glutamate receptors (iGluRs; see Ionotropic receptor 8a) mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. This paper characterizes a family of iGluR-related genes in Drosophila, which have been named ionotropic receptors (IRs). These receptors do not belong to the well-described kainate, AMPA, or NMDA classes of iGluRs, and they have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Misexpression of IRs in different olfactory neurons is sufficient to confer ectopic odor responsiveness. Together, these results lead to the proposal that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues (Benton, 2009).

    Species as diverse as bacteria, plants, and humans have the capacity to sense small molecules in the environment. Chemical cues can transmit the presence of food, alarm signals, and messages from conspecifics that signify mating compatibility. Peripheral chemical recognition largely relies on membrane receptor proteins that interact with external ligands and convert this binding into intracellular responses. The vast majority of identified chemosensory receptors in multicellular organisms belong to the seven transmembrane domain G protein-coupled receptor (GPCR) superfamily, including odorant, gustatory and pheromone receptors in mammals, birds, reptiles, amphibians, fish, and nematodes. Unicellular organisms also use GPCRs for chemoreception, such as the pheromone receptors in budding yeast (Benton, 2009).

    Insects can detect a wide range of environmental chemicals: bitter, sweet, and salty tastants, odors, pheromones, humidity, carbon dioxide, and carbonated water. Most of these chemosensory stimuli are recognized by members of two evolutionarily related insect-specific chemosensory receptor families, the Odorant Receptors (ORs) and Gustatory Receptors (GRs). These proteins contain seven predicted transmembrane domains but are evolutionarily unrelated to GPCRs and adopt a distinct membrane topology. Recent analysis has indicated that insect ORs function as odor-gated ion channels (Sato, 2008; Wicher, 2008), setting them mechanistically apart from metabotropic vertebrate ORs (Benton, 2009).

    Comprehensive analysis of the expression of these receptors in Drosophila, has hinted at the existence of other types of insect chemosensory receptors (Couto, 2005; Yao, 2005). This is particularly apparent in the major olfactory organ, the third segment of the antenna, which bears three types of olfactory sensory hairs (sensilla): basiconic, trichoid, and coeloconic. All olfactory sensory neurons (OSNs) innervating basiconic and trichoid sensilla generally express one OR, along with the OR83b co-receptor. However, with the exception of OR35a/OR83b-expressing neurons (Yao, 2005), OSNs housed in coeloconic sensilla do not express OR83b or members of the OR or GR gene families. Nevertheless, electrophysiological analysis has revealed the existence of multiple types of coeloconic OSNs tuned to acids, ammonia and humidity (Yao, 2005), suggesting that other types of insect chemosensory receptors exist (Benton, 2009).

    A bioinformatic screen has been carried out for insect-specific genes enriched in OSNs (Benton, 2007). Among these, a large expansion was found of the ionotropic glutamate receptor (iGluR) gene family of unknown biological function (Littleton, 2000). This study provides evidence that these variant iGluRs represent a novel class of chemosensory receptor (Benton, 2009).

    The screen identified 6 antennal-expressed genes encoding proteins annotated as ionotropic glutamate receptors (iGluRs) (Littleton, 2000). Using these novel receptor sequences as queries, exhaustive BLAST searches of the Drosophila genome identified a family of 61 predicted genes and 1 pseudogene. These genes are distributed throughout the genome, both as individual sequences and in tandem arrays of up to four genes. This family was named the Ionotropic Receptors (IRs), and individual gene names were assigned to the IRs using nomenclature conventions of Drosophila ORs (Benton, 2009).

    Phylogenetic analysis of predicted IR protein sequences revealed that they are not closely related to members of the canonical families of iGluRs (AMPA, kainate, NMDA, or delta). However, they appear to have a similar modular organization to iGluRs, comprising an extracellular N-terminus, a bipartite ligand-binding domain, whose two lobes (S1 and S2) are separated by an ion channel domain, and a short cytoplasmic C-terminus. It is noted that the gene structure and protein sequence of most receptors are presently only computational predictions. Nevertheless, the family is extremely divergent, exhibiting overall amino acid sequence identity of 10-70%. The most conserved region between IRs and iGluRs spans the ion channel pore, suggesting that IRs retain ion-conducting properties (Benton, 2009).

    The ligand-binding domains are considerably more variable, although alignment of small regions of the S1 and S2 lobes of IRs and iGluRs allowed examination of conservation in amino acid positions that make direct contact with glutamate or artificial agonists in iGluRs. While all iGluRs have an arginine (R) residue in S1 that binds the glutamate α-carboxyl group, only 19/61 (31%) IRs retain this residue. In the first half of the S2 domain, 9/61 (15%) of IRs retain a threonine (T), which contacts the glutamate γ-carboxyl group in all AMPA and kainate receptors. Interestingly, the iGluRs that lack this T residue (NR1, NR3A, delta) have glycine or serine and not glutamate as a preferred ligand (Mayer, 2006; Naur, 2007). Finally, in the second half of the S2 domain, 100% of the iGluRs have a conserved aspartate (D) or glutamate (E) that interacts with the α-amino group of the glutamate ligand, compared with 10/61 (16%) IRs. Of 61 IRs, only three (IR8a, IR75a, IR75c) retain the R, D/E, and T residues characteristic of iGluRs, although these residues lie within a divergent structural backbone. Other IRs have a diversity of different amino acids at one or more of these positions. Thus, the ligand-binding specificity of most or all IRs is likely to be both distinct from that of iGluRs and varied within the IR family (Benton, 2009).

    The expression of the IR family was determined by both tissue-specific RT-PCR and RNA in situ hybridization. Fifteen IR genes are expressed in the antenna. Transcripts of these genes were not detected elsewhere in the adult head, body or appendages, except for IR25a and IR76b, which are also expressed in the proboscis. Expression of the remaining 46 IR genes was not reproducibly detected in any adult tissue. It is unclear whether these genes are not expressed, expressed at different life stages, or expressed in at levels below the detection threshold of these assays (Benton, 2009).

    Analysis of where in the antenna IR genes are expressed compared to ORs was performed by double RNA in situ hybridization with probes for the OR co-receptor OR83b and one of several IR genes, including IR64a, IR76b, IR31a, and IR40a. IRs are not expressed in basiconic and trichoid sensilla, as they are not co-expressed with OR83b, and IR expression persists in mutants for the proneural gene absent md neurons and olfactory sensilla (amos), which completely lack these sensilla types. However, expression of these IRs is dependent upon the proneural gene atonal, which specifies the coeloconic sensilla as well as a feather-like projection called the arista, and a three-chambered pocket called the sacculus. Thus, ORs and IRs are expressed in developmentally distinct sensory lineages in the antenna. One exception is the subpopulation of coeloconic OSNs that expresses both IR76b and OR35a and OR83b. It was confirmed that IR-expressing cells in the antenna are neurons by demonstrating that they co-express the neuronal marker elav (Benton, 2009).

    A comprehensive map was generated of IR expression. Each IR was observed to have a topologically-defined expression pattern that is conserved across individuals of both sexes. IR8a and IR25a, which encode closely related receptors, are broadly expressed, detected in overlapping populations of neurons around the sacculus and in the main portion of the antenna. IR25a but not IR8a is also detected in the arista. IR21a is expressed in approximately 6 neurons in the arista, as well as 5-10 neurons near the third chamber of the sacculus. Three IRs display specific expression in neurons surrounding the sacculus: IR40a and IR93a are co-expressed in 10-15 neurons adjacent to the first and second sacculus chambers, while IR64a is found in 10-15 neurons surrounding the third chamber (Benton, 2009).

    The remaining 9 IRs are expressed in coeloconic OSNs distributed across the antenna. Double and triple RNA in situ hybridization revealed that individual neurons express between 1 and 3 different IR genes and are organized into specific clusters of two or three neurons. Four distinct clusters (cluster A-cluster D), containing two (cluster C) or three (cluster A, B, and D) neurons, could be defined by their expression of stereotyped combinations of IR genes. Cluster C includes a coeloconic neuron that expresses OR35a and OR83b in addition to IR76b. Although each cluster is distinct, there is overlap between the IRs they express. IR76b is expressed in one neuron in all four clusters, IR75d in three clusters and IR75a in two clusters. In additional to these selectively-expressed receptors, individual neurons are likely to express one or both of the broadly-expressed IR8a and IR25a. The combinatorial expression patterns of the IRs raise the possibility that these genes define specific functional properties of these neurons (Benton, 2009).

    Definition of four distinct clusters of IR-expressing neurons in the antenna is consistent with the identification of four types of coeloconic sensilla, named ac1-ac4, which have distinct yet partially overlapping sensory specificities (Yao, 2005). To examine whether IR expression correlates with the chemosensory properties of these OSNs, the spatial organization of IR-expressing neurons was compared using probes for unique IR markers for each cluster type to these functionally distinct sensilla types. As a unique molecular marker for Cluster B is lacking, this cluster was defined as those containing IR75a-expressing OSNs (present in Cluster B and Cluster C) that are not paired with OR35a-expressing cluster C neurons. It was found that each cluster has a different, though overlapping, spatial distribution in the antenna. For example, Cluster A neurons (marked by IR31a) are restricted to a zone at the anterior of the antenna, just below the arista, while cluster C neurons (marked by IR75b) are found exclusively in the posterior of the antenna. These stereotyped IR neuron distributions were observed in antennae from over 20 animals (Benton, 2009).

    The initial description of the coeloconic sensilla classes did not describe their spatial distribution (Yao, 2005). This study therefore recorded odor-evoked responses in >100 coeloconic sensilla in several dozen animals across most of the accessible antennal surface, using a panel of odorants that allowed identification unambiguously of each sensilla type (ammonia for ac1, 1,4-diaminobutane for ac2, propanal and hexanol for ac3, and phenylacetaldehyde for ac4) (Yao, 2005). After electrophysiological identification, the location of the sensilla on the antennal surface was noted (Benton, 2009).

    This mapping process allowed a correlation of the electrophysiological and molecular properties of the coeloconic sensilla. For example, ac1 sensilla were detected only in a region on the anterior antennal surface just ventral to the arista, and therefore are most likely correspond to cluster A, containing IR31a-IR75d-IR76b/IR92a-expressing neurons. The data fit well with the previous assignment of the OR35a-expressing neuron to the ac3 sensillum (Yao, 2005), which is found on the posterior of the antenna and is the only coeloconic sensillum class that unambiguously houses two neurons (Yao, 2005). While these results allow initial assignment of IRs to different coeloconic sensilla classes, it is noted that assignment of specific odor responses to individual IR-expressing OSNs is not possible from these data alone (Benton, 2009).

    All neurons expressing a given OR extend axons that converge upon a single antennal lobe glomerulus, resulting in the representation of a cognate odor ligand as a spatially-defined pattern of neural activity within the brain. To ask whether IR-expressing neurons have the same wiring logic, the targeting of OSNs expressing IR76a was investigated by constructing an IR76a-promoter GAL4 driver that recapitulates the endogenous expression pattern. Labeling of these neurons with mCD8:GFP revealed convergence of their axons on to a single glomerulus, ventral medial 4 (VM4), in the antennal lobe. This glomerulus is one of approximately eight that was previously unaccounted for by maps of axonal projections of OR-expressing OSNs (Benton, 2009).

    To determine where IRs localize in sensory neurons, antibodies were generated against IR25a. Broad expression of IR25a protein was detected in sensory neurons of the arista, sacculus, and coeloconic sensilla. All anti-IR25a immunoreactivity was abolished in an IR25a null mutant. Low levels of IR25a could be detected in the axon segment adjacent to the cell body in some neurons but no staining was observed along the axons as they entered the brain, or at synapses within antennal lobe glomeruli. In coeloconic neurons, prominent anti-IR25a staining was detected both in the cell body and in the distal tip of the dendrite, which corresponds to the ciliated outer dendritic segment innervating the sensory hair. Relatively low levels were detected in the inner dendrites, suggesting the existence of a transport mechanism to concentrate receptor protein in cilia. A similar subcellular localization was observed in sacculus and aristal sensory neurons. The specific targeting of an IR to sensory cilia suggests a role for these proteins in sensory detection (Benton, 2009).

    To test the hypothesis that IR genes encode chemosensory receptors, whether ectopic IR expression could induce novel olfactory specificities was investigated. Three IRs expressed in ac4 sensilla (IR84a, IR76a and IR75d) were individually mis-expressed in ac3 sensilla using the OR35a-GAL4 driver. Single sensillum recordings were used to examine which, if any, of these three IRs, could confer sensitivity to phenylacetaldehyde, the only known robust ligand for ac4 but not ac3 sensilla (Yao, 2005). Mis-expression of IR84a conferred a strong response to phenylacetaldehyde that was not observed in control strains or in animals mis-expressing either IR76a or IR75d. Ectopically-expressed IR84a did not confer sensitivity to the structurally related odor, phenylacetonitrile, which does not activate either ac3 or ac4 neurons (Yao, 2005). This indicates that mis-expressed IR84a does not simply generate non-specific ligand sensitivity in these neurons (Benton, 2009).

    Next the novel odor responses conferred by IR84a mis-expression were compared to the endogenous phenylacetaldehyde responses of ac4 sensilla by generating dose-response curves. Stimulus evoked spike frequencies of ac3 sensilla ectopically expressing IR84a are quantitatively very similar to those in ac4 sensilla, even exceeding the endogenous ac4 responses at higher odor concentrations. These elevated responses are likely to be due to the contribution of weak endogenous phenylacetaldehyde responses that were observed in ac3 sensilla at high stimulus concentrations, as subtraction of these values produces an IR84a-dependent phenylacetaldehyde dose-response curve that is statistically the same as that of ac4 sensilla. Thus, ectopic expression of a single IR in ac3 is sufficient to confer a novel ligand- and receptor-specific odor sensitivity that is physiologically indistinguishable from endogenous responses (Benton, 2009).

    To extend this analysis to a second IR, whether mis-expression of one of the IR genes uniquely expressed in ammonia-sensitive ac1 neurons (IR31a and IR92a) was sufficient to confer ectopic responsiveness to this odor was examined. Because ac3 sensilla neurons display endogenous ammonia-evoked responses at modest stimulus concentrations, these experiments used the IR76a-promoter GAL4 transgene to mis-express these receptors in ammonia-insensitive ac4 sensilla (Yao, 2005). ac4 sensilla mis-expressing IR92a, but not IR31a, displayed responses to ammonia. 1,4-diaminobutane, a control stimulus that does not activate either ac1 or ac4 neurons (Yao, 2005), did not stimulate ac4 sensilla mis-expressing IR92a. It was noted that the magnitude of the ectopic IR92a ammonia response is lower than native ammonia-evoked responses of ac1 sensilla (Yao, 2005). This may be due to the lack of co-factors present in ac1 sensilla but not in ac4 sensilla. Nevertheless, these results suggest that IR92a comprises at least part of an ammonia-specific chemosensory receptor (Benton, 2009).

    The specific combinatorial expression patterns of IRs in sensory neurons and the diversity in their ligand-binding domains is difficult to rationalize with a general role in signal transduction, independent of ligand recognition. More importantly, the novel olfactory sensitivity induced by ectopic expression of IR84a and IR92a provides evidence that IR proteins function directly as ligand-specific, chemosensory receptors. While these experiments demonstrate a sufficiency of IRs for conferring odor-responsiveness, definitive proof of their necessity will require analysis of loss-of-function mutations (Benton, 2009).

    In animal nervous systems, iGluRs mediate neuronal communication by forming glutamate-gated ion channels, and it is speculated that IRs also form ion channels, gated by odors and other chemosensory stimuli. A growing number of ionotropic mechanisms in chemoreception are known. For example, members of the transient receptor potential (TRP) family of ion channels are the primary receptors for nociceptive compounds including capsaicin and menthol and have also been implicated in gustatory detection of acids. Insect ORs also display functional properties of ion channels. Proof that IRs function as ion channels will necessitate electrophysiological characterization of these receptors in heterologous expression systems, and evidence for direct binding of chemosensory ligands to IRs will require biochemical assays in vitro (Benton, 2009).

    iGluRs normally function as heterotetrameric assemblies of variable subunit composition that exhibit differing functional properties such as ligand sensitivity and ion permeability. The current analysis indicates that up to five different IRs may be co-expressed in a single sensory neuron, raising the possibility that these receptors also form multimeric protein assemblies with subunit-dependent characteristics. Of particular interest are the two broadly-expressed members of the family, IR8a and IR25a, which may represent common subunits in many different types of IR complexes. Their function is unclear, but it is possible that they have a co-receptor function with other IRs, analogous to that of OR83b. Preliminary analysis of IR25a mutants revealed no obvious defects in odor-evoked responses in coeloconic sensilla, but this may be due to redundancy of IR25a with IR8a or the existence of homomeric IR receptors without IR8a or IR25a. Other IRs, such as IR75a and IR76b, are expressed in two or more types of coeloconic sensory neurons. In these cases, the response properties may be defined by the combination of IRs expressed in these distinct neuronal populations. However, the present lack of knowledge of relevant ligands for several coeloconic OSNs makes it difficult to match specific ligands to individual IR neurons based on the expression map alone (Benton, 2009).

    The IR repertoire is remarkably similar in size, overall genomic organization and sequence divergence to Drosophila ORs. Like the ORs, individual IRs are specifically expressed in small subpopulations of chemosensory neurons, and this expression is regulated by relatively short (< 1-2 kb) upstream regulatory regions. Furthermore, at least one population of IR-expressing neurons converges on to a single glomerulus in the antennal lobe, similar to the wiring logic established for OR-expressing neurons both in invertebrate and vertebrate olfactory systems. Some differences are observed, however, in the organizational logic of IR and OR expression. Most OR-expressing neurons express a single OR gene, along with OR83b, in distinct clusters that innervate specific olfactory hairs. In contrast, many IR-expressing neurons identified in the antenna express 2 or 3 IR genes, in addition to one or both of the broadly-expressed IR8a and IR25a genes. Moreover, overlap is observed both between the molecular composition of different IR neurons and the combination of neurons that innervate a given sensillum. For example, IR76b is co-expressed with at least two other different IR genes in at least two different sensilla - with IR92a in ac1 and with IR76a in ac4 - as well as being co-expressed with OR35a and OR83b in ac3. While the precise biological logic of IR co-expression awaits the matching of specific chemosensory ligands to IR-expressing neurons, combinatorial expression of IRs may contribute more significantly to their role in sensory detection than for ORs (Benton, 2009).

    Why does Drosophila possess two types of antennal chemosensory receptors? Although both may be ionotropic, IRs and ORs are not simply slight evolutionary variants. The receptor families are molecularly unrelated, are under the control of distinct developmental programs, and housed within sensory structures of radically different morphology. Thus, it seems likely that these chemosensory receptors fulfill distinct functions in chemosensation. Analysis of the chemosensory behaviors mediated by IR sensory circuits (now possible with the identification of specific molecular markers for these pathways) may provide insights into the contributions of these different olfactory subsystems. IRs may also have functions in other chemosensory modalities, as two antennal IRs are also detected in the proboscis, and the expression of 46 members of the repertoire remains unknown (Benton, 2009).

    Chemosensation is an ancient sensory modality that predates the evolution of the eukaryotes. Are there traces of conservation in the molecular mechanism by which prokaryotes and eukaryotes sense external chemicals? iGluRs have long been recognized to have prokaryotic origins. Their ion channel domain is homologous to bacterial potassium channels, and the ligand binding domain is structurally related to bacterial periplasmic binding proteins (PBPs), extracellular proteins that scavenge or sense amino acids, carbohydrates and metal ions by coupling to transporters or chemotaxis receptors. Evolutionary connections between iGluRs and PBP function have not often been considered, perhaps in part due to their very weak primary sequence similarity, the widespread occurrence of the PBP fold -also present, for example, in bacterial transcription regulators - and the dedicated role for iGluRs in mediating or regulating synaptic transmission, a process seemingly distant from bacterial solute uptake and chemotaxis (Benton, 2009).

    This discovery of a family of divergent iGluR-like proteins that may act as peripheral chemosensors provides a link between the disparate functions of these protein modules. While a role for IRs in detecting diverse external ligands is analogous to the function of bacterial PBPs, the primary sequence and neuronal expression of IRs is clearly closer to the properties of iGluRs. Intriguingly, a large family of iGluR-related proteins, the GLRs has also been identified in the plant Arabidopsis thaliana (Lam, 1998; Chiu, 1999). Almost nothing is known about their physiological functions, but bioinformatic analysis of GLRs suggests that glutamate is unlikely to be their natural ligand (Dubos, 2003; Qi, 2006). It is possible that GLRs may have roles as chemosensors, for example in detection of soil nutrients or airborne volatiles. Thus, while iGluRs have been intensely studied for their roles in synaptic communication, this characterization of the IRs leads to the suggestion that the ancestral function of this protein family may have been in detecting diverse chemical ligands to mediate both intercellular communication and environmental chemical sensing (Benton, 2009).

    Farnesol-detecting olfactory neurons in Drosophila

    This study set out to deorphanize a subset of putative Drosophila odorant receptors expressed in trichoid sensilla using a transgenic in vivo misexpression approach. Farnesol was identified as a potent and specific activator for the orphan odorant receptor Or83c. Farnesol is an intermediate in juvenile hormone biosynthesis, but is also produced by ripe citrus fruit peels. This study shows that farnesol stimulates robust activation of Or83c-expressing olfactory neurons, even at high dilutions. The CD36 homolog Sensory neuron membrane protein 1 (Snmp1) is required for normal farnesol response kinetics. The neurons expressing Or83c are found in a subset of poorly characterized intermediate sensilla. It was shown tha these neurons mediate attraction behavior to low concentrations of farnesol and Or83c receptor mutants are defective for this behavior. Or83c neurons innervate the DC3 glomerulus in the antennal lobe and projection neurons relaying information from this glomerulus to higher brain centers target a region of the lateral horn previously implicated in pheromone perception. These findings identify a sensitive, narrowly tuned receptor that mediates attraction behavior to farnesol and demonstrates an effective approach to deorphanizing odorant receptors expressed in neurons located in intermediate and trichoid sensilla that may not function in the classical 'empty basiconic neuron' system (Ronderos, 2014).

    Metabolite exchange between microbiome members produces compounds that influence Drosophila behavior.

    Animals host multi-species microbial communities (microbiomes) whose properties may result from inter-species interactions; however, current understanding of host-microbiome interactions derives mostly from studies in which elucidation of microbe-microbe interactions is difficult. In exploring how Drosophila melanogaster acquires its microbiome, this study found that a microbial community influences Drosophila olfactory behavior and egg-laying behavior differently than individual members. Drosophila prefers a Saccharomyces-Acetobacter co-culture to the same microorganisms grown individually and then mixed, a response mainly due to the conserved olfactory receptor, Or42b. Acetobacter metabolism of Saccharomyces-derived ethanol is necessary, and acetate and its metabolic derivatives are sufficient, for co-culture preference. Preference correlates with three emergent co-culture properties: ethanol catabolism, a distinct volatile profile, and yeast population decline. Egg-laying preference provides a context-dependent fitness benefit to larvae. The study describes a molecular mechanism by which a microbial community affects animal behavior and results support a model whereby emergent metabolites signal a beneficial multispecies microbiome (Fischer, 2017).

    An RNA-seq screen of the Drosophila antenna identifies a transporter necessary for ammonia detection

    Many insect vectors of disease detect their hosts through olfactory cues, and thus it is of great interest to understand better how odors are encoded. However, little is known about the molecular underpinnings that support the unique function of coeloconic sensilla, an ancient and conserved class of sensilla that detect amines and acids, including components of human odor that are cues for many insect vectors. This study generated antennal transcriptome databases both for wild type Drosophila and for a mutant that lacks coeloconic sensilla. These resources were used to identify genes whose expression is highly enriched in coeloconic sensilla, including many genes not previously implicated in olfaction. Among them, an ammonium transporter gene [CG6499, renamed Ammonium transporter (Amt)] was identified that is essential for ammonia responses in a class of coeloconic olfactory receptor neurons (ORNs), but is not required for responses to other odorants. Surprisingly, the transporter is not expressed in ORNs, but rather in neighboring auxiliary cells. Thus, these data reveal an unexpected non-cell autonomous role for a component that is essential to the olfactory response to ammonia. The defective response observed in a Drosophila mutant of this gene is rescued by its Anopheles ortholog, and orthologs are found in virtually all insect species examined, suggesting that its role is conserved. Taken together, these results provide a quantitative analysis of gene expression in the primary olfactory organ of Drosophila, identify molecular components of an ancient class of olfactory sensilla, and reveal that auxiliary cells, and not simply ORNs, play an essential role in the coding of an odor that is a critical host cue for many insect vectors of human disease (Menuz, 2014: PubMed).

