InteractiveFly: GeneBrief

Pickpocket 23, Pickpocket 25 and Pickpocket 29 : Biological Overview | Regulation | Developmental Biology | References

BIOLOGICAL OVERVIEW

Odorants and pheromones as well as sweet- and bitter-tasting small molecules are perceived through activation of G protein-coupled chemosensory receptors. In contrast, gustatory detection of salty and sour tastes may involve direct gating of sodium channels of the DEG/ENaC family by sodium and hydrogen ions, respectively. ppk25, a Drosophila gene encoding a DEG/ENaC channel subunit, is expressed at highest levels in the male appendages responsible for gustatory and olfactory detection of female pheromones: the legs, wings, and antennae. Mutations in the ppk25