    Olfactory proxy detection of dietary antioxidants in Drosophila

    Dietary antioxidants play an important role in preventing oxidative stress. Whether animals in search of food or brood sites are able to judge the antioxidant content, and if so actively seek out resources with enriched antioxidant content, remains unclear. This study shows that the vinegar fly Drosophila melanogaster detects the presence of hydroxycinnamic acids (HCAs)-potent dietary antioxidants abundant in fruit-via olfactory cues. Flies were unable to smell HCAs directly but were found to be equipped with dedicated olfactory sensory neurons detecting yeast-produced ethylphenols that are exclusively derived from HCAs. These neurons were housed on the maxillary palps, expressed the odorant receptor Or71a, and were necessary and sufficient for proxy detection of HCAs. Activation of these neurons in adult flies induced positive chemotaxis, oviposition, and increased feeding. Further, fly larvae also sought yeast enriched with HCAs and used the same ethylphenol cues as the adults but relied for detection upon a larval unique odorant receptor (Or94b), which was co-expressed with a receptor (Or94a) detecting a general yeast volatile. Also, the ethylphenols acted as reliable cues for the presence of dietary antioxidants, as these volatiles were produced-upon supplementation of HCAs-by a wide range of yeasts known to be consumed by flies. For flies, dietary antioxidants are presumably important to counteract acute oxidative stress induced by consumption or by infection by entomopathogenic microorganisms. The ethylphenol pathway described in this study adds another layer to the fly's defensive arsenal against toxic microbes (Dweck, 2015).

    Dietary antioxidants play a fundamental role in preventing oxidative stress by regulating levels of free radicals and other reactive oxygen species. Dietary antioxidants thus constitute a significant nutritional reward. Indeed, for example, frugivorous birds actively seek out fruit with a high content of antioxidants and, furthermore, are able to judge the fruit's antioxidant content by relying on visual cues alone. Whether feeding partiality toward food enriched with dietary antioxidants, as well as the ability to judge antioxidant content, is widespread remains, however, an open question (Dweck, 2015).

    Oxidative stress is of importance not only to long-lived organisms, but also to animals with shorter lifespan, such as insects, in which, apart from aging, oxidative stress has also been shown to accrue from, e.g., cold exposure and through ingestion of environmental toxins. This study examined how Drosophila reacts to the presence of two polyphenolic dietary antioxidants, the hydroxycinnamic acids (HCAs) p-coumaric acid and ferrulic acid. These two HCAs are particularly abundant in fruit, the primary breeding substrate of flies, and therefore are presumably important antioxidants in wild fly populations. In flies, polyphenol antioxidants have been shown to offer protection against induced oxidative stress, and also to prolong lifespan (Dweck, 2015).

    This study demonstrates that flies are able to detect the presence of HCAs via olfactory cues. Flies are, however, unable to smell HCAs directly, but they are equipped with a dedicated olfactory sensory neuron (OSN) class -- localized on the maxillary palps -- that detects volatile ethylphenols, which are exclusively derived from HCAs. Larval flies also do the proxy detection of HCAs via the same ethylphenols, albeit with a different, but similarly tuned, larval unique odorant receptor (OR). These results provide the first indication that animals are able to use olfactory cues to judge content of dietary antioxidants (Dweck, 2015).

    Attempts were made to confirm that a diet supplanted with HCAs remedies the negative effects of induced oxidative stress. Flies with 20 mM paraquat (a pesticide that induces oxidative stress) dissolved either in yeast medium or in HCA-inoculated yeast medium. Flies fed with paraquat dissolved in HCA-inoculated yeast showed a significant enhancement in both survival and locomotor activity compared to flies treated with paraquat dissolved in the yeast medium. Can flies smell HCAs? Three different olfactory assays were used to monitor chemotaxis, oviposition, and feeding, respectively. In none of these assays did flies show any reaction to p-coumaric acid or ferulic acid. A lack of behavior does not, however, mean that flies are unable to smell these substances. Hence, electrophysiology was used, more specifically to single-sensillum recordings (SSRs), to investigate whether stimulation with HCAs induce alterations in spike firing rate. Using the two HCAs as a stimulus (10-2), a system-wide screen was performed across all 48 olfactory sensory neuron (OSN) classes present on the flies' antennae and maxillary palps. Neither HCA yielded any activity from any of the contacted OSNs. It is thus concluded that the olfactory system is unable to detect these two chemicals (Dweck, 2015).

    Although flies are unable to smell the HCAs directly, they could still be able to detect the presence of these chemicals via proxies. Many yeast species, including those consumed by flies, are known to be able to metabolize HCAs into ethylphenols, specifically 4-ethylphenol and 4-ethylguaiacol. Attempts were made to verify that fruits utilized by flies contain HCAs. Indeed, high-performance liquid chromatography (HPLC) analysis of banana pulp revealed the presence of both p-coumaric acid and ferulic acid. Next, whether the HCA amounts present in banana were sufficient to induce production of ethylphenols by yeasts was investigated. Banana-based medium was innoculated with Brettanomyces bruxellensis, a yeast species isolated from wild flies and known for its potent ability to convert HCAs into ethylphenols. Indeed, in yeasts grown on medium mixed with banana pulp, ethylphenols were identifed in the headspace. Similarly, growth of Brettanomyces on medium supplanted with HCAs resulted in the production of ethylphenols, but not when Brettanomyces was grown on standard medium (Dweck, 2015).

    Do flies react to the HCA induced changes in the yeast's volatile headspace? It was first verified that flies reacted to the smell of Brettanomyces yeast, which they did, with flies displaying strong preference for this yeast in the three previously mentioned assays. Next, flies were confronted with a choice between Brettanomyces grown with or without HCAs (henceforth referred as HCA+ and HCA-). In all assays, flies clearly preferred HCA+ yeasts. To verify that this preference is mediated via olfaction, this experiment was repeated with flies lacking Orco, a co-receptor necessary for function in the majority of all OSNs. Indeed, Orco-/- flies did not differentiate between the two treatments in any of the three assays, demonstrating that OSNs expressing ORs are necessary for this behavior. It was next asked whether the preference for HCAs is mediated via ethylphenols. To address this issue, flies were provided with a binary choice of Brettanomyces (grown on standard medium) spiked with either 4-ethylguaiacol and 4-ethylphenol (10-4 dilution) or solvent (mineral oil). Flies preferred the Brettanomyces with added ethylphenols in all three assays. Similarly, flies that were given a choice between HCA+ Brettanomyces and yeasts grown on standard medium, but spiked with ethylphenols, showed no preference either way in all assays. Finally, the behavioral valence of the ethylphenols themselves was examined, and as expected, flies in all three assays showed a strong preference for these yeast metabolites. It is hence concluded that although flies are unable to smell HCAs directly, they are able to detect volatiles derived from HCAs (Dweck, 2015).

    How do flies detect the ethylphenols? A system-wide SSR screen was performed stimulating with the two ethylphenols. Strong responses to these two chemicals (at 10-4 dilution) were exclusively observed from a single OSN class, namely palp basiconic type 1B (pb1B). To determine the specificity of these neurons, a battery of 154 compounds (screened at a higher dose [10-2] was tested to obtain the upper limit of the receptive range). The chosen stimulus included representatives of all relevant chemical classes but focused on substances of structural similarity to the HCA derived ethylphenols. Out of the screened chemicals, none produced a stronger response than 4-ethylguaiacol, and only nine of the compound -- all structurally similar to 4-ethylguaiacol -- yielded a response of >100 spikes/s. Dose-response relationships were examined for the six most efficient agonists using gas chromatography (GC) for controlled stimulus delivery. As suspected, 4-ethylguaiacol was indeed the most efficient ligand, triggering responses already at 10-7dilution. To determine whether the additional ligands for pb1B also activate other OSN classes, an exhaustive SSR screen was performed, this time stimulating with the seven primary agonists for pb1B (at 10-4 dilution) across all 48 OSN classes. With the exception of guaiacol, which also strongly activated antennal basiconic type 6B (ab6B, expressing Or49b), none of the other volatiles triggered significant activity from OSN classes other than pb1B. It is hence concluded that at ecologically relevant concentrations, the ethylphenols and structurally similar phenolic compounds exclusively activate the pb1B pathway (Dweck, 2015).

    The presence of HCAs might also lead to other changes in the yeast's volatile profile, which in turn could activate other subpopulations of OSNs. To control for this eventuality, repeated the system-wide SSR screen was repeated, but employed GC was employed to screen headspace collections from HCA+ and HCA- Brettanomyces. Stimulation with the former activated 12 OSN classes, whereas nine were activated with the latter. The additional OSN classes activated by the HCA+ Brettanomyces headspace were pb1B, ab5B, and ab9A. The pb1B neurons were, as expected, triggered by 4-ethylguaiacol and 4-ethylphenol (as identified via GC-linked mass spectroscopy). The large amount of 4-ethylguaiacol in the HCA+ sample was also sufficient to trigger weak activity from ab9A, whereas the response from ab5B in the HCA+ sample stemmed from greatly increased levels of phenylethanol compared to the HCA- treatment (Dweck, 2015).

    Attempts were then made to determine which of these three OSN classes are necessary for the proxy detection of HCAs. The temperature-sensitive mutant dynamin Shibirets was used to shut down synaptic transmission in the OSN classes specifically activated in the HCA+ sample. At the restrictive temperature (32°C), flies expressing Shibirets from the promoter of the OR expressed in pb1B OSNs--Or71a--displayed no preference toward HCA-inoculated yeasts in any of the three employed assays. The preference of flies with ab9A and ab5A silenced (via Shibirets expression from the promoters of Or69a and Or47a, respectively was, however, not different from that of flies tested at a permissive temperature (25°C) or from parental control lines at restrictive temperatur. It is hence concluded that Or71a alone is necessary for the substitute detection of HCAs. Is activation of pb1B then sufficient to induce the observed preference? Next, expression of the temperature-sensitive cation channel dTRPA1 was driven in the pb1B OSNs, which enabled the conditional activation of this specific OSN population at temperatures above 26°C. Specific activation of pb1B neurons indeed triggered attraction, egg laying, and feeding. In short, the Or71a pathway is both necessary and sufficient for the detection of the HCA derived yeast volatiles (Dweck, 2015).

    In nature, flies are not only confronted with Brettanomyces, but also encounter a wide range of yeast species. If the ethylphenols indeed serve as a general signal enabling identification of HCA enriched substrates, other yeast growing on HCA-containing sources would be expected to also produce these volatiles. To investigate this issue, HCA-induced production of volatile phenols was examined in a range of additional yeast species, namely Wickerhamomyces anomalus, Torulaspora delbrueckii, Hanseniaspora uvarum, Metschnikowia pulcherrima, and Saccharomyces cerevisiae. All of these yeasts have previously been isolated from the surface or guts of drosophilid flies. The conversion of HCAs into volatile phenols involves two steps: first a hydroxycinnamate decarboxylase enzyme converts the HCAs into vinyl derivatives, which are subsequently reduced by a vinyl phenol reductase into the corresponding ethyl derivatives (4-ethylphenol and 4-ethylguaiacol). The examined yeasts ability to complete these synthesis steps differed, with none of the yeasts being able to synthesize 4-ethylphenol. Nevertheless, when stimulated with the HCA+ yeast headspace, the amounts and types of volatile phenols present in were sufficient to activate pb1B OSNs in GC-SSR measurements. Moreover, flies confronted with the same binary choice between HCA+ and HCA- yeasts as before (Dweck, 2015).

    It is, however, not inconceivable that HCAs in combination with other yeast might cause other changes in the volatile profile than does the combination of Brettanomyces and HCAs. To examine this issue, a system-wide GC-SSR screen was again performed, now stimulating with the headspace from the five above mentioned yeasts. Although the other yeast headspace activated a slightly different subset of OSNs than did Brettanomyces, only ab9A and pb1B were additionally recruited by stimulation with the HCA+ yeast headspace compared to HCA-. Hence, it is concluded that ethylphenols serve as a consistent and reliable signal for the presence of HCAs (Dweck, 2015).

    Being able to detect HCA-enriched patches and favorable food yeasts should be important not only for adult flies, but also for larvae. Although essentially confined to their food, the microhabitat of larvae is not uniform, and thus being able to navigate toward suitable pockets within the fruit home should be an important ability. Although Or71a is not expressed in the larval stage, it is possible that among the larval unique OR genes, there are receptors that are able to make the same proxy detection of HCAs as adults do, or, alternatively, to detect HCAs directly. Whether larvae respond behaviorally to HCAs was examined. Larvae confronted with HCAs in a binary-choice larval olfactory preference assay showed no reaction to the HCAs. Although displaying no overt behavior in response to the presence of HCAs, larvae could still be able to smell HCAs. To examine whether larvae can smell HCAs, SSR was performed from the dorsal organ (DO)-the larval nose. The DO is innervated by 21 OSNs, and by gently inserting the recording electrode into this structure, it was possible to simultaneously record the activity of (presumably) all OSNs residing within the DO. Stimulation with HCAs yielded no activity from any of the discernable neurons in multiple recordings. It is thus concluded that larvae, like adults, are unable to detect the presence of HCAs directly (Dweck, 2015).

    Larvae could still, however, make the same proxy detection of HCAs as adults. First whether larvae respond behaviorally to the odor of Brettanomyces grown with or without HCAs-was examined. Both HCA+ and HCA-Brettanomyces triggered positive chemotaxis from the larvae in the olfactory preference assay. Larvae confronted with a binary choice between HCA+ and HCA- cultured Brettanomyces clearly preferred the odor of the former. Orco-/- larvae presented with the same choice did not show any preference, verifying that ORs indeed mediate this preference. Which volatiles do the larvae rely on? Larval GC-SSR measurements were performed, stimulating with HCA+ and HCA- Brettanomyces headspace collections. Compared with HCA-, stimulation with HCA+ samples yielded additional responses toward 4-ethylguaiacol and phenethyl alcohol, the latter again most likely due to the increased amounts in the HCA+ samples. Larvae also displayed increased spike firing rate in response to stimulation with the other primary ligands for Or71a, and, similarly to the situation in the adults, 4-ethylguaiacol elicited the strongest response. In GC-SSR dose-response trials, larvae were, however, less sensitive to 4-ethylguaiacol than were adults, with discernable responses to 4-ethylguaiacol requiring a 3-fold larger dose in larvae than in adults. How do larvae react behaviorally to 4-ethylguaiacol? Application of 4-ethylguaiacol in the larval olfactory choice assay resulted in positive chemotaxis. Moreover, larvae given a choice between HCA+ Brettanomyces and HCA- Brettanomyces spiked with 4-ethylguaiacol showed no preference either way, suggesting that the presence of 4-ethylguaiacol in the HCA+ samples indeed confers the attraction. It is thus concluded that the larvae perform the same proxy detection of HCAs as adults, relying on the presence of ethylphenols to identify antioxidant-enriched patches (Dweck, 2015).

    Attempts were made to determine which OR(s) in the larva detect the ethylphenols. In a recent study, 19 out of the 21 expressed larval ORs were deorphaned using a panel of ~500 chemicals. Although the ethylphenols were not included in the test panel, chemicals of structural proximity were. To identify candidate OR(s) detecting the ethylphenols, a chemometric approach was undertaken. The ethylphenols were plotted in a 32-dimensional odorant space together with the primary larval OR ligands. A principal component analysis (PCA) plot revealed that the primary Or71a ligands clustered closest with the aromatic ligand for Or94a and Or94b, namely guaiacol acetate (or 2-methoxyphenyl acetate). Thermogenetic silencing of the OSNs expressing Or94a and Or94b by expression of Shibirets from the promoter of the latter (the two ORs are co-expressed in the same OSN) indeed abolished preference in a binary-choice test between HCA+ and HCA-Brettanomyces. Furthermore, optogenetic activation of the Or94a/Or94b pathway induced attraction in larvae expressing Channelrhodopsin-2 (ChR-2) from the Or94b promoter, with larvae preferring the side illuminated with blue light (470 nm, activating the ChR-2 molecules, in contrast to parental lines and wild-type (WT) larvae, which are all repelled by blue light. Similarly, larvae confronted with a choice of HCA+ and HCA- Brettanomyces--the latter illuminated with blue light--showed no preference either way (Dweck, 2015).

    To verify that Or94a/Or94b respond to the ethylphenols, the 'empty-neuron' system was used to determine the response properties of these two receptors. Heterologous expression of Or94a and Or94b, respectively, in ab3A OSNs conferred responsiveness toward the ethylphenols. Out of the nine primary ligands of Or71a, Or94b responded most strongly to 4-ethylguaiacol. This compound, however, only elicited minor responses from Or94a, which instead was strongly activated by guaiacol. Moreover, GC dose-response trials showed that these ligands induced responses already at very low concentrations from the respective ORs. Both Or94a and Or94b were also activated by stimulation with the Brettanomyces headspace in GC-SSR experiments. It is noted with interest that guaiacol--similar to 4-ethylguaiacol--activates a different receptor than in the adults, although with similar tuning. Guaiacol is a common microbial volatile (produced, e.g., by all the yeasts examined in this study), and its presence in nature would reliably indicate the occurrence of microbes, to larvae as well as adults (Dweck, 2015).

    Given that Or94a and Or94b are co-expressed in the same neurons, how do larvae distinguish HCA- from HCA+Brettanomyces when the headspace activates the same neural pathway? A possible explanation could be that the dual activation of Or94a and Or94b by the HCA+Brettanomyces sample would lead to a stronger signal into the central nervous system, in turn causing the behavioral preference. To test this notion, the larvae were challenged with a mixture of 4-ethylguaiacol and guaiacol (10-4 dilution, total volume 10 μl) against guaiacol (10-4 dilution, 10 μl volume), a situation chemically mimicking the HCA-/HCA+Brettanomyces choice. Indeed, larvae displayed a significant preference for the mixture over the single component Preference for the mixture remained even when double amounts (i.e., 20 μl) of guaiacol were tested against 10 μl of the mixture, a treatment that would presumably compensate for any effects stemming from an increased volatility of the mix. Next, an available Or94b null mutant (no expression of Or94b was detected in RT-PCR experiments with larval cDNA was tested. As expected, Or94b-/- larvae showed no response to stimulation with 4-ethylguaiacol in SSR experiments nor did these larvae show any reaction to 4-ethylguaiacol in behavioral tests, whereas the response to guaiacol was no different from that of WT larvae. Furthermore, Or94b-/- larvae confronted with a choice between HCA+ and HCA-Brettanomyces displayed no preference either way. In summary, larvae, like adults, identify the presence of HCAs via ethylphenols. Curiously, detection is done via a separate receptor from adults, albeit with similar tuning, which moreover is co-expressed with a receptor detecting a general yeast signal. The larval Or94a/Or94b OSNs thus offers coincidence detection of two distinct, but ecologically related, volatiles (Dweck, 2015).

    This study has shown that flies are able to recognize substrates enriched with HCAs. Flies--adults as well as larvae--do so by relying on specific volatile ethylphenols (4-ethylphenol and 4-ethylguaiacol), which are exclusively derived from HCAs. In adult flies, the ethylphenols are detected by maxillary palp OSNs that express Or71a. This neuron population is both necessary and sufficient for the proxy detection of HCAs. It was demonstrated that the ethylphenols are generated by a wide range of yeasts consumed by flies and thus act as a consistent and reliable signal for the presence of HCAs. It was further shown that larvae perform the same proxy detection of HCAs via the ethylphenols as the adults, but do so via a different OR (Or94b) only expressed in the larval stage (Dweck, 2015).

    In humans, oxidative stress has been implicated in triggering or enhancing a range of diseases typically associated with aging, inter alia cancer and neurodegenerative disorders. For a short-lived species like the fly, the need to prevent the onset of aging related diseases would appear to be an unlikely reason for having a dedicated proxy detection system for dietary antioxidants. For flies, antioxidants could play an important role in counteracting acute oxidative stress induced by immune defense responses and detoxification processes upon consumption or infection by entomopathogenic microorganisms, which co-occur with beneficial food yeasts in the flies' habitat. The importance played by toxic microbes in the fly's ecology is also illustrated by the remarkably sensitive and selective detection system for geosmin, a volatile indicating the presence of harmful microorganisms. The ethylphenol pathway described here thus adds another layer to the fly's defensive arsenal against toxic microbes (Dweck, 2015).

    This study proposes that the ecological significance of the pb1B circuit is to alert flies to the presence of dietary antioxidants. Proxy detection of non-volatile nutrients and health-promoting compounds is most likely an important function of the olfactory system. Many volatiles that humans perceive as having a positive impact on flavor are in fact derived from essential nutrients or from other compounds having direct health benefits. These volatiles are accordingly attractive to humans precisely because they reliably signal the presence of their health-promoting precursors. For a generalist species such as the fly, having dedicated OSNs tuned to volatiles indicating the presence of essential nutrients would make sense. Further research will surely reveal more instances of proxy detection of nutrients in the fly's olfactory system, as well as in other organisms (Dweck, 2015).

    The pb1B pathway joins a growing number of non-pheromonal OSN classes for which dedicated and non-redundant functions has been assigned. Functionally segregated pathways identified so far include the above-mentioned geosmin circuit fed by Or56a, CO2 avoidance mediated via Gr21a and Gr63a, aversion toward select acids via Ir64a, oviposition preference for citrus-like fruits via Or19a, attraction toward farnesol (exact ecological function unclear) via Or83c, attraction toward vinegar via Or42b and Or92a, preference for the yeast metabolites phenylacetic acid and phenylacetaldehyde via Ir84a, and attraction to ammonia and select amines through Ir92a. It is thought that precise and non-redundant functions, linked to ecologically relevant behaviors can be assigned to most, if not all, of the flies' (known) 48 classes of OSNs. Thus, in contrast to the widespread notion that individual odorants are predominantly decoded via combinatorial patterns of glomerular activation, the fly's olfactory system appears to mainly extract information from its chemical surrounding via dedicated olfactory pathways. Although functionally segregated, the respective pathways would still function in concert, with behavioral decisions arising based on the relative input-or lack thereof-into combinations of dedicated circuits, each carrying a distinct ecological message (Dweck, 2015).

    Drosophila avoids parasitoids by sensing their semiochemicals via a dedicated olfactory circuit

    Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes (Ebrahim, 2015).

    A role for a phospholipid intermediate in insect olfactory transduction

    Mechanisms by which G-protein-coupled odorant receptors transduce information in insects still need elucidation. This study shows that mutations in the Drosophila gene for Gqα (dgq) significantly reduce both the amplitude of the field potentials recorded from the whole antenna in responses to odorants as well as the frequency of evoked responses of individual sensory neurons. This requirement for Gqα is for adult function and not during antennal development. Conversely, brief expression of a dominant-active form of Gqα in adults leads to enhanced odor responses. To understand signaling downstream of Gqα in olfactory sensory neurons, genetic interactions of dgq were tested with mutants in genes known to affect phospholipid signaling. dgq mutant phenotypes were further enhanced by mutants in a PLCβ (phospholipase Cβ) gene, plc21C. Interestingly although, the olfactory phenotype of mutant alleles of diacylglycerol kinase (rdgA) was rescued by dgq mutant alleles. These results suggest that Gqα-mediated olfactory transduction in Drosophila requires a phospholipid second messenger the levels of which are regulated by a cycle of phosphatidylinositol 1,4-bisphosphate breakdown and regeneration (Kain, 2008).

    The dgq gene is located at 49B on the second chromosome and encodes the only known functional Gq-like α subunit of heterotrimeric G-proteins in Drosophila. The gene encodes at least two functional isoforms, which arise by alternative splicing. Among these, dgqα1 is expressed primarily in the adult eye (Lee, 1990), whereas dgqα3 mRNA is expressed widely through development including the adult head and appendages (Talluri, 1995; Ratnaparkhi, 2002). Based on RNA expression analysis and behavioral studies with an RNAi construct (Kalidas, 2002), it has been suggested that a Gqα isoform might function downstream of olfactory G-protein-coupled receptors in antennal sensory neurons. To test this idea directly, mutations were generated in the dgq gene by excision of a P-element in the 5'-UTR of dgq, and by ethyl methane sulfonate mutagenesis. Molecular analysis demonstrated that the P-excision allele, dgq221c, carries a lesion in the 5' end of dgq spanning the translation start site in exon 3, thus rendering it null. Exons located 3' to exon 3 are intact (Banerjee, 2006). Both dgq221c and dgq1370 (induced by chemical mutagenesis) are lethal as homozygotes and as heterozygotes with Df(2R)vg-C, which uncovers the dgq locus. The lethality in dgq1370 fails to complement that of dgq221c. Homozygous dgq1370 mutants produce a transcript with a G->A mutation at base pair 1933. This would result in a change from arginine (AGA) to lysine (AAA) at residue 207, which lies in the switch II helix region that is highly conserved among α subunits of all heterotrimeric G-proteins. Structural studies show that this region undergoes a conformational switch on GTP binding, which is considered essential for effector function. Specifically, the arginines at positions 204 and 207 of Dgqα3 correspond to Arg205 and Arg208 of Gαi. Both these residues have been shown to provide important stabilizing contacts after GTP binding with glutamates located further downstream. Presumably, the mutation in dgq1370 destabilizes the GTP-bound state of Gq and renders it effectively dead by preventing activation of the downstream effector. Normal levels of the mutant protein are present in dgq1370 homozygous larvae (Kain, 2008).

    Animals homozygous for the null alleles, dgq221c and dgq1370, die as either first- or early second-instar larvae. To study the odorant response from adult antennas, animals were generated with homozygous mutant clones on the antenna using the MARCM method. Mitotic recombination at the FRT42B target site was induced using Flipase (Flp) driven by the eyeless (ey) promoter, which has been previously demonstrated to generate clones mostly in visual and olfactory sensory neurons (OSNs) and only minimally in the central brain. Mutant clones identified by GFP driven by the OSN-specific 'driver' Or83bGAL4 were obtained in >80% of progeny of the appropriate genotypes (FRT42B, dgqnull/FRT42B, tubPGAL80; ey-Flp/OR83bGAL4, UAS2XEGFP). Animals with strong GFP fluorescence in their antennas were chosen for the experiment, whereas animals with no visible GFP expression (i.e., no mutant clones) were control for effects attributable to background genotypes. Confocal sectioning of antenna bearing large clones, revealed significant mixing of marked and nonmarked cells suggesting that ey-FLP induces FRT-mediated recombination at multiple times during antennal development. The olfactory response was measured after pulsed odor delivery. EAGs were obtained after each successive pulse of a concentration of each of five different odorants. Odorant concentrations used were chosen such that they lay within the range of the log linear response to that specific odorant, when tested on wild-type flies. The response of wild-type (CS) and control flies bearing no detectable GFP-positive cells gave a comparable range of amplitudes for each of the odors tested. Antennas bearing clones of homozygous dgq221c or dgq1370 tissue showed a response reduced to 3-10 mV compared with 8-16 mV in the controls. The mutation did not affect the morphology of the antenna. Antennal sensory neurons and their projections also appear normal as judged by immunohistochemical staining with mAb22C10 and anti-GFP (Kain, 2008).

    It is known that a mutation in dgq affecting the visual splice variant (dgqα1) compromises the light response as measured by electroretinograms (ERGs) (Scott, 1995). As expected, ey-Flp also generated clones of dgq nulls in the retina. As an independent confirmation of the clonal analysis, ERGs were measured from flies with visible antennal clones and it was found that responses were reduced to the same level as seen with the dgq1 allele. Moreover, dgq221c/dgq1 and dgq1370/dgq1 flies are viable and show compromised phototransduction, although not to the same extent as shown previously by dgq1/Df flies (Scott, 1995). The residual visual response observed in dgq221c/dgq1 and dgq1370/dgq1 animals may arise from different genetic backgrounds of the three dgq alleles tested. Because the dgq1 allele affects only the visual splice variant of dgq (Scott, 1995; Ratnaparkhi, 2002), neither dgq221c/dgq1 nor dgq1370/dgq1 flies exhibited any defects in EAG measurements. These data together with EAG recordings suggest that light and odor transduction in Drosophila exploit common mechanisms albeit using distinct transcripts of the dgq gene (Kain, 2008).

    In the canonical receptor and G-protein-coupled signaling pathway, the enzyme phospholipase Cβ (PLCβ) is activated on GTP binding to Gqα, which cleaves the membrane bound phospholipid, PIP2, to generate soluble InsP3 and membrane-bound diacylglycerol (DAG). The Drosophila genome has two genes encoding for PLCβ -norpA and plc21C. A previous study found that dgq221c and plc21CP319 mutant alleles have synergistic effects on larval viability and adult flight (Banerjee, 2006), indicating that PLCβ21C can function together with Drosophila Gqα. The plc21CP319 mutant allele is a hypomorph, with a P-insert in the first intron, and is homozygous viable, whereas plc21Cp60a is a small deficiency that removes the 5' end of plc21C plus a neighboring essential gene p60. A reduction of plc21C RNA levels in plc21CP319 homozygotes and plc21Cp60a heterozygotes was confirmed by RT-PCR. UASplc21C557 is a recently generated UASRNAi strain reported as specific for plc21C (Kain, 2008).

    EAG responses of plc21CP319 homozygotes and plc21CP319/p60A combinations were reduced to <6 mV for all chemicals tested. The EAG responses to ethyl acetate (from basiconic sensilla) and propionic acid (from coeloconic sensilla) were found to be reduced at several concentrations. plc21C/+ and dgq/+ heterozygotes resulted in a small but significant decrease in EAG responses compared with wild-type (CS) controls (Kain, 2008).

    A combination of plc21C/dgq resulted in a 7-9 mV reduction in EAG responses compared with wild-type controls. This reduction is significantly greater than the value expected to arise from a mere additive effect of plc21C/+ and dgq/+ heterozygotes. Although other explanations for this haploinsufficent interaction are formally possible, the explanation is favored that PLCβ21C interacts with Gqα in normal olfactory transduction. This observation was supported by the significantly reduced EAG response to all five odorants obtained in an RNAi-driven OSN-specific downregulation of plc21C, and by downregulation of plc21c in the dgq expression domain (GqGAL4/RNAiplc21c557). EAG responses of the plc21C RNAi strain were reduced further on introduction of UASGq1F1 (RNAiGq). In the double mutant dgq-plc21C RNAi strain, EAG responses were reduced to ~2 mV but are still not abolished completely. Moreover, expression of plc21C RNAi in the OSNs resulted in a significant reduction of the spike frequency of ab2a neurons on stimulation by ethyl acetate. Thus, odor transduction in ab2a neurons requires both Gqα and Plc21C (Kain, 2008).

    Because small, but significant, odor responses remain in all dgq and plc21C mutant genotypes tested, these data further highlight the likelihood of other signaling mechanisms that collaborate with Gqα during odor detection. As reported previously, EAG responses recorded from a null allele for the second PLCβ, norpAP24, appear close to normal for the five odorants tested. The introduction of norpAP24 in plc21C/dgq animals had no additional effect in reducing EAG amplitudes. norpA thus has no significant effect on antennal physiology in contrast to its proposed effect on signaling in OSNs located on the maxillary palp and the major role played by this gene in visual transduction (Kain, 2008).

    The results so far show that signaling downstream of olfactory receptors in the Drosophila antennas requires Dgqα3 and PLCβ21C. To identify which of the two second messengers generated by Gqα activation of PLCβ21C, DAG or InsP3, are required for the electrical response to odorants, mutants were tested that function in the two arms of the pathway. The rdgA gene codes for an ATP-dependant DAG kinase that converts DAG to phosphatidic acid, which in turn is a precursor for PIP2 formation. Homozygotes for two rdgA mutant alleles rdgA1 and rdgA3 show reduced EAG responses to all five odorants tested, whereas heterozygotes appear close to normal. rdgA1 homozygotes also show reduced EAG responses to multiple concentrations of two odorants tested, ethyl acetate and propionic acid. Interestingly, unlike previously observed effect of rdgA mutants on ERG termination kinetics, slower termination was not observed of the EAG response in rdgA mutants. Moreover, unlike eyes of 1-d-old rdgA1/rdgA1 adults, which exhibit strong retinal degeneration, both rdgA alleles have normal antennal morphology even 6 d after eclosion. Thus, the reduced EAGs observed are not a consequence of antennal neuron degeneration (Kain, 2008).

    Next, the EAG response was tested after introducing a single mutant copy of dgq in rdgA homozygotes (rdgA/rdgA;dgq/+). Interestingly, it was found that reduction of Dgq levels to 50% by introduction of a single copy of the dgqnull allele rescued the EAG responses of rdgA homozygotes bringing them back to near normal. The extent of rescue varies with specific dgq and rdgA mutant allele combinations. Strongest effects were seen in rdgA1/rdgA1; dgq1370/+ animals. Reducing dgq function by expression of the RNAi construct UASGq1F1 also rescued the EAG defects produced by both rdgA mutant homozygous alleles. The rescue of EAG responses in rdgA mutants by dgq mutants was further verified by testing RNAi lines specific for rdgA and dgq in appropriate combinations. EAG responses were significantly higher in the presence of RNAi constructs for both rdgA and dgq compared with the response from individual RNAi lines. A possible explanation for these data is that failure of conversion of DAG back to phosphatidic acid and PIP2 through rdgA encoded DAG kinase compromised the generation of an odor-evoked response but does not cause degeneration of OSNs. Reduction in the signaling flux through Gqα (by lower Dgq levels in dgqnull/+ genotypes or by Gqα RNAi expression) compensates for this defect and rescues the olfactory defect of rdgA (Kain, 2008).

    Because there is a significant body of data from studies with other organisms suggesting that InsP3 is a second messenger in invertebrate olfactory transduction (Ache, 2005), EAG responses were measured from single copies of the two dgq alleles with two different mutations of the InsP3 receptor as heterozygotes (dgqnull/+; itprEMS/+). itprsv35 is a null, whereas itprka901 can act as a gain-of-function allele. The effect of these alleles on antennal olfactory responses has not been investigated before. No significant change in the response to any chemical was observed in the eight strains tested. These data are in agreement with a previous study from Drosophila, in which EAGs measured from the antennas of viable alleles for the InsP3 receptor were normal. Thus, primary olfactory responses from the Drosophila antennas do not require the InsP3 receptor but are dependent on a DAG kinase (rdgA) (Kain, 2008).

    This study has shown that mutations in the G-protein Gqα result in reduced sensitivity of Drosophila antennal sensory neurons to several different chemical stimuli. Genetic analysis suggested that its downstream effector is phospholipase Cβ encoded by the plc21C gene. PLCβ enzymes catalyze breakdown of the membrane-bound lipid PIP2 to generate DAG and InsP3. Reduced EAG amplitudes in DAG kinase mutants (rdgA) and their rescue by lowering Gqα levels, argue that the extent of PIP2 depletion after Gqα and PLCβ activation is responsible for this arm of olfactory transduction. Altered PIP2 levels have been shown to gate or modulate membrane conductances in other contexts. The presence of a residual olfactory response in antennal neurons that are null for dgq supports existence of an additional transduction mechanism (Kain, 2008).

    Odorant detection in animals occurs through a diverse class of G-protein-coupled receptors. In mammalian olfactory sensory neurons, it appears that all odorant receptors activate a single G-protein Golf and generate one second messenger, cAMP. In Drosophila dgq mutants, a reduced response was found to all five odorants tested, indicating that multiple odorant receptors activate Gqα. Ethyl acetate, isoamyl acetate, and benzaldehyde stimulate receptors located in the basiconic sensilla, whereas butanol and propionic acid activate neurons innervating the coeloconic sensilla. It has been proposed that the basiconic sensilla primarily detect food odors and the coeloconics express receptors that respond to certain specialized odors. The results suggest that these functional distinctions are not based on a change in signal transduction machinery (Kain, 2008).

    The residual odor-induced response observed in Gqα mutant antennas and in single ab2a neurons argues for the presence of an alternate transduction mechanism(s) in OSNs. Odorants could stimulate either other G-proteins, which alter cyclic nucleotide levels, or a direct odor-gated ion channel, as in heteromers of Manduca OR/OR83b or both. Precisely how the alternate mechanism of odor transduction interacts with Gqα-mediated transduction described here requires additional study. The strong genetic evidence presented here, supporting a role for a Gqα-mediated signaling cascade during olfactory transduction, needs to be reconciled with recent data suggesting that Drosophila odorant receptors do not conform to the structure or topology typical of other G-protein-coupled receptors (Kain, 2008).

    Based on the quality and strength of the odor stimulus it receives, each odorant receptor could stimulate different transduction pathways to varying extents. It is proposed that firing frequency of the OSN is an integration of this information, perhaps by cross-sensitization or desensitization of membrane conductances gated by the individual pathways. Interestingly, it has been demonstrated that a single odorant receptor (e.g., Or59b), present within the same neuron can mediate both excitatory and inhibitory responses as a consequence of stimulation by two different odorants. Precedents for multiple signaling pathways in odor detection within a single organism are present in invertebrates (Ache, 2005), although their existence within the same olfactory neuron has not been demonstrated so far (Kain, 2008).

    The requirement of phospholipase C for odor-induced activity of OSNs in the maxillary palp highlights an overlap in signaling mechanisms during olfaction and vision. The data further highlight parallels in these two sensory systems. In Drosophila photoreceptors, signal amplification begins with activation of a number of G-protein molecules by a single photoisomerised rhodopsin molecule, followed by activation of one PLC molecule for each G-protein . Although the current methods of measuring olfactory responses in Drosophila do not allow direct measurement of signal amplification, the enhanced EAG amplitudes observed in AcGq3-expressing animals and synergism between dgq and plc21C mutant alleles observed in this study supports the idea of signal amplification by Gqα (Kain, 2008).

    The rdgA gene has a role in both amplification and termination of the visual response. The observation that rdgA1 and rdgA3 homozygotes exhibit reduced EAG responses, in the absence of antennal neuron degeneration, suggest that the rate of DAG turnover to generate other intermediates leading to PIP2 is important for membrane depolarization of OSNs. The absence of antennal degeneration predicts a low level of basal PLCβ activity in OSNs, and perhaps strong adaptive mechanisms that prevent activation of membrane channels in normal odor-rich environments. This needs to be investigated further. The fact that reduced EAGs can be rescued by a single copy of dgq mutant alleles, supports the existence of a cycle of phosphoinositide turnover in OSNs, in which reduced levels of Gqα help to slow down this cycle and thus balance the rate of PIP2 regeneration in rdgA mutants. The observed suppression appears qualitatively similar to the interaction of rdgA and dgq during the amplification phase of Drosophila visual transduction in which rdgA mutants enhance the sensitivity of dgqα1 mutants as measured by the amplitude of quantum bumps. Finally, either PIP2 depletion, or a lipid intermediate presumably gates a depolarizing membrane channel. The identity of this channel can only be conjectured on at this stage. However, based on studies from other G-protein-coupled lipid signaling pathways, a transient receptor potential (TRP) family channel might be a possible candidate. Among this family, the two most well studied members in Drosophila are TRP and TRPL (TRP-like), both of which are enriched in photoreceptors. A previous study on the olfactory responses of trp mutants ruled out a role for this gene in primary sensory transduction of odor stimuli. Several members of this family exist in Drosophila and need to be investigated for their role in olfactory transduction (Kain, 2008).

    Current source density mapping of antennal sensory selectivity reveals conserved olfactory systems between tephritids and Drosophila

    Ecological specialization of insects involves the functional and morphological reshaping of olfactory systems. Little is known about the degree to which insect sensitivity to odorant compounds is conserved between genera, tribes, or families. This study compared the olfactory systems of six tephritid fruit fly species spanning two tribes and the distantly related Drosophila melanogaster at molecular, functional, and morphological levels. Olfaction in these flies is mediated by a set of olfactory receptors (ORs) expressed in different functional classes of neurons located in distinct antennal regions. A phylogenetic analysis was performed that revealed both family-specific OR genes and putative orthologous OR genes between tephritids and Drosophila. With respect to function, a current source density (CSD) analysis was used to map activity across antennae. Functional maps mirrored the intrinsic structure of antennae observed with scanning electron microscopy. Together, the results revealed partial conservation of the olfactory systems between tephritids and Drosophila. It was also demonstrated that the mapping of olfactory responses is necessary to decipher antennal sensory selectivity to olfactory compounds. CSD analysis can be easily applied to map antennae of other species and therefore enables the rapid deriving of olfactory maps and the reconstructing of the target organisms' history of evolution (Jacob, 2017).

    Differential contributions of olfactory receptor neurons in a Drosophila olfactory circuit

    The ability of an animal to detect, discriminate, and respond to odors depends on the functions of its olfactory receptor neurons (ORNs). The extent to which each ORN, upon activation, contributes to chemotaxis is not well understood. It was hypothesized that strong activation of each ORN elicits a different behavioral response in the Drosophila melanogaster larva by differentially affecting the composition of its navigational behavior. To test this hypothesis, Drosophila larvae were exposed to specific odorants to analyze the effect of individual ORN activity on chemotaxis. Two different behavioral paradigms were used to analyze the chemotaxis response of larvae to odorants. When tested with five different odorants that elicit strong physiological responses from single ORNs, larval behavioral responses toward each odorant differed in the strength of attraction as well as in the composition of discrete navigational elements, such as runs and turns. Further, behavioral responses to odorants did not correlate with either the strength of odor gradients tested or the sensitivity of each ORN to its cognate odorant. Finally, evidence is provided that wild-type larvae with all ORNs intact exhibit higher behavioral variance than mutant larvae that have only a single pair of functional ORNs. It is concluded that individual ORNs contribute differently to the olfactory circuit that instructs chemotactic responses. The results, along with recent studies from other groups, suggest that ORNs are functionally nonequivalent units. These results have implications for understanding peripheral odor coding (Newquist, 2016).

    Olfactory control of blood progenitor maintenance

    Drosophila hematopoietic progenitor maintenance involves both near neighbor and systemic interactions. This study shows that olfactory receptor neurons (ORNs) function upstream of a small set of neurosecretory cells that express GABA. Upon olfactory stimulation, GABA from these neurosecretory cells is secreted into the circulating hemolymph and binds to metabotropic GABAB receptors expressed on blood progenitors within the hematopoietic organ, the lymph gland. The resulting GABA signal causes high cytosolic Ca2+, which is necessary and sufficient for progenitor maintenance. Thus, the activation of an odorant receptor is essential for blood progenitor maintenance, and consequently, larvae raised on minimal odor environments fail to sustain a pool of hematopoietic progenitors. This study links sensory perception and the effects of its deprivation on the integrity of the hematopoietic and innate immune systems in Drosophila (Shim, 2013).

    Niche-dependent mechanisms of hematopoietic progenitor development and maintenance have been extensively described in both vertebrate and invertebrate literature. Mechanisms independent of the niche that operate at a more systemic level but affect progenitor development have recently started to emerge (Shim, 2013).

    This study describes a signal that originates from the brain and regulates blood progenitor maintenance. This pathway is independent of the nutritional signal that involves Drosophila insulin and TOR. Olfaction-dependent sensory stimulation relays systemic cues from the central nervous system to the undifferentiated blood progenitors by regulating physiological levels of GABA secreted into the blood stream. GABA is expressed in a small number of neurosecretory cells of the brain, and the release of GABA from this class of neurosecretory cells is critically dependent on olfactory stimulation. Olfactory dysfunction decreases GABA expression in neurosecretory cells and also reduces systemic GABA levels in the circulating blood. Blood progenitors express the metabotropic GABAB receptor, which enables them to respond to GABA, raising the concentration of their cytosolic calcium essential for inhibition of premature differentiation and maintenance of the progenitors. This control is lost when either the olfactory neurons or their network partners in the olfactory glomeruli are disrupted. A consequence of the above mechanism is that wild-type Drosophila larvae reared on odor-limited media have dramatically reduced systemic GABA levels, and consequently, their blood progenitors precociously differentiate. Upon blocking olfaction, GABA levels in the entire central brain region are reduced, but it is the two GABA-expressing neurosecretory cells in each lobe of the central brain that are important in controlling GABA secreted into the aorta that controls hematopoiesis (Shim, 2013).

    Within the lymph gland, the GABAB receptor is expressed in the blood progenitors and is downregulated as the cells differentiate. Binding of GABA to GABABR maintains high cytosolic Ca2+ in the progenitors, a prerequisite for their remaining undifferentiated. The differentiated blood cells have very low or undetectable levels of Ca2+ and are also unresponsive to its alterations. Downstream of elevated Ca2+, the functions of Calmodulin and CaMKII are essential for progenitor maintenance. Events further downstream currently remain unclear. In principle, Ca2+ could directly or indirectly interact with either ROS or Wg-related pathways shown to be important for progenitor maintenance (Shim, 2013).

    Accumulating evidence has shown that the mammalian nervous system also regulates innate immune responses through hormonal and neuronal routes. Sympathetic and parasympathetic nervous systems directly innervate into immune organs, whereas neuroendocrine factors control inflammation at a systemic level. Furthermore, immune cells express receptors for various neuronal factors, supporting the idea that there are contributions of the nervous system to immunity. Brain dysfunction, including certain neurodegenerative diseases, generate heightened immune reaction, as the central nervous system is generally thought to inhibit immune responses. The mammalian hematopoietic niche is innervated, and cells within the niche express a Ca2+-sensing receptor on their surface that they utilize to home toward the periendosteal compartment. However, a direct involvement of secreted GABA or olfaction in the maintenance of hematopoietic progenitors has not been demonstrated in any other system (Shim, 2013).

    GABA is conserved from bacteria to plants and animals. In plants, GABA functions as a metabolite, a signaling molecule, and in stress response. In vertebrates, GABA function has been primarily studied in neurotransmission, but it also functions as a metabol and in developmental signaling in both embryonic tissue and in adult regeneration. This study could readily detect GABA secreted into the Drosophila hemolymph. This is not unprecedented, as GABA can be measured in the bloodstream of many mammals, including humans. Interestingly, GABABR is expressed in primary human HSCs, and its expression is higher in immature stem cells than in more mature progenitors. GABA function in human HSCs remains unclear, and it is not known whether its function is controlled through a sensory signal as has been described in this study in Drosophila (Shim, 2013).

    In addition to the universally used developmental pathways such as Hh, Dpp, and Wg, Drosophila blood precursors utilize several unusual pathways for their development. For example, physiologically generated ROS functions as a signaling molecule that allows the blood progenitors to differentiate, whereas increased ROS, resulting from infection, is a stress signal that causes rapid expansion of this differentiation process. It was shown that insulin maintains the progenitor population during normal development, and starvation is a stress condition that causes a drop in insulin levels and allows premature differentiation. Similarly, Hif-alpha, stabilized under normoxic conditions by physiologically generated NO, binds Notch and maintains a class of blood cells, whereas hypoxic conditions sensed as a stress stabilize additional amounts of Hif-alpha and increase the number of these blood cells. To summarize, in all the above instances, examples are seen of signals that are used by the myeloid precursors for their normal development in a programmed manner, and the same signals cause rapid expansion of these blood cells upon conditions of stress. In the fly, the conditions that favor blood differentiation, including reduced olfaction, are normally initiated during pupariation when the need for increased numbers of macrophages is critical. As a bonus, these same pathways can cause increased differentiation earlier in larval life when activation of these pathways is perceived as a stress response. This response is reflected in mutational studies (Shim, 2013).

    Overall, this study describes the mechanism for coordinating inputs from olfactory stimulation to maintain blood progenitors via regulation of systemic GABA levels. As olfaction is an important sensory input for the larva, inability to sense odor could be interpreted as an important stress response. Anosmic larvae cannot survive in a competitive environment due to lack of food-searching behavior. Furthermore, a recent study has shown that OR56a senses a microbial odorant to avoid unsuitable breeding and feeding sites. Thus, proper olfaction promotes survival, both by allowing improved competition within a brood and through avoidance of infectious organisms. Increased hematopoietic differentiation in the absence of odor input could also be beneficial to the larva in mounting an immune response, although this remains to be proven in future studies. In humans, loss of olfaction has been associated with abnormalities in many parts of the brain, and impaired olfaction leads to amplified inflammation in mammals. The current data in Drosophila highlight that sensory stress response can directly influence developmental and cell fate decisions of blood progenitors. Whether this is also relevant to higher organisms with more complicated blood lineages remains to be explored (Shim, 2013).

    Or47b plays a role in Drosophila males' preference for younger mates

    Reproductive behaviour is important for animals to keep their species existing on Earth. A key question is how to generate more and healthier progenies by choosing optimal mates. In Drosophila melanogaster, males use multiple sensory cues, including vision, olfaction and gustation, to achieve reproductive success. These sensory inputs are important, yet not all these different modalities are simultaneously required for courtship behaviour to occur. Moreover, the roles of these sensory inputs for male courtship choice remain largely unknown. This study demonstrates that males court younger females with greater preference and that olfactory inputs are indispensable for this male courtship choice. Specifically, the olfactory receptor Or47b is required for males to discriminate younger female mates from older ones. In combination with previous studies indicating that gustatory perception is necessary for this preference behaviour, these data demonstrates the requirement of both olfaction and gustation in Drosophila males' courtship preference, thus providing new insights into the role of sensory cues in reproductive behaviour and success (Zhuang, 2016).

    A CD36 ectodomain mediates insect pheromone detection via a putative tunnelling mechanism

    CD36 transmembrane proteins have diverse roles in lipid uptake, cell adhesion and pathogen sensing. Despite numerous in vitro studies, how they act in native cellular contexts is poorly understood. A Drosophila CD36 homologue, Sensory neuron membrane protein 1 (SNMP1), was previously shown to facilitate detection of lipid-derived pheromones by their cognate receptors in olfactory cilia. This study investigated how SNMP1 functions in vivo. Structure-activity dissection demonstrates that SNMP1's ectodomain is essential, but intracellular and transmembrane domains dispensable, for cilia localization and pheromone-evoked responses. SNMP1 can be substituted by mammalian CD36, whose ectodomain can interact with insect pheromones. Homology modelling, using the mammalian LIMP-2 structure as template, reveals a putative tunnel in the SNMP1 ectodomain that is sufficiently large to accommodate pheromone molecules. Amino-acid substitutions predicted to block this tunnel diminish pheromone sensitivity. A model is proposed in which SNMP1 funnels hydrophobic pheromones from the extracellular fluid to integral membrane receptors (Gomez-Diaz, 2016).

    The structural biology of olfactory organs

    (1) Organization and function of maxillary palp olfactory receptor neurons and brain olfactory glomeruli

    There are two olfactory organs on the adult fly: the third segment of the antenna and the maxillary palp. In both organs, ORNs are housed in sensory hairs called sensilla. In contrast to the antenna, the organization of the 120 ORNs of the maxillary palp is simpler. There are 60 sensilla basiconica on the maxillary palp, each housing two ORNs. The 120 ORNs fall into six different classes based upon their odorant response profiles. Neurons of the six ORN classes are always found in characteristic pairs in three functional types of sensilla basiconica, with the total number of neurons in each class being equal. Each class is distributed broadly over all, or almost all, of the olfactory surface of the maxillary palp.

    Olfactory receptor neurons of the adult fly are located in both the antenna and the maxillary palp. To ask whether any of the DOR genes are expressed in these neurons, in situ hybridization to RNA was carried out in adult tissue sections. Of 11 genes examined, 7 show detectable expression, which in every case is observed only in the olfactory organs. The 46F.1 probe hybridizes to a subset of ORNs in the maxillary palp. A count of labeled ORNs in serial sections reveals that the total number of 46F.1-staining ORNs per maxillary palp is 18, or 15% of the 120 olfactory neurons in the maxillary palp. A similar number of neurons, 17, was labeled by another probe, 33B.3. The neuronal identity of the labeled cells is apparent from the presence in many cases of a well-defined axon projecting from the labeled cell body and joining the maxillary nerve. For both probes, the labeled neurons are distributed broadly over the olfactory surface of the organ, and are interspersed among unlabeled neurons. Staining in many cells appears annular, which is interpreted to reflect a perinuclear distribution of mRNA, as expected of an mRNA present at highest concentrations in the cell bodies of these ORNs. The 33B.3 and 46F.1 genes are evidently expressed in different subsets of ORNs, because the number of neurons hybridizing with a mixed probe is greater than the number of neurons that hybridized when either probe is used individually. For neither probe was hybridization detected in the antenna, head, or thorax (Clyne, 1999a)

    The number and broad distribution of maxillary palp neurons expressing 46F.1 and 33B.3 are intriguing in light of electrophysiological studies. There are about 120 ORNs on the palp, which fall into six different classes based on their odorant response profiles. Each class contains roughly equal numbers of neurons, distributed broadly over the olfactory surface of the palp. Thus, if an individual receptor gene is expressed in all ORNs of a functional class, one might expect a gene to be expressed in a broad distribution, in about 20 neurons, in good agreement with the distribution and numbers observed for both 46F.1 and 33B.3 (Clyne, 1999).

    Three DOR genes that are expressed in the maxillary palp come from the 16% of the genome analyzed. Since these three genes, like most DOR genes, are not clustered in the genome, linear extrapolation suggests that the entire genome contains on the order of 18 DOR genes expressed in the maxillary palp, an organ that has six functional classes of neurons. If all neurons within a functional class, i.e., with the same odor specificity, are identical in terms of their receptor expression, then the ratio of expressed genes to neuronal classes (three genes per neuronal class) in this organ would be consistent with a model in which an individual ORN expresses a small number of odorant receptors (Clyne, 1999a and Vosshall, 1999).

    ORNs in Drosophila and other insects project to an olfactory processing center, the antennal lobe, which is much like the olfactory bulb of vertebrates. Like its vertebrate counterpart, the antennal lobe contains olfactory glomeruli, of which the antennal lobe of Drosophila has a total of 40 olfactory glomeruli. In vertebrates, there is an approximate equivalence between the estimated number of odorant receptor genes and the number of glomeruli. If, in fact, the number of Drosophila odorant receptor genes is on the order of 100, then the ratio of odorant receptor genes to glomeruli would exceed two to one, and would rise if additional families of odorant receptor genes were discovered. It is noted that the number of glomeruli receiving input from the maxillary palp has been variously estimated as three and five; if the estimate of 18 genes expressed in the maxillary palp is correct, then the ratio of these receptor genes to their corresponding glomeruli would fall in the range of three to six (Clyne, 1999a).

    2) Organization of antennal olfactory receptor neurons

    Whereas the maxillary palp has approximately 120 ORNs, the antenna has 1200. The organization of the 1200 ORNs of the antenna is complex but ordered. On the antenna, there are different morphological categories of sensilla: sensilla trichodea, sensilla coeloconica, large sensilla basiconica, and small sensilla basiconica. The different morphological categories of sensilla are distributed in overlapping patterns across the surface of the antenna. Electrophysiological studies show that each morphological category of sensilla can be divided into different functional types, defined by the characteristic response profiles of their ORNs. For sensilla trichodea, the different functional types are segregated into zones on the surface of the antenna; segregation is also observed for the different functional types of sensilla coeloconica. This zonal organization is less conspicuous for large and small sensilla basiconica, of which different functional types are intermingled. Electrophysiological data suggest that there are on the order of 30 different classes of ORNs in the antenna, a rough estimate based on the odor response profiles of individual ORNs (Clyne, 1999a and references).

    Many of the DOR genes are expressed in the antenna and not in the maxillary palp. The 47E.1 probe hybridizes to 40 cells in a broad area across the antenna, including both anterior and posterior faces, similar to the distribution pattern of small sensilla basiconica. A probe from the 25A.1 gene hybridizes to fewer cells (16) but in a region of the antenna similar to that of 47E.1 staining, as judged by reconstruction of serial sections. The 22A.2 probe hybridizes to 22 cells in a different distribution, clustered in the dorsomedial region of the antenna. This pattern matches the distribution of the large sensilla basiconica (Clyne, 1999).

    The Vosshall (1999) study reveals topographically defined subpopulations of olfactory sensory neurons in either the antenna or the maxillary palp. In situ hybridization experiments reveal that each receptor is expressed in a spatially restricted subpopulation of neurons in the antenna or maxillary palp. The total number of cells expressing each receptor per antenna was obtained by counting the positive cells in serial sections of antennae from multiple flies. DOR67 and DOR53, for example, are expressed by about 20 neurons on the medial-proximal edge of the antenna, whereas DOR62 and DOR87 are expressed by subpopulations of 20 cells at the distal edge of the antenna. Approximately ten cells in the distal domain express DOR64. Each of three linked genes, dor71, dor72, and dor73, is expressed in different neurons. dor72 is expressed in approximately 15 antennal cells, while dor73 is expressed in 1 to 2 cells at the distal edge of the antenna. In contrast, dor71 is expressed in approximately ten maxillary palp neurons but is not detected in the antenna. The three sensillar types are represented in a coarse topographic map across the third antennal segment. The proximal-medial region, for example, contains largely basiconic sensilla. Receptors expressed in this region (dor53 and dor67) are therefore likely to be restricted to the large basiconic sensilla. More distal regions contain a mixture of all three sensilla types, and it is therefore not possible from these data to assign specific receptors to specific sensillar types. The spatial pattern of neurons expressing a given receptor is conserved between individuals. In situ hybridization with two receptor probes to three individual flies reveals that both the frequency and spatial distributions of the hybridizing neurons are conserved in different individuals. At present, the precision of this topographic map cannot be determined and it can only argued that given receptors are expressed in localized domains.

    Receptors for mate recognition in Drosophila

    Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila. The trichoid sensilla, one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, were systematically examined by electrophysiological analysis. None respond strongly to food odors but all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone transferred from males to females during mating. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin-female extract as well. Thus, males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. All members of the Odor receptor (Or) gene family that are expressed in trichoid sensilla were then systematically tested by using an in vivo expression system. Four receptors respond to fly odors in this system: Two respond to extracts of both males and virgin females, and two respond to cVA. A model is proposed describing how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic (van der Goes van Naters, 2007).

    The responses of ORNs in trichoid sensilla of the antenna were measured by single-unit electrophysiology. All three trichoid-sensilla subtypes, T1, T2, and T3, which contain one, two, and three ORNs, respectively, were tested. These three subtypes occupy distinct but overlapping regions of the antennal surface and together comprise more than 20% of the sensilla in the antennae. Initially, 86 compounds were tested, most of which are found in fruits or are fermentation products. These compounds were tested on 60 trichoid sensilla, 30 from males and 30 from females. The compounds were tested in mixtures, and no mixture elicited a response greater than 20 impulses/s, which represents less than 10% of the maximal response of these ORNs. Some mixtures inhibited the spontaneous activity of T2 and T3 sensilla and produced decreases of 10–20 impulses/s in the action-potential rate. The three most inhibitory odors were subsequently determined to be 1-hexanol, hexyl acetate, and butyl acetate. The paucity of strong excitatory responses to food odors is consistent with the results of an earlier screen with a limited number of chemicals; in this earlier screen, no strong responses were found, although modest responses were elicited by trans-2-hexenal and cis-vaccenyl acetate (cVA) (van der Goes van Naters, 2007).

    The odor of live flies was tested. 50 flies were placed in a glass tube that was closed at both ends with a cotton mesh. Air was puffed through the tube toward the antenna of a fly mounted for electrophysiological recording. 75 individual trichoid sensilla, of all three subtypes, were tested for responses to the odors of both males and virgin females. Air passing over male flies elicited a strong response from ORNs in a large group of trichoid sensilla. These ORNs did not respond to the odor of virgin females. These sensilla correspond to the T1 subtype, each of which houses a single ORN. T1 sensilla are found on both male and female antennae; in both cases they respond to the odor of males but not of virgin females. The T2 and T3 sensilla did not produce responses to fly odors when they were tested in this paradigm (van der Goes van Naters, 2007).

    These experiments showed that at least some trichoid sensilla respond to fly odors. However, whether other trichoid sensilla might show responses to fly odors was tested in a more sensitive assay. A new paradigm was developed. Because flies approach each other closely during courtship, it was reasoned that some pheromone-sensitive sensilla might be adapted for short-range information reception. Some of the chemical cues that influence courtship behavior in Drosophila are present in the cuticle, i.e., on the surface of the fly, and are long-chain unsaturated hydrocarbons of very limited volatility. Although some of these cues are believed to be detected via the taste system, it seemed possible that the olfactory system might also contribute to the reception of cuticular components at very close range during courtship (van der Goes van Naters, 2007).

    Accordingly, rather than adding odor stimuli to an air stream directed at the fly from a distance, stimuli were presented by approaching the antenna with the tip of a glass capillary carrying the odor. This procedure was designed to simulate the proximity of two interacting flies. As an initial test of the feasibility of this paradigm, 500 pl of a solution of cVA was draw into the capillary. cVA has been shown to act as an anti-aphrodisiac pheromone in Drosophila; there is also evidence for its playing a role as an aggregation pheromone. As the capillary tip approached certain trichoid sensilla, the impulse rates of certain ORNs increased and reached a maximum of >200 impulses/s upon physical contact of the capillary tip with the sensillum shaft. Control stimuli prepared with the hexane solvent alone gave no response (van der Goes van Naters, 2007).

    Having established a short-range delivery paradigm, the responses, initially to cVA, of trichoid sensilla were systematically examined across the entire antennal surface. Mature male flies contain approximately 1 μg of cVA, primarily in the ejaculatory bulb. A capillary tip was loaded with 5 ng of cVA (0.005 fly equivalent) and 189 trichoid sensilla were approached individually. Strong responses of >100 impulses/s in were observed 169 of the 189 sensilla. Previous reports had shown that the ORN in T1 sensilla responds to cVA, and this study confirmed this finding. Responses to 5 ng of cVA exceeded 200 impulses/s in T1 sensilla. Also in agreement with the previous reports, some sensilla immediately adjacent to the zone containing T1 did not respond to cVA. However, it was determined that, in addition to the T1 subtype, a large number of sensilla more distolateral on the antennal surface also contained ORNs that are sensitive to cVA in this paradigm. Neurons in the distolateral sensilla responded to the cVA stimulus with a rate increase of more than 100 impulses/s. Thus, there appear to be at least two populations of sensilla with ORNs that respond to this pheromone (van der Goes van Naters, 2007).

    To expand the scope of this analysis from a single defined pheromone, cVA, to a broad representation of the cuticular pheromone profile, hexane extracts of males and virgin females were prepared. Approximately 500 pl of extract was drawn into the capillary tip; this amount is equal to 0.25% of the material extracted from a single fly (van der Goes van Naters, 2007).

    When a male extract was used as the odor source, all 147 trichoid sensilla tested, from all regions of the antennal surface, yielded responses. Different ORNs began to respond to the approaching odor source at different distances. The T1 sensilla, which house a single ORN, appeared to be particularly sensitive; they showed responses greater than 20 impulses/s when the odor source came within a 1 cm radius. As the odor source became still closer, the impulse rates increased rapidly. ORNs in T2 and T3 sensilla appeared to be less sensitive and had impulse rates increasing only after the odor source approached a distance of 200 μm, as determined with an ocular micrometer. The responses were dose dependent; when the dose was increased from 0.25% fly equivalent to 5% fly equivalent, the response radius increased from 200 μm to 500 μm (van der Goes van Naters, 2007).

    When an extract from virgin females was used as the stimulus, strong responses were observed in ORNs of all trichoid sensilla except T1. Thus, T1 sensilla appear to be tuned to male odor, whereas T2 and T3 sensilla yield strong responses to both males and virgin females. Sensitivity to male and virgin-female extracts was comparable in T2 and T3 sensilla. These in vivo recordings, taken together, demonstrate that trichoid sensilla respond to fly odors and that the odors of males and virgin females are registered differently across the ensemble of trichoid sensilla. A limitation of the analysis is that it is difficult to ascribe responses to individual ORNs within trichoid sensilla. With the exception of T1, trichoid sensilla contain multiple ORNs. In recordings, this is evident from summation and cancellation events between impulses in the traces. In most cases it was not possible to discriminate the activities of the individual ORNs because the action potentials, as recorded extracellularly, did not differ significantly in size or shape. Because of the inability to classify action potentials with confidence, it was not possible to determine whether there is a functional subdivision among the ORNs sharing a sensillum. To address this limitation, advantage was taken of another experimental system, the “empty neuron” system, in an effort to analyze the responses of trichoid sensilla at a higher resolution (van der Goes van Naters, 2007).

    Drosophila contains a family of 60 Or (Odor receptor) genes, and the following 12 family members have been reported to map to individual ORNs of trichoid sensilla: Or2a, Or19a, Or19b, Or23a, Or43a, Or47b, Or65a, Or65b, Or65c, Or67d, Or83c, and Or88a. Each of these 12 Or genes were expressed in the “empty neuron” system, an in vivo expression system based on a mutant ORN, ab3A, that resides in a basiconic sensillum. The endogenous receptor genes of this ORN, Or22a and Or22b, are deleted, and the promoter of Or22a drives ectopic expression of another odor receptor in ab3A via the UAS-GAL4 system. The odor responses conferred upon ab3A by the ectopically expressed receptor are then measured by single-unit electrophysiology (van der Goes van Naters, 2007).

    The 12 trichoid receptors were systematically tested in the empty-neuron system with a panel of fly-derived chemicals: hexane extracts of males and virgin females, material from the genital regions of flies (males, virgin females, and mated females), and cVA. The genital odors were obtained by drawing a glass capillary, with a tip pulled to a diameter of 3 μm, across the genital region of a fly such that material visibly coated the tip. Preliminary experiments showed that the responses could be quantified most reproducibly not during the approach of a stimulus to the antenna but after the capillary tip contacted the sensillum. Responses mediated by the trichoid receptors were were therefore quantified by determining impulse rates of the ORN after contact. The 12 receptors were expressed and tested in both male and female recipients with all six stimuli, and no differences between the responses of male and female flies were identified (van der Goes van Naters, 2007).

    Of the 12 receptors, four mediated responses to fly odors in this system. All four, Or47b, Or65a, Or67d, and Or88a, responded to male extract, and their action-potential frequencies increased by 50–200 impulses/s. Two of these receptors, Or65a and Or67d, did not respond to extract from virgin females. The sex specificity of Or65a and Or67d is consistent with a role for these receptors in the detection of male-specific pheromones. The other two receptors, Or47b and Or88a, responded to extract from virgin females; these responses were comparable to those they gave to male extracts. It was noted that both Or47b and Or88a were previously tested in the empty-neuron system with a panel of 110 odors, most of which were present in fruits and were of widely varying chemical structures, and no excitatory responses were recorded. These results are consistent with the hypothesis that Or47b and Or88a detect a pheromone secreted by both males and females (van der Goes van Naters, 2007).

    Male genital material elicited strong responses from Or65a, Or67d, and Or88a. Genital material from virgin females did not elicit a strong response from any of the 12 receptors. However, material from the genital region of females that were mated 1–4 hr previously produced responses from these three receptors, which, yielded firing rates comparable to those observed with male genital material. These results suggest that during copulation the male transfers compounds that activate these receptors (van der Goes van Naters, 2007).

    One compound that the male transfers to the female during copulation is cVA. The sensitivity of Or67d to cVA is consistent with previous observations; expression studies have shown that Or67d is expressed in T1 sensilla, which are sensitive to cVA, and ectopic expression of Or67d in other trichoid sensilla conferred sensitivity to cVA. However, the results indicate that there are multiple receptors for cVA. Both Or67d and Or65a responded most strongly to cVA among a panel of six related compounds. The two receptors differed in their specificities, however; Or67d gave a relatively stronger response than did Or65a to cis-vaccenyl alcohol, for example. It is noted that the detection of a second cVA receptor, which has not been reported previously, may reflect the sensitivity of the short-range delivery paradigm that was designed (van der Goes van Naters, 2007).

    The response specificity of Or67d, as measured in the empty-neuron system, is nearly identical to that of the ORN in the T1 sensillum. However, it is noted that the magnitude of the response to cVA in the expression system is approximately half that in T1. Dose-response curves show that the response threshold is also lower in the native T1 sensillum; it appears as though the T1 neuron can detect a dose of approximately 10−4 ng, whereas the expressed Or67d receptor may require a dose of approximately 10−2 ng for detection. Slower rise and decay rates were also found, along with higher levels of spontaneous firing in the expression system. These results suggest that the expression system may lack a component that is present in the endogenous context; for example, the odorant-binding protein LUSH was found to be required for normal response to cVA in T1 sensilla (van der Goes van Naters, 2007).

    Whereas Or67d mediates responses to cVA in T1 sensilla, Or65a is expressed in the ORNs of trichoid sensilla that are more distolateral on the antenna and that also respond to cVA. It is noted that the Or65a gene is in close proximity to Or65b and Or65c and that the three genes are coexpressed in a single ORN. Although neither Or65b nor Or65c mediated responses to any of the fly odors tested in the empty-neuron system, the possibility was considered that they might contribute to the response of the ORN if they were coexpressed with Or65a, perhaps via heterodimer formation. Accordingly, all pairwise combinations of the three receptor genes were co-expressed. It was found that coexpression of Or65b or Or65c with Or65a did not increase the response mediated by Or65a to any stimulus or change the level of spontaneous activity. Coexpression of Or65b and Or65c yielded little, if any, response to any stimulus (van der Goes van Naters, 2007).

    Finally, it is noted with interest that although Or88a conferred responses to male genital material, it did not mediate responses to cVA, suggesting that it detects an additional pheromone that is also transferred from males to females upon mating (van der Goes van Naters, 2007).

    This study has identified four receptors that mediate responses to fly odors. Or47b and Or88a mediate responses to the odors of both males and virgin females. Or65a and Or67d mediate responses to cVA, a male-specific lipid that is present in male genital material, is presumably extracted in hexane extracts, and is transferred to females upon mating. Or88a also responds to a compound in male genitalia, but this compound is distinct from cVA (van der Goes van Naters, 2007).

    The responses of these receptors suggest a working model of the olfactory basis of mate recognition by males. In this model, neural activity mediated by Or47b and Or88a reports the proximity of a fly, either male or female. This olfactory recognition may contribute to the recognition mediated by other sensory modalities; recognition of conspecifics is a prerequisite to successful courtship. The activity of Or65a, Or67d, or both would indicate that the partner is a male or a recently mated female; thus, when the antenna of a male is in close proximity to another fly, the activation of Or65a and/or Or67d would report that the other fly is unsuitable as a mate. The lack of a signal from these receptors would permit continued courtship activity by the male (van der Goes van Naters, 2007).

    A well-documented phenomenon can be interpreted in terms of this model. Mature males not only court virgin females but also vigorously court newly eclosed males. Young males, like virgin females, lack cVA and would not be expected to activate Or65a and Or67d, allowing courtship to proceed (van der Goes van Naters, 2007).

    Why would Or65a and Or67d not be activated in the antenna of a male by material in its own genital region? Perhaps very little of the internal genital material is released to the air unless the region is manipulated by a capillary tip or washed in hexane, and perhaps what little is released under natural conditions can normally be detected only at very close range; if cVA were released in large amounts and inhibited mating over a long range, then mating might be inhibited at sites where flies congregate and often mate, such as rich food sources. It is also possible that the fly adapts to the ambient level of cVA, produced by its own genital region, and is sensitive to increases above that level (van der Goes van Naters, 2007).

    Why are there two cVA receptors, expressed in two distinct ORNs, in different subtypes of trichoid sensilla? There is evidence that cVA serves two functions as a pheromone in Drosophila. (1) cVA has been shown to act as an anti-aphrodisiac, detering males from courting with a recently mated female. (2) cVA is deposited by females during egg laying, and there is evidence that it enhances the attractiveness of the oviposition substrate to other flies. Perhaps Or65a and Or67d activate two distinct behavioral circuits and thereby separately mediate two functions of cVA in conjunction with other cues (van der Goes van Naters, 2007).

    Interestingly, no receptor for female-specific odors was identifed, although there is evidence that 7,11-heptacosadiene and 7,11-nonacosadiene, two female-specific hydrocarbons, act as aphrodisiacs. It is possible that some of the trichoid receptors respond to these compounds, which were not tested individually, or other female-specific compounds but do not function efficiently in the expression system. It is also possible that these compounds are detected by gustatory receptors, perhaps members of the Gr family. One class of gustatory neuron, which expresses Gr68a, has been shown to be required for normal courtship. Finally, the possibility is noted that some of the receptors that did not respond to the tested stimuli might detect pheromones of other Drosophila species (van der Goes van Naters, 2007).

    It is striking that no differences were observed between males' and females' antennal responses to any of the fly odors tested. This similarity is in stark contrast to the extreme sexual dimorphism in antennal responses to pheromones in moths, such as Bombyx mori and Manduca sexta. The similarity in Drosophila peripheral olfactory responses suggests that in the fly, differences in male and female behavioral responses may be determined by differences in reception of other classes of sensory input, such as taste information, or by differences in the transmission or processing of olfactory information. It is possible that cVA, for example, is sensed through the same peripheral mechanisms in males and females but that only in males is the primary representation transformed in a way that accords it a negative valence (van der Goes van Naters, 2007).

    In summary, a systematic analysis was carried out of the trichoid sensilla, one of the three major types of sensilla on the Drosophila antenna. These sensilla appear to be specialized for sensing fly odors, as opposed to food odors. The differential activity of ORNs in trichoid sensilla provides an olfactory basis for a male's ability to discriminate suitable from unsuitable mating partners. The molecular basis of these responses was further explored and four odor receptors were identified that mediate responses to fly odors. A model is proposed in which olfactory information flows through these receptors according to a simple logic. Although the full repertoire of pheromones and receptors has yet to be characterized, it is possible that the model may be richly elaborated without undergoing an alteration in its fundamental logic (van der Goes van Naters, 2007).

    Li, Z., Ni, J. D., Huang, J. and Montell, C. (2014). Requirement for Drosophila SNMP1 for rapid activation and termination of pheromone-induced activity. PLoS Genet 10: e1004600. PubMed ID: 25255106

    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). This study 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. This study 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). The bombykol receptor complex was expressed in Xenopus oocytes in the presence or absence of the silk moth SNMP1 (BmSNMP), and it was found that addition of BmSNMP accelerated receptor activation and deactivation. These results thus clarify SNMP1 as an important player required for the rapid kinetics of the pheromone response in insects (Li, 2014: PubMed).

    Transcriptional regulation of odorant receptors; Mechanisms of odor receptor gene choice in Drosophila

    A remarkable problem in neurobiology is how olfactory receptor neurons (ORNs) select, from among a large odor receptor repertoire, which receptors to express. Computational algorithms and mutational analysis were used to define positive and negative regulatory elements that are required for selection of odor receptor (Or) genes in the proper olfactory organ of Drosophila, and an element was identified that is essential for selection in one ORN class. Two odor receptors are coexpressed by virtue of the alternative splicing of a single gene, and dicistronic mRNAs were identified that each encode two receptors. Systematic analysis reveals no evidence for negative feedback regulation, but provides evidence that the choices made by neighboring ORNs of a sensillum are coordinated via the asymmetric segregation of regulatory factors from a common progenitor. Receptor gene choice in Drosophila also depends on a combinatorial code of transcription factors to generate the receptor-to-neuron map (Ray, 2007).

    Sequences that dictate expression in a specific ORN lie close to an Or gene: In order to identify sequences that dictate the expression of Or genes in particular ORNs, the minimal promoter regions that are sufficient to drive faithful expression of an Or gene were defined. This analysis with the Or85e gene. It had been found that the 3.1 kb genomic sequence upstream of the Or85e translational start site was capable of driving faithful expression of the yeast GAL4 gene in pb2A ORNs, as indicated by the expression of UAS-lacZ and UAS-GFP reporter genes that are activated by GAL4 (Ray, 2007).

    A series of deletions of this 3.1 kb region were generated, each deletion progressively removing an additional ~500 bp from the 5' end of the region. Each truncated Or85e-GAL4 construct was then used to drive expression of UAS-lacZ and UAS-GFP reporters, and at least five independent lines were examined for each deletion construct. A construct containing only 450 bp upstream of the Or85e translational start site was found, and all larger constructs in the series, gave expression patterns similar to that of the initial 3.1 kb construct. However, a construct containing only 350 bp of upstream DNA showed no expression. To determine whether the cells labeled by the Or85e-GAL4 construct containing 450 bp of upstream DNA are in fact of the pb2A class, a double-labeling experiment was carried out. It was found that all GFP+ cells were also labeled with an in situ hybridization probe for Or85e RNA; moreover, nearly all cells labeled with the Or85e RNA probe were also GFP+. The simplest interpretation of these results is that 450 bp of DNA upstream of the Or85e translational start site contain the information that dictates specific expression in the pb2A neurons (Ray, 2007).

    It was surprising that the information dictating expression in a single ORN class was so economically packed. To determine whether the organization of the Or85e gene is representative of other Or genes, another Or gene, Or46a, which is expressed in the ORN that neighbors pb2A, pb2B, were analyzed. Consistent with the results found for Or85e, it was found that 400 bp upstream of the Or46a translational start site is sufficient to confer specific expression in the pb2B class of ORN (Ray, 2007).

    To determine the distance between the transcriptional start site and the translational start site, rapid amplification of 5' cDNA ends (5' RACE) from maxillary palp RNA was carried out for both Or85e and Or46a, as well as for two other Or genes, Or59c and Or71a. In all cases, the predicted transcriptional start sites, as determined by the longest RACE products, lie within 50 bp of the predicted translational start sites (Ray, 2007).

    To investigate the mechanism by which the selection of an Or gene in an olfactory organ is restricted to an organ-specific subset of Or genes, it was asked whether there are regulatory elements that are shared among maxillary palp Or genes but not antennal Or genes. Such elements might dictate the organ-specific expression of the maxillary palp genes, perhaps by binding maxillary palp-specific transcription factors. Focused was initially placed on the 500 bp upstream of the translational start sites, as regions of this size were sufficient to confer faithful GAL4 expression patterns in the cases analyzed. As a first means of searching for sequence elements shared among maxillary palp Or genes sequence alignments were generated, but this analysis revealed remarkably little conservation among the 500 bp regions upstream of the seven maxillary palp Or genes, much less than has been observed upstream of mammalian OR genes. Therefore, some powerful computational algorithms were used to identify sequence motifs shared among maxillary palp Or genes. Algorithms were used that detect both of the major types of short DNA elements to which transcription factors bind: unipartite, or oligonucleotide, motifs and bipartite motifs. Specifically, OLIGO-ANALYSIS, which searches for oligonucleotide motifs of 6-8 nucleotides shared by coregulated genes, and DYAD-ANALYSIS, which searches for shared bipartite sequence motifs consisting of either two trimers or two tetramers, separated by 1-20 nucleotides, was used. These algorithms were used to seek motifs that are overrepresented upstream of maxillary palp Or genes, as compared to upstream of all annotated Drosophila genes, but that are not overrepresented upstream of antennal Or genes (Ray, 2007).

    A bipartite motif, termed Dyad-1, was identified that consists of two trimers, CTA and TAA, separated by nine nucleotides, that is, CTA(N)9TAA. This motif was of special interest in that all seven maxillary palp Or genes contain at least one iteration of this sequence in the 500 bp region upstream of the predicted translational start site, and most of these genes contain more than one. The frequency of occurrence upstream of maxillary palp Or genes exceeds that found upstream of the ensemble of Drosophila genes by a factor of 7.2. By contrast, its frequency upstream of 32 Or genes shown to be expressed in the antenna exceeds that of the ensemble of Drosophila genes by a factor of only 1.2 (Ray, 2007).

    A heptamer sequence, CTTATAA, which was termed Oligo-1, was identifed. This motif attracted attention, in part because it contains a 6 bp palindromic core sequence, which is characteristic of many transcription factor binding sites. The frequency of occurrence of Oligo-1 in the 500 bp upstream region of maxillary palp Or genes exceeds that of the ensemble of Drosophila genes by a factor of 10.0. By contrast, upstream of the antennal Or genes, it is overrepresented by a factor of only 1.3. The degree of overrepresentation was calculated using a different approach: the iteration frequency in the 500 bp upstream region of each maxillary palp Or gene was compared to that in the entirety of Drosophila noncoding genomic DNA. It was found that by this measure, Dyad-1 is overrepresented by a factor of 13.7 upstream of the maxillary palp genes, and Oligo-1 is overrepresented by a factor of 10.0. By contrast, these elements are overrepresented by factors of only 2.0 and 1.0, respectively, upstream of antennal Or genes (Ray, 2007).

    Thus, these two sequence elements are present at unexpectedly high frequencies upstream of maxillary palp Or genes, but not antennal Or genes, suggesting the possibility that these sequences play a role in the process by which the choice of Or genes by ORNs is restricted to an organ-specific subset (Ray, 2007).

    The function of Dyad-1 elements was tested in the context of three Or genes using two approaches. First, a series of deletions in the Or71a locus was generated by imprecise excision of a P element located 1.0 kb upstream of the Or71a translational start site. Chromosomes retaining 170 bp or more of the most proximal upstream DNA sequences continued to express Or71a, as determined by in situ hybridization with an Or71a probe. Chromosomes that contain 121 bp or less of this sequence (Δ3, Δ4, and Δ5) did not express Or71a. There are two Dyad-1 elements upstream of Or71a; the chromosomes that retained one of these elements therefore retained Or71a expression, but those that retained no Dyad-1 elements lost expression (Ray, 2007).

    Second, the Dyad-1 elements located upstream of Or46a were mutated. Specifically, a 1.9 kb region of DNA upstream of Or46a has previously been shown to drive expression of GAL4 in pb2B ORNs of the maxillary palp. Within this 1.9 kb region are two Dyad-1 elements, located 5 bp apart. A construct that carries alterations in the sequences of both elements and the spacing between them was tested, and it was found that this mutated construct no longer drives expression in the maxillary palp (Ray, 2007).

    Third, the function of the Dyad-1 elements at Or85e was tested using both ablation and deletion approaches. The 0.45 kb Or85e-GAL4 construct described above was used and base pairs of each of its two Dyad-1 elements were mutated without altering their relative positions. This ablation abolished expression in the maxillary palp. Truncated constructs that do not contain Dyad-1 elements did not drive expression. The shortest of these constructs contains 90 bp of sequence, which is predicted to contain the basal promoter of the Or85e gene (see Promoter prediction program for the neural network promoter prediction algorithm). The two Dyad-1 sequences were added to the 90 bp construct; the addition of these Dyad-1 elements did not restore expression in the maxillary palp. It was demonstated (see below) that the 90 bp sequence drives expression when different elements are added to it (Ray, 2007).

    The simplest interpretation of these results is that Dyad-1 is a positive regulatory element that is necessary but not sufficient for expression in the maxillary palp (Ray, 2007).

    To investigate the function of the Oligo-1 element, it was mutated in the context of the Or85e promoter. The mutation was found to cause misexpression in the antenna. A marked decrease was observed in labeling of maxillary palp ORNs (Ray, 2007).

    These results were confirmed and extended in an analysis of the Or71a gene. There is no Oligo-1 element in the 500 bp upstream of the Or71a gene, but there are two clustered Oligo-1 elements downstream of the gene, located 426 bp and 458 bp downstream from the translational stop site. When the 2.3 kb of upstream region was used alone to drive GAL4 expression, misexpression was found in the antenna. Reduced labeling was also observed in the maxillary palp: only 48% of cells labeled with an Or71a in situ hybridization probe were also GFP+ and the intensity of the GFP+ labeling appeared weak in a limited experiment (n = 9 maxillary palps). When both the 2.3 kb of upstream DNA and a 1.4 kb region of downstream DNA that contains both Oligo-1 elements were used, the antennal misexpression was not observed. Moreover, expression in the maxillary palp was increased: nearly 100% of cells labeled with an Or71a in situ hybridization probe were also GFP+ (n = 9 maxillary palps), and the intensity of the GFP+ labeling appeared strong. To determine whether the repression of antennal labeling and the enhancement of maxillary palp labeling were in fact due to the presence of the Oligo-1 elements in the downstream sequences, a third construct was generated, similar to the second but in which both Oligo-1 sequences were mutated. This construct again produced misexpression in the antenna, and reduced expression in the maxillary palp: again only 48% of cells labeled with an Or71a in situ hybridization probe were also GFP+. Moreover, expression was highly variable, and the intensity of the GFP+ labeling appeared weak (n = 9 maxillary palps) (Ray, 2007).

    The simplest interpretation of these results is that the Oligo-1 elements repress expression of maxillary palp Or genes in the antenna and enhance their expression in the maxillary palp. Moreover, Oligo-1 elements appear capable of acting either upstream or downstream of an Or gene (Ray, 2007).

    The Dyad-1 and Oligo-1 elements were identified in a search for sequences that act in the process of receptor gene choice by dictating in which organ an Or gene is expressed. Beyond organ-specific regulation, however, Or genes require an additional level of control to generate the receptor-to-neuron map: they must contain information dictating their precise expression in a single ORN class (Ray, 2007).

    In an effort to identify a neuron-specific regulatory element, advantage was taken of the discovery that one maxillary palp ORN, pb2A, coexpresses two unlinked Or genes, Or85e and Or33c. It was reasoned that because both Or genes are expressed in the same neuron, they are likely to share regulatory elements that dictate expression in this neuron. Accordingly, the 500 bp upstream regions of both genes were examined for sequence elements of at least 6 bp in length that are shared by these two genes, but not by other maxillary palp Or genes (Ray, 2007).

    Two elements were identified that meet these criteria: a 12 bp element, TTTATTTGCATA, which was designated the pb2A-1 element, and an 8 bp element, AGTTTTTA, which was designated pb2A-2. pb2A-1 is located at −320 bp relative to the translational start site of Or85e and at −206 bp relative to Or33c; pb2A-2 is located at −102 bp relative to Or85e and −274 bp relative to Or33c. It was noted that although these elements were identified by examining the 0.5 kb upstream regions, their specificity extends farther: they are not found in the 1 kb region upstream of any other maxillary palp Or gene, nor in the 0.5 kb downstream region of any maxillary palp Or gene (Ray, 2007).

    To test the function of these elements, they were mutated in the context of the 450 bp Or85e promoter. No effect of mutating pb2A-1 was found: the mutant construct produced a pattern of GFP expression that appeared identical to that of the wild-type control construct. Moreover, it was confirmed that these GFP+ cells are in fact pb2A cells by showing that they hybridize to an Or85e probe in a double-label experiment. Mutation of pb2A-2, however, abolished expression in pb2A cells, indicating that the pb2A-2 element is necessary for expression in pb2A. It was also found that when seven copies of pb2A-2 and two copies of Dyad-1 were fused to the 90 bp basal promoter of Or85e, expression was observed in the maxillary palp. As a further test, seven copies of pb2A-2 and two copies of Dyad-1 were inserted upstream of a second minimal promoter; in this case, the pb2A-2 elements were 45 bp upstream of a TATA box. Again, expression was found in the maxillary palp. Although expression was too weak to allow double-label in situ hybridization, in each case at least some of the cells could be identified as neurons by the presence of dendrites, and their patterns of expression and distribution on the maxillary palp are consistent with those of pb2A. These results indicate that not only is pb2A-2 necessary for Or85e expression but that artificial promoters containing pb2A-2 sequences and Dyad-1 sequences can drive expression in maxillary palp ORNs. It was noted that two Dyad-1 sequences alone did not drive expression of Or85e, as indicated above (Ray, 2007).

    Two functional odor receptors from one alternatively spliced Or gene: One mechanism by which an ORN may select two Or genes thus appears to depend on the location of common ORN-specific elements, such as pb2A-2, upstream of two different Or genes. Another instance was found of receptor coexpression that occurs through a different mechanism. This coexpression was revealed by a detailed analysis of the Or46a locus, which has been shown to be expressed in pb2B cells (Ray, 2007).

    The Or46a locus has been proposed to contain two coding regions, Or46aA and Or46aB, separated by less than 100 bp and expressed by alternative splicing. It was confirmed that the locus produces two alternatively spliced mRNAs; both mRNAs were identified in multiple independent experiments. These two splice forms were observed at comparable levels in RT-PCR analysis, and were the major products detected; the possibility that minor species of functional significance may also be produced cannot be excluded. A detailed analysis of the two major mRNAs revealed surprising structures. The shorter mRNA is spliced from an internal position within exon 3 to exon 5, bypassing exon 4, and contains the Or46aA coding region. The longer mRNA lacks the splice between exons 3 and 5 and is a dicistronic message, containing two coding regions. The first of these two coding regions, which was termed Or46a1, is identical to that of Or46aA except at the 3' end, which encodes C-terminal residues that lie immediately beyond the seventh predicted transmembrane domain. Or46a1 encodes 18 terminal amino acids that show no identity to the 17 terminal amino acids of Or46aA. The second of the two coding regions, Or46aB, shares only 36% amino acid identity with Or46aA and 31% identity with Or46a1 (Ray, 2007).

    This unexpected splicing pattern predicts that multiple receptors from this locus may be coexpressed in the same cell. A double-label in situ hybridization experiment was carried out using probes for Or46aA, consisting of sequences from within exon 1, and Or46aB, consisting of sequences within exon 4. It was found that all cells labeled by Or46aA sequences were also labeled by Or46aB sequences; thus all cells that express the short transcript also express the long transcript. Likewise, all cells labeled with Or46aB were also labeled by Or46aA, but this latter result is less informative, on account of the expected crosshybridization between Or46aA and Or46a1. Thus, from this experiment alone, it cannot be determined whether there are any cells that express the long transcript but not the short transcript. In any case, these experiments reveal that pb2B cells express sequences corresponding to more than one Or open reading frame (Ray, 2007).

    In order to determine whether more than one of the distinct ORFs in fact encodes a functional odor receptor, an in vivo expression system, the 'empty neuron' system, was used. Using this method, it was found that Or46aA and Or46aB both encode functional odor receptors that respond to phenols, while Or46a1 did not impart responses to any odor (Ray, 2007).

    The Or46a locus thus encodes three predicted odor receptors. Their expression appears limited to a single class of neuron, pb2B. To investigate the significance of this one receptor gene-multiple receptor organization, it was asked whether it is conserved in evolution. The genomes of five additional Drosophila species were examined: D. simulans, D. yakuba, D. erecta, D. pseudoobscura, and D. grimshawi. It was found that orthologs of the ORFs encoding Or46aA, Or46a1, and Or46aB are present in all species and thus have been maintained for tens of millions of years. Moreover, the organization of the locus is well conserved; for example, the distance between the stop codon of Or46a1 and the start codon of Or46aB is in all cases between 69 bp and 164 bp. Double-label in situ hybridization with D. pseudoobscura sequences representing the orthologs of Or46aA and Or46aB showed coexpression in cells of the D. pseudoobscura maxillary palp (Ray, 2007).

    In the mosquito Anopheles gambiae, the genes most closely related to Or46aA and Or46aB are AgOr34 and AgOr37, whose predicted products both show 22% amino acid identity to Or46aB and 19% and 20% identity to Or46aA, respectively. Interestingly, these two mosquito genes are also tightly linked, but in inverted fashion, with only 1.5 kb of intervening noncoding DNA, suggesting the possibility that they may share common regulatory sequences and hence be coexpressed (Ray, 2007).

    It is noted, finally, that in order to express a functional Or46aB protein from a dicistronic RNA, an internal ribosome entry site (IRES) is required. Although there is precedent for functional IRES sequences in Drosophila, they have not been well defined. IRES sequences are poorly conserved across phylogeny, but dicistronic RNAs identified from polio virus and the human ornithine decarboxylase gene share a common UUUC sequence approximately 26-34 bp upstream of the distal AUG. The presence was noted of a UUUC sequence 38 bp upstream of the AUG of Or46aB, a sequence that is conserved at the same position in D. yakuba and D. ananassae (Ray, 2007).

    Mechanisms of Or gene coexpression in the antenna: Evidence has been presented for two distinct mechanisms by which a single maxillary palp ORN can express two odor receptors, one mechanism that depends on the localization of a common cis-regulatory element upstream of two unlinked Or genes, and one that depends on alternative splicing. To determine whether these mechanisms are singularities, unique to the maxillary palp, the analysis was expanded to include the entire olfactory system. All pairs of Or coding regions were examined that are separated by <1 kb of intervening DNA: (Or22a, Or22b), (Or33a, Or33b), (Or33b, Or33c), (Or42a, Or42b), (Or59b, Or59c), (Or65b, Or65c), (Or69aA, Or69aB), and (Or85b, Or85c). Also examined were (Or10a, Gr10a), which are also tightly linked (Ray, 2007).

    It was surprising to find a dicistronic message that encodes both Or10a and Gr10a, in both of two strains analyzed, Oregon-R and w1118. Or10a is an odor receptor for methyl salicylate, while Gr10a is a member of the gustatory receptor gene family. The dicistronic mRNA was identified in each of six independent experiments, each using a different combination of primer pairs. It was noted that there are two UUUC motifs upstream of the Gr10a translational start codon, at positions −32 and −52, suggesting the possibility that one may act as an IRES. These results provide a molecular mechanism to explain the coexpression of these two genes in ab1D ORNs (Ray, 2007).

    It was found that although Or22a and Or22b are each encoded by independent transcripts, two transcripts were identified that are likely to have the same 5' ends, based on the positions of the longest cDNAs isolated. These results suggest that Or22a and Or22b can be expressed from the same promoter, which could in principle explain the coexpression of the two genes in ab3A ORNs. In the longer transcript, encoding Or22b, the second intron is not removed, creating a frameshift mutation and a nonsense codon in Or22a; thus a functional Or22a receptor would be encoded only by the shorter transcript. The presence is noted of a UUUC at position −37 upstream of the Or22b translational start codon, suggesting a means by which Or22b could be translated from a long mRNA that also includes an Or22a translational start site. At the same time, there is a TATA box 70 bp upstream of the Or22b translational start codon, and a 1.3 kb region upstream of Or22b was found to drive faithful reporter gene expression, suggesting the existence of a third, Or22b-specific transcript. In the Canton-S strain, Or22a has been shown to be a functional odor receptor, whereas Or22b is nonfunctional. However, in an Oregon-R strain, both genes encode functional odor receptors (Ray, 2007).

    Or69a also produces two distinct mRNAs, which contain identical, or nearly identical, 5'ends. The shorter mRNA encodes Or69aA; the longer transcript encodes Or69aB. The mRNA encoding Or69aB contains many of the codons of Or69aA but the Or69aA ORF is terminated by a stop codon following the first splicing event. These results provide a molecular explanation for the coexpression of Or69aA and Or69aB in ORNs of ab9 sensilla; however, a 0.9 kb region upstream of the Or69aB ATG was found to drive reporter gene expression in ab9 sensilla, suggesting the possibility of an additional Or69aB-specific transcript (Ray, 2007).

    In addition to these three cases of antennal coexpression, recent mapping studies have identified 6 additional antennal ORN classes that coexpress Or genes among the 36 antennal ORN classes to which Or genes were mapped. Four of these coexpressed Or gene pairs are unlinked: (Or33b, Or85a); (Or33a, Or56a); (Or33b, Or47a); and (Or49a, Or85f), raising the possibility that they might, like (Or33c, Or85e), contain a common regulatory element. Using the same bioinformatics approach used to identify pb2A-1 and pb2A-2, common motifs were identified for each pair. These elements were neuron specific in that they were not found in the 500 bp upstream of any other Or gene in the genome. Shared motifs were likewise identified for (Or22a, Or22b) and (Or69aA, Or69aB), suggesting an additional mechanism by which the upstream and downstream ORFs could be coexpressed. A common element was found for a pair of Or genes that are coexpressed in a larval ORN (Or94a, Or94b). As a control, the same bioinformatics approach was carried on two genes that are not coexpressed, Or85f and Or56a, and no elements were found that met these criteria, that is, no common elements were found unique to these genes. Or19a and Or19b are coexpressed but appear to have duplicated recently and there is extensive identity in their upstream regions (Ray, 2007).

    Odor receptor expression is permissive: lack of negative feedback regulation in Drosophila: In mammals, analysis of nonfunctional receptor genes has led to the proposal that the expression of one odor receptor inhibits the expression of others in the same ORN by negative feedback regulation. To investigate directly whether expression of an odor receptor inhibits the expression of any others in Drosophila, each of three receptors was ectopically expressed in the maxillary palp, and the effects on expression of others were tested. Expression of Or85e, which in wild-type is expressed only in pb2A, was driven in all or almost all ORNs of the maxillary palp using the C155-GAL4 driver, which initiates expression before the onset of normal Or expression. Then the expression of other maxillary palp Or genes was tested by in situ hybridization and electrophysiology (Ray, 2007).

    It was confirmed by in situ hybridization that Or85e was in fact expressed in most if not all ORNs of the maxillary palp in C155-GAL4; UAS-Or85e flies. Then expression of the Or genes that are normally expressed in each of the other five maxillary palp ORN classes was examined. It was found that each tested gene was expressed in what appeared to be a normal pattern, in the presence of ectopic Or85e expression. Thus, expression of Or85e does not inhibit transcription of other Or genes (Ray, 2007).

    It was then asked whether the other Or genes were functionally expressed, by carrying out electrophysiological recordings from C155-GAL4; UAS-Or85e flies, using a diagnostic odor panel that distinguishes between Or85e-expressing ORNs and all other classes of maxillary palp ORNs. Or85e responds strongly to fenchone; the pb1A neuron, by contrast, responds strongly to 2-heptanone, on account of Or42a expression, but not fenchone. When Or85e is overexpressed, neurons were detected that respond strongly to both fenchone and 2-heptanone. The simplest interpretation of these results is that misexpression of Or85e in pb1A cells does not inhibit functional expression of Or42a. Similar physiological evidence was found to indicate that expression of Or85e does not inhibit the functional expression of Or71a, Or46a, Or59c, and Or85d. In a more limited experiment, C155-GAL4 was used to drive the early expression of two other receptors, Or42a and Or10a (an antennal gene). By performing a similar electrophysiological analysis, it was found that in each ORN class in which a novel odor response was conferred by misexpression of Or42a or Or10a, the response conferred by the endogenously expressed Or gene was still present. These results, taken together, indicate that expression of one Or gene does not repress that of others by feedback regulation (Ray, 2007).

    Coordination of receptor choice between two neurons of a sensillum: Each sensillum of the maxillary palp contains two ORNs, combined according to a strict pairing rule. For example, each pb1 sensillum contains a pb1A neuron that expresses Or42a, paired with a pb1B neuron that expresses Or71a; for the present analysis, this cellular expression pattern was designated as the (Or42a+; Or71a+) configuration. Such stereotyped pairing of ORNs has been documented in diverse insects but is not observed in mammals. It raises the problem of how the choice of a receptor in one ORN is coordinated with that in a neighboring ORN. In principle, the choice made by one ORN could induce a specific choice in the neighboring ORN; alternatively, the choices of two neighboring maxillary palp ORNs could be made simultaneously and be coordinated by virtue of the asymmetric segregation of regulatory proteins from a common progenitor cell (Ray, 2007).

    In the development of antennal sensilla there is evidence that a single progenitor, or founder cell, recruits three secondary progenitor cells of which one, PIIc, divides to give rise to two neurons. Mastermind (Mam), a nuclear protein in the Notch pathway, is essential for asymmetric cell division of embryonic neuroblasts in the developing Drosophila nervous system: both loss and gain of mam function result in altered identities of the daughter cells. The expression of Or genes was systematically investigated by paired ORNs in maxillary palps that misexpress mastermind (mam) (Ray, 2007).

    First UAS-mam was expressed in the developing olfactory organs using the eyeless-GAL4 driver. Then receptor gene expression was examined by double-label in situ hybridization. It was found that the coordination of receptor gene expression between neighboring ORNs was abnormal in all three types of sensilla, pb1, pb2, and pb3. A substantial fraction of sensilla expressing Or42a showed expression of this gene in both neurons: these sensilla contained one ORN that expressed Or42a alone and another that expressed both Or42a and Or71a, designated the (Or42a+; Or42a+Or71a+) configuration. Likewise, sensilla were found in (Or85e+; Or85e+Or46a+) and (Or59c+; Or59c+Or85d+) configurations; in wild-type, these genes are expressed only in (Or85e+; Or46a+) and (Or59c+; Or85d+) configurations. No other abnormal configurations were identified: those ORNs that misexpressed an Or gene always misexpressed the Or gene of the neighboring ORN (Ray, 2007).

    This abnormal partitioning of receptor expression was confirmed by an independent method, electrophysiological recordings. Specifically, in the wild-type pb2 sensillum, the A cell produces large spike amplitudes and strong responses to fenchone, whereas the B cell produces small spike amplitudes and strong responses to 4-methyl phenol. In eyeless-GAL4; UAS-mam, sensilla were identified containing one neuron with a large spike amplitude and a strong fenchone response, paired with a neuron that produces a small spike amplitude and strong responses to both fenchone and 4-methyl phenol, as expected of (Or85e+; Or85e+Or46a+) sensilla. It was also confirmed the abnormal partitioning of receptor expression in another genotype, by driving a truncated dominant-negative form of mam, UAS-mamH, with the pan-neuronal driver elav-GAL4. Double-label in situ hybridization was carried out with Or85e and Or46a probes and identified (Or85e+; Or85e+Or46a+) sensilla (Ray, 2007).

    These results are consistent with a model in which the A and B neurons of a maxillary palp sensillum are siblings that derive from a common progenitor by a Mam-dependent asymmetric cell division. Perturbation of Mam function might lead to the abnormal segregation of a regulatory protein, which in turn leads to misexpression of a receptor (Ray, 2007).

    The observation that misexpression in an ORN was restricted to the Or gene of its neighbor, and not to any other Or genes, is consistent with a model in which the progenitor has undergone a restriction that limits its daughter ORNs to the expression of the Or genes of one sensillum type. It is noted that the data are also consistent with a model in which the progenitor, and both daughter ORNs, contain a positive regulatory factor that binds to a site shared by, and specific to, the Or genes expressed in a particular sensillum type. Computational analysis has in fact revealed such elements -- for example, Or42a and Or71a both contain an upstream AAATCAATTA element that is not found adjacent to other Or genes of the maxillary palp or antenna; however, genetic analysis of this element has not revealed a functional requirement for it in receptor gene expression, and no support was found for the existence of a sensillum-specific determinant of receptor gene expression (Ray, 2007).

    Thus, these data, taken together, are consistent with a model in which the coordination of receptor gene expression in a sensillum is achieved through the Mam-dependent segregation of regulatory factors. The results underline the importance of identifying regulatory proteins whose proper distribution in the ORNs of the maxillary palp is essential to the proper distribution of receptor gene expression (Ray, 2007).

    Different Or genes depend on different combinations of the transcription factors Lz and Acj6: In a complementary analysis of the mechanisms of receptor gene choice, transcription factors were investigated whose expression had been reported in at least one olfactory organ and whose mutations had been shown to cause olfactory defects. One such protein, the Runx domain-containing transcription factor Lozenge, was found had predicted binding sites (RACCRCA, R = purine) adjacent to four maxillary palp Or genes. Specifically, it was found that two maxillary palp Or genes, Or59c and Or85d, had two Lz binding sites, and two genes, Or71a and Or85e, had one Lz binding site, within 1 kb upstream or downstream of the coding region. Lz is required for the specification of cell fate in the eye and for normal numbers of olfactory sensilla in the antenna. In the maxillary palp the numbers of sensilla are normal, but electropalpogram recordings showed large reductions in odor responses (Ray, 2007).

    To investigate the possibility that Lz is required for normal receptor gene expression, it was first asked whether it is expressed in ORNs of the maxillary palp. Lz is coexpressed with Elav, indicating that it is expressed in the nuclei of all maxillary palp ORNs. Then the expression of six maxillary palp Or genes was examined, one from each ORN class, in lz3, a strong hypomorphic mutant. The four genes that are flanked by predicted Lz binding sites all showed reduced levels of expression; the two genes that contain two Lz binding sites, Or59c and Or85d, showed particularly severe reductions (of 47% and 87%, respectively) in the number of labeled cells. The mildest reduction, 18%, was observed for Or85e; consistent with this result, a 14% reduction was observed when DNA including the predicted Lz binding site was removed from an Or85e-GAL4 driver (the construct containing 3 kb of upstream DNA labeled 13.4 ± 0.4 cells, whereas the construct containing 0.45 kb labeled 11.5 ± 0.3 cells; n = 12). The two genes that did not contain Lz binding sites did not show a reduction in labeling in lz3. These results demonstrate that lz is required for the expression of a subset of Or genes in the maxillary palp (Ray, 2007).

    Next a weaker, temperature-sensitive allele, lzts1, was used to investigate the possibility that levels of Or gene expression are susceptible to modulation during the adult stage. It was found that Or85d is expressed in 18% fewer cells (p < 0.05) when lzts1 flies are raised at the restrictive temperature (29°) than when raised at the permissive temperature (18°). When flies were raised at the restrictive temperature and then shifted to the permissive temperature for 24 hr, 1 week after eclosion, the number of Or85d-expressing cells showed an increase of 19%, to a level indistinguishable from that of flies that had been cultured continuously at the permissive temperature. These results confirm the finding of a functional role for lz in Or expression, provide direct evidence that levels of Or expression can be altered after eclosion, and invite investigation of epigenetic modulation of odor receptor expression in Drosophila (Ray, 2007).

    Only one other transcription factor, the POU domain protein Acj6, has previously been demonstrated to be required for odor receptor expression in Drosophila. Specifically, expression of Or33c, Or42a, Or46a, Or59c, and Or85e was severely reduced by the null allele acj66, whereas expression of Or71a and Or85d was unaffected. It has been shown in this study that expression of Or59c, Or71a, Or85e, and Or85d was reduced by lz3, but expression of Or42a and Or46a was not. Thus, the maxillary palp Or genes can be divided into three classes based on their sensitivity to these mutations: those sensitive to both acj66 and lz3 (Or59c and Or85e), to acj66 alone (Or42a and Or46a), or to lz3 alone (Or71a and Or85d). These results support a model in which Or gene expression depends not only on a combinatorial code of regulatory elements but also on a combinatorial code of transcription factors (Ray, 2007).

    In summary, in mammals, it is thought that transcriptional regulatory mechanisms direct expression of OR genes in specific zones of the olfactory epithelium, but that within a zone, OR gene choice is based on a stochastic selection mechanism. A third mechanism, negative feedback, could then operate to limit the number of OR genes expressed in individual neurons (Ray, 2007).

    In Drosophila, the process of receptor gene choice achieves a conceptually simple end: it produces a highly stereotyped receptor-to-neuron map. However, the large number of receptors and neurons presents a regulatory problem of great complexity. To achieve such a precise and highly ordered organization, Drosophila has evolved a sophisticated suite of regulatory mechanisms. This study has documented organ-specific and neuron-specific levels of transcriptional control, including both positive and negative mechanisms. A posttranscriptional mechanism, alternative splicing, was identified and the system has even evolved a relatively rare innovation, dicistronic mRNAs (Ray, 2007).

    The worm Caenorhabditis elegans has a much larger repertoire of odor receptor genes than Drosophila, but the number of ORNs to which it allocates them is very limited. Thus the number of receptor genes per neuron is increased, but the complexity of the regulatory problem is decreased. In vertebrates, however, the repertoire is very large and the number of receptor genes expressed per neuron is very low. Perhaps as the receptor gene repertoire expanded in vertebrate evolution, the complexity of the regulatory problem eventually exceeded the ability of the system to execute a deterministic plan with sufficient fidelity, and deterministic mechanisms were replaced by a stochastic mechanism and a negative feedback mechanism. In any case, the ultimate result of receptor gene choice in Drosophila is the same as in vertebrates: a spectacular diversity of ORNs that underlie the detection and discrimination of odors (Ray, 2007).

    A regulatory code for neuron-specific odor receptor expression

    Olfactory receptor neurons (ORNs) must select (from a large repertoire) which odor receptors to express. In Drosophila, most ORNs express one of 60 Or genes, and most Or genes are expressed in a single ORN class in a process that produces a stereotyped receptor-to-neuron map. The construction of this map poses a problem of receptor gene regulation that is remarkable in its dimension and about which little is known. By using a phylogenetic approach and the genome sequences of 12 Drosophila species, regulatory elements were systematically identfied that are evolutionarily conserved and specific for individual Or genes of the maxillary palp. Genetic analysis of these elements supports a model in which each receptor gene contains a zip code, consisting of elements that act positively to promote expression in a subset of ORN classes, and elements that restrict expression to a single ORN class. A transcription factor, Scalloped, was identifed that mediates repression. Some elements are used in other chemosensory organs, and some are conserved upstream of axon-guidance genes. Surprisingly, the odor response spectra and organization of maxillary palp ORNs have been extremely well-conserved for tens of millions of years, even though the amino acid sequences of the receptors are not highly conserved. These results, taken together, define the logic by which individual ORNs in the maxillary palp select which odor receptors to express (Ray, 2008).

    The spatial organization was examined of ORN classes in the maxillary palp. First, an anti-Elav antibody was used to illustrate the distribution of the entire population of ORN nuclei of the maxillary palp. Second, a multiple-label experiment was carried out to differentially mark ORNs of the three types of sensilla: ORNs of the pb1A class were labeled in green, pb2B in yellow, and pb3A in red. The three classes of ORNs show extensive spatial overlap. These results are consistent with the intermingling of sensillum types that are observed when recordings are taken from sensillar shafts. The spatial overlap of ORN nuclei indicates that the identity of an ORN and, by extension, its choice of a receptor gene, are not dictated solely by its spatial position in a field (Ray, 2008).

    The upstream regions of the two Or genes coexpressed in pb2A have been compared to identify regulatory sequences shared by these two genes, but not by any other maxillary palp Or gene. To identify upstream regulatory elements for the other five maxillary palp Or genes, a different strategy was used based on phylogenetic analysis (Ray, 2008).

    D. melanogaster and D. pseudoobscura diverged tens of millions of years ago and contain orthologous receptor genes. The upstream regions of orthologous Or genes were examined for conserved elements shared by the members of each orthologous pair, but not by any of the other maxillary palp Or genes. Accordingly, all conserved upstream sequences were identified greater than 6 base pairs (bp) in length for each pair of orthologs using DOT-PLOT analysis, and from these conserved elements, those were selected that were specific to each gene. The analysis was focused on the 500 bp that are upstream of the translational start site, because in a previous study, this extent of DNA was sufficient to confer faithful expression to a GAL4 reporter gene in the case of each of two maxillary palp Or genes analyzed in detail. One pair of orthologs, Or85d and its D. pseudoobscura counterpart, was exceptionally well-conserved in the 500-bp upstream region, showing 80% identity. To identify discrete conserved elements within the region upstream of Or85d, the analysis was expanded to include a more divergent species, D. virilis (Ray, 2008).

    Conserved, gene-specific elements were identified for each of the five Or genes analyzed. The number of such elements varies: Or59c contains one, whereas Or42a contains six. In the special case of Or85d, two elements are shared by D. virilis and D. melanogaster upstream of Or85d, but are not found upstream of any other maxillary palp Or gene (Ray, 2008).

    To identify the best candidate for a regulatory element for each of these receptor genes, a powerful bioinformatic approach was used that takes advantage of the recent sequencing of the genomes of ten other Drosophila species: D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. persimilis, D. willistoni, D. virilis, D. mojavensis, and D. grimshawi. The upstream regulatory regions of the orthologous receptor genes from all 12 species were aligned using the genome browser at the University of California Santa Cruz, and each of the elements was mapped onto the alignment. Using this approach, it was possible to identify the gene-specific element with the highest sequence conservation for each of the receptor genes; in the case of Or42a, two elements were nearly identical in their extent of conservation, and both were analyzed (Ray, 2008).

    To determine whether the evolutionarily conserved, gene-specific elements have a regulatory function, they were tested in vivo using two complementary approaches, one based on a loss of function and one on a gain of function. For each gene, the element was analyzed with the highest sequence conservation. Or85d elements were not analyzed because no faithful Or85d-GAL4 driver was available (Ray, 2008).

    Or46a is expressed in the pb2B neuron, and its upstream region contains two conserved, gene-specific elements. One of these elements, 46a1, is more highly conserved. It is 10 bp long, its sequence shows 93% identity across the 12 species, and its position is conserved. A 1.9-kb region of DNA upstream of Or46a drives faithful expression of a GAL4 reporter in pb2B. However, when the 46a1 element is mutated, the 1.9-kb region no longer drives expression. In most cases, no cells are labeled; in rare cases, a single ORN is labeled. The simplest interpretation of these results is that the 46a1 element is necessary for Or46a expression in pb2B (Ray, 2008).

    It was then asked whether the 46a1 element can drive expression in the context of a minimal promoter. Four copies of 46a1 upstream were placed of a TATA box, and it was found that this small construct can in fact drive expression in maxillary palp cells. Many, if not all, of the cells could be identified as ORNs, because they contain dendrites and axons; their identity is considered further below. Expression from this artificial promoter could also be detected in a small subset of neurons in the main gustatory organ, the labellum (Ray, 2008).

    Or71a is expressed in pb1B. Its upstream region contains multiple gene-specific elements, of which the longest and best conserved is 71a3, consisting of 16 bp and showing 97% sequence identity. This element was tested in the context of the Or71a 5' + 3' construct, which contains sequences both upstream and downstream of Or71a. This construct drives faithful expression of GAL4 when the 71a3 element is intact, but not when it is mutated. When multiple copies of 71a3 were placed upstream of a TATA box, the construct drove GAL4 expression in maxillary palp cells that can be identified as ORNs by virtue of their dendrites and axons. Low levels of expression could also be detected in a small subset of cells in the labellum (Ray, 2008).

    Or59c is expressed in pb3A, and its upstream region contains a single gene-specific conserved element, 59c1, which is 11 bp long and shows 97% sequence identity across nine species; the region containing the 59c1 sequences could not be identified in three of the most distantly related species, D. virilis, D. mojavensis and D. grimshawi. Its function was tested by placing multiple copies upstream of a TATA box and it was found that this minimal promoter drives robust expression of GAL4 in the maxillary palp. Expression was not detected in the labellum (Ray, 2008).

    Earlier studies have shown that the expression of a subset of the maxillary palp Or genes requires the POU domain transcription factor Acj6, which is expressed in all ORNs of the maxillary palp. Acj6 also controls axon targeting specificity of a subset of maxillary palp ORNs . The 46a1, 71a3, and 59c1 elements do not contain predicted Acj6 binding sites, and the transcription factors that act on these sequences are unknown. To test whether the factors that act on these neuron-specific elements are dependent on acj6, the expression of the minimal promoter constructs was examined in an acj66 background (Ray, 2008).

    In the acj66 mutant, although the expression of the Or46a-GAL4 driver is lost, which is consistent with the loss of Or46a mRNA observed previously, the expression of the 46a1 minimal promoter construct is still strong. These results suggest that the factors that direct expression from the 46a1 motif are independent of acj6 for their expression and function. An alternative possibility is that another transcription factor can compensate for the loss of acj6 (Ray, 2008).

    Expression of the Or71a-GAL4 driver can be detected in acj6, and the expression of the 71a3 minimal promoter construct can also be detected. These results suggest that the factors binding to 71a3 do not require acj6 for their expression or function (Ray, 2008).

    In the case of Or59c, it was found that acj6 is required both for expression of the gene and for the minimal promoter. These results suggest that acj6 is required directly or indirectly for the expression of the 59c1 binding factor or for its function at the 59c1 site (Ray, 2008).

    Or42a is expressed in pb1A, and 4.1 kb of upstream DNA drives faithful expression of GAL4 in maxillary palp ORNs. Two elements are nearly identical in their high conservation: 42a4 (98%) and 42a6 (98%), and the function of both elements was tested in vivo. 42a6 maps only three bp from 42a5. A small deletion was constructed that eliminates both 42a6 and 42a5 elements, and no effect was found on Or42a-GAL4 expression (Ray, 2008).

    The longer of the two most highly conserved elements at Or42a, 42a4, contains an inverted repeat: AGTGTAAAAGTTTACACTT. Surprisingly mutation of this element led to a 2-fold increase in the number of labeled maxillary palp cells, from 18.2 ± 1.8 to 33.2 ± 3.7. The simplest interpretation of this result is that 42a4 is a negative regulatory element that represses Or42a in a subset of ORNs. To test this interpretation, a double-label experiment was carried out using probes for the endogenous Or42a mRNA and for the green fluorescent protein (GFP) that is driven by the mutant promoter via GAL4. It was found that all Or42a+ cells express GFP, but that GFP is also expressed in an additional subset of cells (Ray, 2008).

    To identify the cells that ectopically express GFP, a series of additional double-label experiments was undertaken. It was found that the GFP+ cells do not express Or59c mRNA, indicating that they are not pb3A neurons, nor are they paired with cells that express Or59c mRNA, indicating that they are not pb3B neurons. In another experiment, GFP+ cells did not label with an Or33c probe, indicating that they are not pb2A neurons; however, GFP+ cells were often found paired with Or33c+ cells, indicating that many GFP+ cells are pb2B neurons. The identity of these GFP+ cells as pb2B neurons was confirmed directly in another double-label experiment using a probe for Or46a mRNA (Ray, 2008).

    The simplest interpretation of these results is that positive regulatory elements in the Or42a upstream region are capable of driving expression not only in the pb1A neuron but also in the pb2B neuron. The 42a4 element represses expression in pb2B neurons, thereby restricting expression to a single ORN class, pb1A (Ray, 2008).

    The ectopic expression of an Or42a promoter in Or46a+ neurons suggested a relationship between these two genes. Further evidence for a relationship came from analysis of the minimal promoter containing multiple copies of 46a1. This promoter drove GFP expression in more ORNs than could be accounted for by Or46a+ neurons alone. A double-label experiment showed that while most of the GFP+ cells are in fact Or46a+, some are Or42a+ (Ray, 2008).

    The reciprocal relationship between Or42a and Or46a misexpression suggests that Or42a may contain an unidentified positive regulatory element, 42ax, that is similar in sequence to 46a1, with both sites able to bind a transcription factor present in both pb1A and pb2B. To test this interpretation, the 500 bp upstream region of Or42a was examined for an element similar, but not identical, to 46a1 (GACATTTTAA). A sequence, TATATTTTAA, was identified identical to 46a1 at the 8 underlined positions, at -455 bp. Moreover, these two sequences share an ATTTTA core, which has been shown to function as a binding site for basic helix-loop-helix transcription factors at other loci. TATATTTTAA is not found upstream of any other maxillary palp Or genes. This 42ax sequence is conserved in sequence (80% identity) and location in seven of the 12 Drosophila species. It will be interesting to identify the transcription factor that binds 46a1 and then test directly its binding to 42ax (Ray, 2008).

    When DNA upstream of Or59c was fused to GAL4, expression of the reporter GFP was not faithful; the same result was obtained when upstream regions of varying lengths were used (either 2.1 kb, which extends to the next upstream gene, or 5.2 kb, which includes upstream coding sequences). Double-label experiments using an Or59c probe revealed misexpression in many Or59c– cells; moreover, many Or59c+ cells did not express GFP. Some of the misexpressing cells are the neighboring pb3B neurons, which can be seen to be paired with Or59c+ pb3A cells. To identify the other ORNs that ectopically express the Or59c-GAL4 construct, double-label experiments were carried out with other Or genes. Misexpression was also observed in pb1A cells, which express Or42a, but not in the pb1B cells, nor in the pb2A or B cells. In summary, misexpression is specific to pb1A and pb3B (Ray, 2008).

    Because neither of the varying lengths of upstream DNA sequences were sufficient to restrict GAL4 expression to the Or59c+ cells, 3' sequences were added to the construct. Initially, 500 bp of DNA taken directly from the region immediately downstream from the Or59c stop codon was added downstream of the GAL4 coding region. Between the downstream sequences of Or59c and the GAL4 coding region was the Hsp70 3' untranslated region (UTR), which is present in the GAL4 vector and which is often present in promoter-GAL4 analysis (Ray, 2008).

    This Or59c 5' + 3' construct showed much less misexpression in Or59c cells. The total number of GFP+ cells declined from 49.7 ± 1.3 to 27.3 ± 2.1. However, some misexpression remained, and only 62% of the Or59c+ neurons were GFP+. Then the Hsp70 3' UTR sequences were removed, such that the Or59c downstream sequences were in close proximity to the 3' end of the GAL4 coding region and the Or59c 3' UTR is used. This construct drove faithful expression. Thus, there is a negative regulatory element downstream of Or59c that restricts expression of this gene to pb3A neurons, and either there is a requirement that the native 3' UTR be used, or else there is a regulatory factor that acts on this element in a context-dependent fashion in order to achieve this negative regulation. It is noted with interest that the inclusion of the downstream sequences, without the Hsp70 sequences, also drove expression in Or59c+ neurons that had previously failed to express the reporter, suggesting that the downstream sequences are required for positive as well as negative regulation of Or59c (Ray, 2008).

    Inspection of the sequences downstream of Or59c that repressed misexpression revealed a binding site for the transcription factor Scalloped (Sd), AAATATTT. This site is well-conserved among a number of other species. Sd has been shown to be expressed in olfactory organs. To confirm and extend the description of sd expression an enhancer trap line, sdETX4 was used, and it was confirmed that sd is expressed in a subset of cells in the maxillary palp (Ray, 2008).

    To test whether sd represses Or59c, in situ hybridizations were carried to the maxillary palp of a hypomorphic sd mutant, sd1. A 40% increase was found in the number of Or59c+ neurons. By contrast, there was no increase in the number of Or42a+ neurons. There was, however, an increase in the number of Or85d+ cells, and it is noted with interest that there is another type of Sd binding site, TAAAATTA, 737 bp downstream from the stop codon of Or85d (Ray, 2008).

    The Or59c-GAL4 construct that contains only upstream sequences, Or59c 5', misexpresses in two ORN classes, the neighboring pb3B cell (Or85d+) and pb1A (Or42a+). It was asked whether sd is expressed in these two ORN classes. Using an Or59c probe, which labels the pb3A cell, it was found that sd is in fact expressed in neighboring cells, but not in pb1A cells, which express Or42a. These results suggest that Sd may repress the Or59c gene in pb3B. If so, it would be expected that in an sd mutant, cells would be observed that coexpress Or59c and Or85d. This possibility was tested by carrying out double-label in situ hybridizations in two different hypomorphic alleles of sd, sd1, and sdSG29.1. In both alleles, Or59c+ Or85d+ cells were found, but not Or59c+ Or42a+. Thus repression of Or59c in the neighboring pb3B cell requires both a Sd binding site and Sd (Ray, 2008).

    Since Sd represses Or59c in pb3B, why doesn't Sd also repress Or85d in pb3B, given that both Or genes have Sd binding sites? The simplest explanation is that the two Sd binding sites are distinct. There are several potential interacting partners with which Sd may interact to form a functional transcription factor, and the pb3B cell may contain a partner necessary for repression at the Or59c binding site but not a partner necessary for repression at the Or85d binding site. If a faithful Or85d-GAL4 construct becomes available, it will be interesting to replace the Or85d-type Sd binding site with the Or59c-type Sd binding site, to determine whether the Or59c-type site confers repression in the pb3B cell (Ray, 2008).

    It is noted that Or85d-GAL4 constructs containing only the 5' regions of Or85d, which lack the Sd binding site, drive misexpression in a number of non-neuronal cells of the maxillary palp. Most of the labeled cells lack dendrites and axons, and when labeled with a membrane-bound GFP, as opposed to with RNA probes that label the cell bodies, these cells appear larger than ORNs. These results suggest that Sd may interact with a binding partner in non-neuronal cells to repress Or85d expression in these cells (Ray, 2008).

    Or42a is expressed in the larval olfactory system as well as in the maxillary palp. The Or42a-GAL4 construct shows expression in one ORN in each of the bilaterally symmetric larval olfactory organs, the dorsal organs. Expression was also observed in two neurons of the labellum, the taste organ on the adult head. To determine whether the conserved elements identified in analysis of maxillary palp receptor choice can act in these other chemosensory organs, Or42a-GAL4 constructs were examined in which these elements were mutated. A mutation that affects both 42a6 and 42a5, which did not affect expression in the maxillary palp, had no effect on expression in these other organs. However, mutation of 42a4, which relieved repression of Or42a in other maxillary palp ORNs, also relieved repression of Or42a-GAL4 in the larval olfactory organs and the labellum: in both cases supernumerary neurons were labeled. In the labellum, ~8-10 pairs of neurons were labeled. These results suggest that the molecular mechanisms underlying receptor gene choice in the maxillary palp overlap with those specifying receptor expression in other chemosensory organs (Ray, 2008).

    This study has identified and functionally characterized a number of regulatory elements that operate in directing the formation of the receptor-to-neuron map of D. melanogaster. Because the newly defined elements analyzed in this study are conserved in sequence and position among Drosophila species, it is predicted that the programmed regulation leading to the formation of receptor-to-neuron maps would be conserved as well. To test this prediction, a physiological analysis of the D. pseudoobscura maxillary palp was carrie out . Although each of the seven Or genes expressed in the maxillary palp has an ortholog expressed in the D. pseudoobscura maxillary palp, it is expected that their odor response profiles would have diverged a great deal over the course of tens of millions of years. It was not known a priori whether it would be possible to correlate D. pseudoobscura ORNs with D. melanogaster counterparts (Ray, 2008).

    It was surprising to find that the profiles of the maxillary palp ORNs are remarkably well conserved between these two species. Despite the tens of millions of years of separation, each ORN class in D. melanogaster has a counterpart in D. pseudoobscura, and their responses to a panel of ten diverse odorants are strikingly similar. Not only are the magnitudes of the responses well conserved, but the modes of the responses, i.e., excitation versus inhibition, are conserved. For example, both the pb2B ORN of D. melanogaster and its D. pseudoobscura counterpart are excited by 4-methyl phenol and inhibited by 3-octanol. The orthologous receptors show amino acid identity as low as 59% in the case of Or71a, and in no case exceeded 84%, the identity determined for Or42a. Thus pb1B in D. melanogaster, which expresses Or71a, shows the same specificity for 4-methyl phenol and 4-propyl phenol as the corresponding ORN in D. pseudoobscura, although Or71a is only 59% identical between the two species (Ray, 2008).

    The conservation of odor response spectra allows determination of whether the stereotyped pairing of ORNs is also conserved in the two species. The results suggest that not only are the response spectra of the odor receptors conserved with respect to a diverse panel of odorants, but that the program of receptor gene expression is also conserved between these distantly related species (Ray, 2008).

    Given the success in identifying gene-specific elements required for the expression of individual Or genes in individual classes of ORNs, it was asked whether the same approach could be used to identify sensillum-specific elements required uniquely by the Or genes that are expressed in the neighboring ORNs of a common sensillum. Sensillum-specific elements were sought conserved in the upstream regions of D. melanogaster and D. pseudoobscura Or genes. Only one element, AAATCAATTA, was found upstream of all orthologs expressed in a particular sensillum type. Mutational analysis of this element in the Or42a promoter did not, however, appear to affect expression. Furthermore, expression was not affected by mutation of the more proximal of the two copies of this element in the Or71a upstream region. These results suggest that this element is not required for expression in the pb1 sensillum (Ray, 2008).

    This study has concentrated on receptor gene choice in the maxillary palp, on account of its numerical simplicity. Does a system of molecular zip codes also underlie the process of receptor gene choice across the entire odor receptor repertoire? In addition to the seven maxillary palp receptors, the Or gene family contains 53 other members expressed in the antenna or the larval olfactory system. Using a comparative bioinformatic approach, a large-scale analysis was performed of sequence conservation in the 500 bp upstream of each of 42 Or genes across all 12 Drosophila species. Great diversity was found in the number, lengths, and distribution of highly conserved upstream regions. Within the most highly conserved of these regions a variety of elements were identified that are shared among subsets of Or genes. This analysis, then, reveals a combinatorial structure to the organization of shared elements upstream of these receptor genes. This pattern supports a model in which a combinatorial code of positive and negative regulatory elements dictates the proper expression of each Or gene (Ray, 2008).

    What kind of proteins accomplish this regulation? In C. elegans, several kinds of transcription factors have been elegantly shown to play roles in specifying ORN identity and receptor expression. In the mouse, a LIM-homeodomain protein, Lhx2, is required for normal ORN differentiation and expression of OR genes. In Drosophila the POU domain protein Acj6 is required for the expression of a subset of Or genes. This study has shown that Sd, a TEA domain-containing transcription factor, is critical in restricting the expression of some Or genes to their proper ORNs. Sd has been shown to act as a repressor in other systems and in fact is required for normal taste behavior in both larvae and adults. Another aspect of receptor gene choice depends on proteins of the Notch pathway: receptor choice in neighboring ORNs of a sensillum appears to be coordinated via asymmetric segregation of regulatory factors from a common progenitor (Ray, 2008).

    Some elements that are essential to odor receptor gene choice are also located upstream of genes required for axon guidance and sorting. The presence and positions of these elements have been conserved for tens of millions of years of evolution. The presence of Or regulatory elements upstream of ORN axon-guidance genes could reflect a relationship between receptor gene choice and axon targeting. In addition to selecting particular Or genes for expression, ORNs send axons to particular glomeruli in the antennal lobe of the brain. ORNs that express the same Or gene send axons to the same glomerulus. Thus the olfactory system contains both a stereotyped receptor-to-neuron map and a stereotyped connectivity map in the antennal lobes. The tight coordination between receptor gene choice and axonal projection could in principle arise in part from overlap in the mechanisms underlying these processes. In mammals, odor receptors play a role in ORN targeting. In Drosophila, ORN targeting does not require the receptors, but could require the regulatory apparatus used to express the receptors. Acj6 provides an example of a link between the two processes: it acts both in receptor expression and ORN axon targeting. Moreover, it was found that Acj6 is required for the activity of one of the regulatory elements identified in this study (Ray, 2008).

    This study found a remarkable similarity of function between the maxillary palp ORNs of two species that diverged more than tens of millions of years ago. It had been expected that over this time interval, the odor specificities of the ORNs would have diverged markedly to serve differing needs of the two evolving species. Instead, every ORN class showed strikingly similar responses, with few exceptions. The results show that two odor receptors can differ a great deal in amino acid sequence and still exhibit a very similar odor specificity (Ray, 2008).

    The organization of the organ in the two species is also identical, in that corresponding ORNs are combined according to the same pairing rules. This high degree of conservation suggests a critical role for the maxillary palp in odor coding and in the generation of olfactory-driven behavior. The conservation of regulatory elements and organization also suggests that the two species use common mechanisms to specify the receptor-to-neuron map (Ray, 2008).

    The regulatory challenge confronted by the Drosophila olfactory system represents an extreme among problems of gene regulation. It requires the storage and deployment of a great deal of information. These data support a model in which Or gene expression is controlled by a system of molecular zip codes. Each Or gene contains elements that dictate expression in the proper olfactory organ, positive regulatory elements that specify expression in a subset of ORN classes, and negative regulatory elements that restrict expression to a single ORN class. This logic and the components that execute it have solved such a challenging problem with such efficiency that they have apparently been well conserved for tens of millions of years (Ray, 2008).

    Combinatorial rules of precursor specification underlying olfactory neuron diversity

    Sensory neuron diversity ensures optimal detection of the external world and is a hallmark of sensory systems. An extreme example is the olfactory system, as individual olfactory receptor neurons (ORNs) adopt unique sensory identities by typically expressing a single receptor gene from a large genomic repertoire. In Drosophila, about 50 different ORN classes are generated from a field of precursor cells, giving rise to spatially restricted and distinct clusters of ORNs on the olfactory appendages. Developmental strategies spawning ORN diversity from an initially homogeneous population of precursors are largely unknown. This study has unraveled the nested and binary logic of the combinatorial code that patterns the decision landscape of precursor states underlying ORN diversity in the Drosophila olfactory system. The transcription factor Rotund (Rn) is a critical component of this code that is expressed in a subset of ORN precursors. Addition of Rn to preexisting transcription factors that assign zonal identities to precursors on the antenna subdivides each zone and almost exponentially increases ORN diversity by branching off novel precursor fates from default ones within each zone. In rn mutants, rn-positive ORN classes are converted to rn-negative ones in a zone-specific manner. This study provides a model describing how nested and binary changes in combinations of transcription factors could coordinate and pattern a large number of distinct precursor identities within a population to modulate the level of ORN diversity during development and evolution (Li, 2013).

    Neuronal diversity is a common characteristic of all sensory systems throughout the animal kingdom. Among these, the olfactory system demonstrates an extreme case in its diversity of ORN classes. In Drosophila, each of the 50 adult ORN classes is defined by the unique expression of typically a single olfactory receptor from a pool of around 80 genes. How this ORN diversity is generated from a field of homogeneous precursor cells during development remains elusive. Combinatorial control of transcription factors has been proposed as an important mechanism that complex systems utilize to create cellular diversity. This study demonstrates the nested and binary combinatorial rules by which transcription factors interact with each other to guide decisions regarding ORN precursor identities. The results suggest that nesting the regulatory relationship of transcription factor combinations allows the concurrent use of the same factors in parallel lineages to generate ORN diversity in a very efficient manner. Under this logic, binary lineage choices in precursor cells are made based on historical contingency, which could serve as an effective strategy for establishing cellular complexity in many other developing systems (Li, 2013).

    In both vertebrates and invertebrates, each ORN class is spatially restricted to specific zones within the peripheral olfactory organs. In Drosophila, antennal ORNs are housed in three morphologically and topographically different types of sensilla occupying distinct zones, while maxillary palps have only a single type of sensilla. Each of the sensilla type zones on the antenna are subdivided into subzones that are defined by sensilla subtypes, which have similar morphology but differ in the set of olfactory receptors expressed in the ORNs they house. It has been shown that the decision for a given palp-specific olfactory receptor gene to be expressed in maxillary palp ORNs, but not in antennal ORNs, requires both positive and negative regulatory elements around that gene. For antennal ORNs, the proneural genes amos and ato and the prepatterning gene lz were found to assign sensilla type identities to the precursors and determine olfactory receptors expressed by the neurons housed in these sensilla. The loss of Amos or Ato leads to the complete loss of basiconic and trichoid or coeloconic sensilla types, respectively, and corresponding ORNs. Lz diversifies sensilla type identities within the Amos-expressing lineage, where high levels of Lz are associated with basiconic sensilla fates, versus low levels of Lz, which generates ORNs in trichoid sensilla. Hypomorphic alleles of lz result in basiconic-to-trichoid sensilla type conversions. Lz is also required for the expression of Amos, suggesting the existence of regulatory loops among transcription factors in the same network (Li, 2013).

    The current results explain how the next level of diversification occurs following sensilla type specification in the antenna. Rn is expressed in a subset of antennal sensilla precursors and splits precursors of each zone into Rn-positive and Rn-negative subtypes. In rn mutants, ORN diversity decreases almost by half as ORN classes from rn-positive subtypes are switched to rn-negative identities within the same zone. The results suggest that Rn is required to branch off novel precursor identities from default ones, resulting in the generation of new ORN classes in a zone-specific manner. It should be noted that some rn-negative sensilla subtypes, for example at2 and ac3, neither decrease nor increase in their numbers in rn mutants, suggesting that there are additional factors driving the diversification of the ORN classes in these sensilla. Similarly, further diversification of rn-positive ORN precursors should also be under the control of additional factors, such as En, operating in concert with Rn function (Li, 2013).

    These results along with others suggest a two-step mechanism for ORN diversification: (1) successive restrictions on precursor differentiation potentials by spatiotemporal factors, such as proneural/prepatterning gene products and Rn, and (2) segregation of restricted fates through Notch-mediated asymmetric divisions and local transcription factor networks for directly turning on olfactory receptor expression. Hypothetically, the sensilla precursor differentiation potentials can be represented by distinct sets of olfactory receptor genes being organized into euchromatic regions in a lineage-specific manner. The aforementioned combinations of transcription factors may influence the dynamics of such epigenetic states, resulting in limited combinations of receptors transcriptionally accessible for later stages of ORN differentiation. Examples of chromatin modulation in OR expression have been demonstrated in both flies and mice. Once precursor potentials are set, the Notch signaling pathway could continue to bifurcate alternate sensory identities into ORNs generated through asymmetric precursor cell divisions. Transcription factor networks expressed later in development, including the well-characterized Acj6, Pdm3, and Scalloped, could then directly regulate olfactory receptor expression during these divisions based on their genomic accessibility, giving rise to terminally differentiated ORNs (Li, 2013).

    In comparison with the Drosophila olfactory system, mammals exhibit remarkable organizational similarities in the olfactory circuitry, even though the numerical complexity far exceeds that of their insect counterparts. For example, the zonal pattern of olfactory receptor expression in the mammalian olfactory epithelium is analogous to the topographic segregation of sensilla type-dependent olfactory receptor expression in the antenna. A number of transcription factors were reported to regulate the zone-specific expression of a subset of olfactory receptors, yet no mutants resulting in ORN sensory conversion have been described. Despite the consensus on the stochastic nature of mammalian olfactory receptor expression within each zone, it would be interesting to see whether the zones are defined by a similar developmental strategy. The model presented in this study also provides an ancestral precursor decision landscape that reveals the interaction pattern among factors to maintain and modify phenotypic complexity and diversity within sensory neural circuits on evolutionary timescales. New regulatory nodes might be added to the combinatorial code at distinct stages of precursor cell development to change ORN specification programs. For example, addition of mir-279, a negative regulator of the transcription factor nerfin-1 expressed in maxillary palp ORN precursors, results in the elimination of CO2-sensory ORNs from specific maxillary palp sensilla (Hartl, 2011). Furthermore, olfactory receptor genes have been shown to be fast evolving across and within genomes. Incremental addition of individual regulatory modules to preexisting lineage-specific combinations operating in binary ON/OFF mode could facilitate the coordination of novel ORN fates with the evolution of receptor genes, which can be modified in response to changes in the quantity, quality, and context of the olfactory environment (Li, 2013).

    Odorant receptors of Drosophila are sensitive to the molecular volume of odorants

    Which properties of a molecule define its odor? This is a basic yet unanswered question regarding the olfactory system. The olfactory system of Drosophila has a repertoire of approximately 60 odorant receptors. Molecules bind to odorant receptors with different affinities and activate them with different efficacies, thus providing a combinatorial code that identifies odorants. This study hypothesized that the binding affinity of an odorant-receptor pair is affected by their relative sizes. The maximum affinity can be attained when the molecular volume of an odorant matches the volume of the binding pocket. The affinity drops to zero when the sizes are too different, thus obscuring the effects of other molecular properties. A mathematical formulation of this hypothesis was developed and verified using Drosophila data. The volume and structural flexibility of the binding site of each odorant receptor were also predicted; these features significantly differ between odorant receptors. The differences in the volumes and structural flexibilities of different odorant receptor binding sites may explain the difference in the scents of similar molecules with different sizes (Saberi, 2016).

    The corepressor Atrophin specifies odorant receptor expression in Drosophila

    In both insects and vertebrates, each olfactory sensory neuron (OSN) expresses one odorant receptor (OR) from a large genomic repertoire. How a receptor is specified is a tantalizing question addressing fundamental aspects of cell differentiation. This study demonstrates that the corepressor Atrophin (Atro) segregates OR gene expression between OSN classes in Drosophila. The knockdown of Atro results in either loss or gain of a broad set of ORs. Each OR phenotypic group correlated with one of two opposing Notch fates, Notch responding, Nba (Non), and nonresponding, Nab (Noff) OSNs. The data show that Atro segregates ORs expressed in the Nba OSN classes and helps establish the Nab fate during OSN development. Consistent with a role in recruiting histone deacetylates, immunohistochemistry revealed that Atro regulates global histone 3 acetylation (H3ac) in OSNs and requires Hdac3 to segregate OR gene expression. It was further found that Nba OSN classes exhibit variable but higher H3ac levels than the Nab OSNs. Together, these data suggest that Atro determines the level of H3ac, which ensures correct OR gene expression within the Nba OSNs. A mechanism is proposed by which a single corepressor can specify a large number of neuron classes (Alkhorn, 2013).

    Vibrational detection of odorant functional groups by Drosophila melanogaster

    A remarkable feature of olfaction, and perhaps the hardest one to explain by shape-based molecular recognition, is the ability to detect the presence of functional groups in odorants, irrespective of molecular context. Previous work has shown that Drosophila trained to avoid deuterated odorants could respond to a molecule bearing a nitrile group, which shares the vibrational stretch frequency with the CD bond. This study reproduces and extends this finding by showing analogous olfactory responses of Drosophila to the chemically vastly different functional groups, thiols and boranes, that nevertheless possess a common vibration at 2600 cm(-1). Furthermore, it was shown that Drosophila do not respond to a cyanohydrin structure that renders nitrile groups invisible to IR spectroscopy. It is argued that the response of Drosophila to these odorants which parallels their perception in humans, supports the hypothesis that odor character is encoded in odorant molecular vibrations, not in the specific shape-based activation pattern of receptors (Maniati, 2017).

    Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity

    Insect olfactory sensory neurons (OSN) express a diverse array of receptors from different protein families, i.e. ionotropic receptors (IR), gustatory receptors (GR) and odorant receptors (OR). It is well known that insects are exposed to a plethora of odor molecules that vary widely in both space and time under turbulent natural conditions. In addition to divergent ligand specificities, these different receptors might also provide an increased range of temporal dynamics and sensitivities for the olfactory system. To test this, different Drosophila OSNs were challenged with both varying stimulus durations (10-2000 ms), and repeated stimulus pulses of key ligands at various frequencies (1-10 Hz). The results show that OR-expressing OSNs responded faster and with higher sensitivity to short stimulations as compared to IR- and Gr21a-expressing OSNs. In addition, OR-expressing OSNs could respond to repeated stimulations of excitatory ligands up to 5 Hz, while IR-expressing OSNs required ~5x longer stimulations and/or higher concentrations to respond to similar stimulus durations and frequencies. Nevertheless, IR-expressing OSNs did not exhibit adaptation to longer stimulations, unlike OR- and Gr21a-OSNs. Both OR- and IR-expressing OSNs were also unable to resolve repeated pulses of inhibitory ligands as fast as excitatory ligands. These differences were independent of the peri-receptor environment in which the receptors were expressed and suggest that the receptor expressed by a given OSN affects both its sensitivity and its response to transient, intermittent chemical stimuli. OR-expressing OSNs are better at resolving low dose, intermittent stimuli, while IR-expressing OSNs respond more accurately to long-lasting odor pulses. This diversity increases the capacity of the insect olfactory system to respond to the diverse spatiotemporal signals in the natural environment (Getahun, 2012).

    Olfactory receptor neurons use gain control and complementary kinetics to encode intermittent odorant stimuli

    Insects find food and mates by navigating odorant plumes that can be highly intermittent, with intensities and durations that vary rapidly over orders of magnitude. Much is known about olfactory responses to pulses and steps, but it remains unclear how olfactory receptor neurons (ORNs) detect the intensity and timing of natural stimuli, where the absence of scale in the signal makes detection a formidable olfactory task. By stimulating Drosophila ORNs in vivo with naturalistic and Gaussian stimuli, this study shows that ORNs adapt to stimulus mean and variance, and that adaptation and saturation contribute to naturalistic sensing. Mean-dependent gain control followed the Weber-Fechner relation and occurred primarily at odor transduction, while variance-dependent gain control occurred at both transduction and spiking. Transduction and spike generation possessed complementary kinetic properties, that together preserved the timing of odorant encounters in ORN spiking, regardless of intensity. Such scale-invariance could be critical during odor plume navigation (Gorur-Shandilya, 2017).

    Insect odorant response sensitivity is tuned by metabotropically autoregulated olfactory receptors

    Insects possess one of the most exquisitely sensitive olfactory systems in the animal kingdom, consisting of three different types of chemosensory receptors: ionotropic glutamate-like receptors (IRs), gustatory receptors (GRs) and odorant receptors (ORs). Both insect ORs and IRs are ligand-gated ion channels, but ORs possess a unique configuration composed of an odorant-specific protein OrX and a ubiquitous coreceptor (Orco). In addition, these two ionotropic receptors confer different tuning properties for the neurons in which they are expressed. Unlike IRs, neurons expressing ORs are more sensitive and can also be sensitized by sub-threshold concentrations of stimuli. What is the mechanistic basis for these differences in tuning? This study shows that intrinsic regulation of Orco enhances neuronal response to odorants and sensitizes the ORs. It was also demonstrated that inhibition of metabotropic regulation prevents receptor sensitization. These results indicate that Orco-mediated regulation of OR sensitivity provides tunable ionotropic receptors capable of detecting odors over a wider range of concentrations, providing broadened sensitivity over IRs themselves (Getahun, 2013).

    The independent evolution of these two different ionotropic receptor families (ORs/GRs and IRs) has become a great topic of speculation for the field. Why do these multiple families persist among all higher insect orders? And why do they possess such radically different molecular conformations? Initially, it was suggested that these multiple families expand the affinity of the olfactory palette to different chemical classes. However, a recent study also revealed that olfactory sensory neurons (OSNs) expressing ORs, GRs, or IRs exhibit intrinsic differences in temporal kinetics to brief or intermittent stimuli (Getahun, 2012). Specifically, OR-expressing neurons respond faster and with higher sensitivity to brief stimulation, while IR-expressing neurons do not adapt to long stimulations. This implies that OR-expressing neurons are more accurate at detecting the low-concentration, punctate plume packets received at long distances from the odor source, while IR-expressing neurons can better track the high-concentration, long lasting stimulation received when on or near the source. This diversity offers both broader ligand specificity and expanded spatiotemporal dynamics with which to parse the odor world, and is particularly important for insects challenged by the high-speed performance of flight. Interestingly, the purported evolution of ORs corresponds well to the evolution of flight during the Carboniferous Era (Getahun, 2013).

    Although both insect ORs and IRs operate as ionotropic receptors, their tuning properties differ fundamentally. While prolonged stimulation leads to adaptation of ORs, there is no adaptation of IRs. On the other hand, ORs but not IRs expand their dynamic range through intrinsic sensitization. This difference in sensitization is apparent even between ORs and IRs expressed in co-localized sensilla. Thus, sensitization must result from intrinsic, rather than extrinsic neuronal properties that are unique to ORs. The most parsimonious explanation for the mechanistic differences between these families, is the use of intracellular signalling to modulate OR activity. Given the previous in vivo evidence for a role of metabotropic signalling in OR function, this study first pursued the metabotropic regulation of Orco in mediating OR activity (Getahun, 2013).

    OR sensitization could be mimicked by manipulations enhancing cAMP production or PKC activity and depressed by inhibition of cAMP production or PLC/PKC activity. These intracellular signalling systems not only influence the OR sensitivity at weak odor stimuli, they also modulate the OR response for stronger stimuli. In detail, microinjection of cAMP or adenylyl cyclase activators into sensilla increased the odorant response and shifted the dose-response curve toward lower odorant concentrations. A previous study has revealed that Orco sensitivity to cAMP is regulated by protein kinase C (PKC)-dependent phosphorylation (Sargsyan, 2011). Inhibition of PLC or PKC also inhibits any effect of cAMP, indicating that the enhanced sensitivity caused by cAMP is regulated by Orco activity. The metabotropic regulation of Orco also lead to sensitization of the OSN to repeated subthreshold odor responses, which is abolished by adenylyl cyclase inhibition. Furthermore, the sensitization of the odor response was blocked in mutant flies with impaired Orco phosphorylation (Orco mut) further indicating that metabotropic regulation of Orco activity is required for the enhanced odorant response. It cannot be excluded that cAMP and PKC activation may regulate OR sensitivity to odors via other mechanisms, such as through modulation of membrane traffick. Nevertheless, the lack of response modulation following injection of forskolin into PKC flies, indicates that the metabotropically-enhanced odor sensitivity is intrinsic to the OR complex and does not result from extrinsic cellular processes (Getahun, 2013).

    The results thus suggest that intracellular signalling, and in particular metabotropic regulation of Orco, plays a vital role in conferring the mechanistic differences between ORs and IRs. Although the mechanistic basis of intracellular signalling in these OSNs cannot yet be machanistically confirmed, it is concluded that modulations that activate Orco when heterologously expressed enhance the odor sensitivity of ORs in vivo and, vice versa, modulations that inhibit Orco reduce OR sensitivity. It must also be kept in mind that the ORs are Ca2+-permeable, constitutively active ion channels, the background activity of which is also able to activate enzymatic activity. Future studies should characterize the composition of the respective signalling subsystems, e.g. those involved in sensitizing receptors vs. those involved in terminating the odorant response (Getahun, 2013).

    The evolution of a highly sensitive and adaptable olfactory system is believed to be a key factor allowing insects to radiate into more or less every environment on earth. Given the importance of OSN dynamics in tracking turbulent odor plumes, olfactory sensitization via Orco regulation can enhance an insect's ability to accurately detect and respond to intermittent, low concentration stimuli. Insect ORs are thought to have evolved from ionotropic gustatory receptors, which detect millimolar ligand concentrations. The results imply that the special heterodimeric design of ORs has likely evolved to quickly detect and respond to volatile compounds at very low concentrations, such as those encountered by flying insects. Regardless of the source of this difference, it is clear that the OR expansion of ionotropic receptors offers the insect olfactory system both broadened ligand affinity as well as expanded spatiotemporal dynamics with which to navigate the olfactory world (Getahun, 2013).

    Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits

    The internal state of an organism influences its perception of attractive or aversive stimuli and thus promotes adaptive behaviors that increase its likelihood of survival. The mechanisms underlying these perceptual shifts are critical to understanding of how neural circuits support animal cognition and behavior. Starved flies exhibit enhanced sensitivity to attractive odors and reduced sensitivity to aversive odors. This study shows that a functional remodeling of the olfactory map is mediated by two parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion at the level of the first synapse. Short neuropeptide F sensitizes an antennal lobe glomerulus wired for attraction, while tachykinin (DTK) suppresses activity of a glomerulus wired for aversion. Thus this study shows parallel neuromodulatory systems functionally reconfigure early olfactory processing to optimize detection of nutrients at the risk of ignoring potentially toxic food resources (Ko, 2015).

    This study demonstrates that shifts in the internal metabolic state of an animal lead to dramatic functional changes in its olfactory circuit and behaviors. Starved flies exhibit enhanced odor sensitivity in odorant receptor neurons (ORNs) that mediate behavioral attraction and decreased sensitivity in ORNs that mediate behavioral aversion. This functional remodeling of the olfactory map is mediated by parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion. An earlier study showed that sNPFR signaling increases sensitivity in Or42b ORNs and thus enhances behavioral attraction (Root, 2011). The current study, however, shows that sNPFR signaling does not account for all changes induced by starvation in behavioral responses to a wider range of odor concentrations. Second, this study shows that starvation leads to a decreased sensitivity in the Or85a ORNs, an odorant channel that mediates behavioral aversion. Third, it was shown that DTKR signaling mediates the reduced sensitivity in the Or85a ORNs and partly accounts for enhanced behavioral attraction to high concentrations of vinegar. Fourth, eliminating DTKR and sNPFR signaling pathways together fully reverses the effect of starvation on behavioral attraction across all odor concentrations tested. Finally, evidence suggests that the same global insulin signal regulating sNPFR expression may also regulate DTKR expression (Ko, 2015).

    In the wild, rotten fruits early in the fermentation process are more attractive to Drosophila than fresh or highly fermented fruits. In the laboratory, well fed flies display very little attraction to apple cider vinegar (Root, 2011). Low levels of vinegar are indicative of fresh fruit of limited nutritional value. Expanding odor sensitivity to lower concentrations of potential food odors may encourage flies to accept food sources of lower value. High odor concentrations typically accompany late stages of fermentation and are often aversive or uninteresting to flies. Starved flies are attracted to high concentrations of vinegar partly due to neuromodulatory mechanisms that enhance sensitivity in Or42b ORNs, an attractive odor channel, and partly through neuromodulatory mechanisms that reduce sensitivity in Or85a ORNs, an aversive odor channel. In the working model, behavioral attraction to higher odor concentrations of vinegar is the sum of the opposing effects of Or42b and Or85a. When flies face starvation, the balance of these inputs shifts to favor Or42b over Or85a inputs, as mediated by selective upregulation of sNPFR and DTKR in these ORNs, respectively. These processes could serve to encourage flies to risk ingestion of potentially toxic foods when under nutritional stress (Ko, 2015).

    Given the broad array of glomeruli that can respond to odors such as vinegar, it may be surprising that the modulation of only two glomeruli is sufficient to significantly impact fly behavioral attraction. Whether these findings extend to a broad array of food associated odors and whether additional glomeruli are modulated by these neuromodulatory systems remain to be determined. In this context, it is noted that a recent correlational analysis predicts DM5 activity is highly correlated with behavioral attraction. However, this prediction has not been confirmed by direct testing of the DM5 glomerulus in behavioral experiments and is contradicted by more recent findings, as well as the data in this paper. Thus the current findings suggest that in starved flies the concentration range over which vinegar odor is attractive expands in both directions, with the acute need for caloric intake apparently outweighing considerations of food quality or risk (Ko, 2015).

    This study highlights the importance of neuromodulators in shaping local neural circuit activity to accommodate the internal physiological state of an organism. The often unique expression patterns of specific GPCRs in sensory systems highlights the flexibility conferred by this evolutionarily ancient mechanism to translate neuroendocrine signals into local shifts in neuronal excitability and network properties that ultimately lead to adaptive behaviors. sNPF shares structural and functional similarities with its vertebrate homolog, NPY. Both neuropeptides show roles in controlling food intake and feeding behaviors in insects and vertebrates. Interestingly, NPY is also expressed in the vertebrate olfactory bulb and is thus positioned to shape olfactory processing during shifts in appetitive states as well. sNPF's broad expression pattern in the fly brain supports the possibility it is widely used to orchestrate changes across many different neuropils to shape appetitive behaviors. Indeed, sNPF and NPF, another NPY homolog in Drosophila, have been shown in the fly gustatory system to control sweet and bitter taste sensitivity, respectively, in parallel but opposing directions (Inagaki, 2014). The similar changes manifested by nutritional stress in both the olfactory and gustatory systems suggests complex networks of neuromodulators may shape sensory processing of aversive and attractive inputs differentially throughout the brain in a hunger state (Ko, 2015).

    DTK and DTKR share homology with substance P and its receptor NK1, respectively. Interestingly, they seem to share roles in shaping the processing of stressful or negative sensory cues in both flies and mammals. For example, in rodents, emotional stressors cause long-lasting release of substance P to activate NK1 in the amygdala to generate anxiety-related behavior. In Drosophila, DTK signaling has also been shown to be critical for aggressive behaviors among male flies (Asahina, 2014). Previous work has shown Drosophila tachykinin mediates presynaptic inhibition in ORNs and detected expression in the LNs. This current study maps the locus of DTK's effects on behavioral responses to vinegar to the Or85a/DM5 ORNs using behavior and functional imaging. It was also confirmed that the source of the peptide is indeed the LNs as previous anatomical data had suggested. Thus, tachykinin's role in modulating stressful sensory inputs appears to extend to a glomerulus hardwired to behavioral aversion in the olfactory system (Ko, 2015).

    The current results here resonate with discoveries in the gustatory system (Inagaki, 2014) and show that starvation changes the perception of both attractive and aversive sensory inputs beginning at the peripheral nervous system. Through the use of parallel neuromodulatory systems, the internal state of the organism functionally reconfigures early olfactory processing to optimize its detection of nutrients at the risk of ignoring potentially toxic food resources. It is certainly likely that neuromodulatory systems also impact and reconfigure central circuits in appetitive contexts. Thus, it will be of great interest to understand the contributions of peripheral and central circuits towards modifying appetitive behaviors (Ko, 2015).

    Decoding of context-dependent olfactory behavior in Drosophila

    Odor information is encoded in the activity of a population of glomeruli in the primary olfactory center. However, how this information is decoded in the brain remains elusive. This question was addressed in Drosophila by combining neuronal imaging and tracking of innate behavioral responses. The behavior is accurately predicted by a model summing normalized glomerular responses, in which each glomerulus contributes a specific, small amount to odor preference. This model is further supported by targeted manipulations of glomerular input, which biased the behavior. Additionally, it was observed that relative odor preference changes and can even switch depending on the context, an effect correctly predicted by the normalization model. These results indicate that olfactory information is decoded from the pooled activity of a glomerular repertoire and demonstrate the ability of the olfactory system to adapt to the statistics of its environment (Badel, 2016).

    Elucidating the neuronal architecture of olfactory glomeruli in the Drosophila antennal lobe

    Olfactory glomeruli are morphologically conserved spherical compartments of the olfactory system, distinguishable solely by their chemosensory repertoire, anatomical position, and volume. Little is known, however, about their numerical neuronal composition. This study therefore characterized their neuronal architecture and correlated these anatomical features with their functional properties in Drosophila melanogaster. All olfactory sensory neurons (OSNs) innervating each glomerulus were quantitatively mapped, including sexually dimorphic distributions. The data reveal the impact of OSN number on glomerular dimensions and demonstrate yet unknown sex-specific differences in several glomeruli. Moreover, uniglomerular projection neurons were quantified for each glomerulus, which unraveled a glomerulus-specific numerical innervation. Correlation between morphological features and functional specificity showed that glomeruli innervated by narrowly tuned OSNs seem to possess a larger number of projection neurons and are involved in less lateral processing than glomeruli targeted by broadly tuned OSNs. This study demonstrates that the neuronal architecture of each glomerulus encoding crucial odors is unique (Grabe, 2016).

    Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster

    Neuromodulation confers flexibility to anatomically-restricted neural networks so that animals are able to properly respond to complex internal and external demands. However, determining the mechanisms underlying neuromodulation is challenging without knowledge of the functional class and spatial organization of neurons that express individual neuromodulatory receptors. This study describes the number and functional identities of neurons in the antennal lobe of Drosophila melanogaster that express each of the receptors for one such neuromodulator, serotonin (5-HT). Although 5-HT enhances odor-evoked responses of antennal lobe projection neurons (PNs) and local interneurons (LNs), the receptor basis for this enhancement is unknown. Endogenous reporters of transcription and translation for each of the five 5-HT receptors (5-HTRs) were used to identify neurons, based on cell class and transmitter content, that express each receptor. Specific receptor types are expressed by distinct combinations of functional neuronal classes. For instance, the excitatory PNs express the excitatory 5-HTRs (5-HT2 type and 5-HT7), the 5-HT1 type receptors are generally inhibitory, and distinct classes of LNs each express different 5-HTRs. This study therefore provides a detailed atlas of 5-HT receptor expression within a well-characterized neural network, and enables future dissection of the role of serotonergic modulation of olfactory processing (Sizemore, 2016).

    Neuromodulators often act through diverse sets of receptors expressed by distinct network elements and in this manner, differentially affect specific features of network dynamics. Knowing which network elements express each receptor for a given neuromodulator provides a framework for making predictions about the mechanistic basis by which a neuromodulator alters network activity. This study provides an 'atlas' of 5-HTR expression within the AL of Drosophila, thus revealing network elements subject to the different effects of serotonergic modulation. In summary, different receptors are predominantly expressed by distinct neuronal populations. For example, the 5-HT2B is expressed by ORNs, while the 5-HT2A and 7 are expressed by cholinergic PNs. Additionally, each receptor was found to be expressed by diverse populations of LNs, with the exception the 5-HT1B. For instance, 5-HT1A is expressed by GABAergic and peptidergic (TKK and MIP) LNs, while 5-HT2A and 2B are not expressed by peptidergic LNs. However, the vPNs are the exception to the general observation that distinct neuronal classes differ from each other in the 5-HTRs and the implications of this are discussed below. Together, these results suggest that within the AL, 5-HT differentially modulates distinct populations of neurons that undertake specific tasks in olfactory processing (Sizemore, 2016).

    A recurring theme of neuromodulation is that the expression of distinct receptor types by specific neural populations allows a single modulatory neuron to differentially affect individual coding features. For instance, GABAergic medium spiny neurons (MSNs) in the nucleus accumbens express either the D1 or D2 dopamine receptor allowing dopamine to have opposite effects on different MSNs via coupling to different Galpha subunits (reviewed in56). MSNs that differ in dopamine receptor expression also differ in their synaptic connectivity. Dopamine activates D1-expressing MSNs that directly inhibit dopaminergic neurons in the ventral tegmental area (VTA), and inhibits D2-expressing MSNs that inhibit GABAergic VTA interneurons thus inducing suppression of dopamine release. In this manner, a single neuromodulator differentially affects two populations of principal neurons via different receptors to generate coordinated network output. This principle also holds true for the effects of 5-HT within the olfactory bulb. For instance, 5-HT enhances presynaptic inhibition of olfactory sensory neurons by 5-HT2C-expressing juxtaglomerular cells57, while increasing excitatory drive to mitral/tufted cells and periglomerular cells via 5-HT2A-expressing external tufted cells. Similarly, distinct classes of AL neurons were observed to differ in their expression of 5-HTRs. For instance, ePNs express the 5-HT2A, 5-HT2B and 5-HT7 receptors, while peptidergic LNs predominantly express the 5-HT1A receptor. This suggests that the cumulative effect of 5-HT results from a combination of differential modulation across neuronal populations within the AL. Interestingly, although it was found that 5-HT2B is expressed by ORNs, previous reports found that 5-HT does not directly affect Drosophila ORNs. In this study, ORNs were stimulated using antennal nerve shock in which the antennae were removed in order to place the antennal nerve within a suction electrode. Thus, if 5-HT2B is localized to the ORN cell body, removal of the antennae would eliminate any effect of 5-HT on ORNs. In several insects, 5-HT within the antennal haemolymph modulates ORN odor-evoked responses. Therefore, it is plausible ORNs are modulated by a source of 5-HT other than the CSD neurons within the AL. Serotonergic modulation of LN activity has widespread, and sometimes odor specific, effects on olfactory processing. LNs allow ongoing activity across the AL to shape the activity of individual AL neurons, often in a glomerulus specific manner creating non-reciprocal relationships. It is fairly clear that 5-HT directly modulates LNs, although 5-HT almost certainly affects synaptic input to LNs. Serotonin modulates isolated Manduca sexta LNs in vitro and, consistent with the current results, a small population of GABAergic LNs in the AL of Manduca also express the 5-HT1A receptor. Furthermore, 5-HT has odor-dependent effects on PN odor-evoked activity, suggesting that odor specific sets of lateral interactions are modulated by 5-HT. Different populations of LNs were found to express different sets of 5-HT receptors, however LNs were categorized based on transmitter type, so it is possible that these categories could be even further sub-divided based on morphological type, synaptic connectivity or biophysical characteristics. Regardless, the results suggest that 5-HT modulates lateral interactions within the AL by selectively affecting LN populations that undertake different tasks. For instance, the TKKergic LNs that express the 5-HT1A receptor provide a form of gain control by presynaptically inhibiting ORNs32. The results suggest that 5-HT may affect TKK mediated gain control differently relative to processes undertaken by other LN populations. Furthermore, the expression of the TKK receptor by ORNs is regulated by hunger, allowing the effects of TKK to vary with behavioral state. It would be interesting to determine if the expression of 5-HTRs themselves also vary with behavioral state as a means of regulating neuromodulation within the olfactory system (Sizemore, 2016).

    Although it was primarily found that individual populations of AL neurons chiefly expressed a single or perhaps two 5-HTR types, the vPNs appear to be an exception. As a population, the vPNs express all of the 5-HTRs and the vPNs that express each 5-HTR did not appear to differ in terms of the proportion of those neurons that were GABAergic or cholinergic (roughly 3:2). Unfortunately, the approach does not allow determination of the degree to which individual vPNs co-express 5-HTRs. However, it is estimated that there are ~51 vPNs and even if this is an underestimate, there is likely some overlap of receptor types as a large number of vPNs expressed the 5-HT1A, 1B, 2B and 7 receptors. It is possible that a single vPN expresses one 5-HTR in the AL and a different 5-HTR in the lateral horn. However, the current approach only allows identification of which neurons express a given 5-HTR, not where that receptor is expressed. The CSD neurons ramify throughout both ALs and both lateral horns, thus vPNs could have differential spatial expression of individual 5-HTRs. Individual neurons expressing multiple 5-HTRs has been demonstrated in several neural networks. For instance, pyramidal cells in prefrontal cortex express both the 5-HT1A and 5-HT2A7. This allows 5-HT to have opposing effects that differ in their time course in the same cell. In terms of the vPNs, the results suggest that the current understanding of the diversity of this neuron class is limited. The expression of receptors for different signaling molecules could potentially be a significant component to vPN diversity (Sizemore, 2016).

    Neuromodulators are often released by a small number of neurons within a network, yet they can have extremely diverse effects depending upon patterns of receptor expression. For the most part, individual populations of AL neurons differed in the receptor types that they expressed. This suggests that 5-HT differentially acts on classes of neurons that undertake distinct tasks in olfactory processing. In the case of the vPNs, this differential modulation may be fairly complex due to the diversity within this neuronal class. The goal of this study was to establish a functional atlas of 5-HTR expression in the AL of Drosophila. This dataset therefore provides a mechanistic framework for the effects of 5-HT on olfactory processing in this network (Sizemore, 2016).

    Identified serotonergic modulatory neurons have heterogeneous synaptic connectivity within the olfactory system of Drosophila

    Modulatory neurons project widely throughout the brain, dynamically altering network processing based on an animal's physiological state. The connectivity of individual modulatory neurons can be complex, as they often receive input from a variety of sources and are diverse in their physiology, structure, and gene expression profiles. To establish basic principles about the connectivity of individual modulatory neurons, a pair of identified neurons was examined, the 'contralaterally projecting, serotonin-immunoreactive deutocerebral neurons' (CSDns), within the olfactory system of Drosophila. Specifically, the neuronal classes were determined providing synaptic input to the CSDns within the antennal lobe (AL), an olfactory network targeted by the CSDns, along with the degree to which CSDn active zones are uniformly distributed across the AL. Using anatomical techniques, the CSDns were found to receive glomerulus-specific input from olfactory receptor neurons (ORNs) and projection neurons (PNs), and network-wide input from local interneurons (LNs). Furthermore, the number of CSDn active zones was quantified in each glomerulus; CSDn output was fount to be not uniform, but rather heterogeneous across glomeruli and stereotyped from animal to animal. Finally, it was demonstrated that the CSDns synapse broadly onto LNs and PNs throughout the AL, but do not synapse upon ORNs. These results demonstrate that modulatory neurons do not necessarily provide purely top-down input, but rather receive neuron class-specific input from the networks that they target, and that even a two cell modulatory network has highly heterogeneous, yet stereotyped pattern of connectivity (Coates, 2017).

    Wiring variations that enable and constrain neural computation in a sensory microcircuit

    Neural network function can be shaped by varying the strength of synaptic connections. One way to achieve this is to vary connection structure. To investigate how structural variation among synaptic connections might affect neural computation, primary afferent connections were examined in the Drosophila olfactory system. Large-scale serial section electron microscopy was used to reconstruct all the olfactory receptor neuron (ORN) axons that target a left-right pair of glomeruli, as well as all the projection neurons (PNs) postsynaptic to these ORNs. Three variations were found in ORN-->PN connectivity. First, a systematic co-variation was found in synapse number and PN dendrite size, suggesting total synaptic conductance is tuned to postsynaptic excitability. Second, PNs were found to receive more synapses from ipsilateral than contralateral ORNs, providing a structural basis for odor lateralization behavior. Finally, evidence was found of imprecision in ORN-->PN connections that can diminish network performance (Tobin, 2017).

    Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila

    Previous electrophysiological and behavioural studies implicate Esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. This study shows that Esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of Esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show Esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. The data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate (Younus, 2017).

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    Zygotically transcribed genes

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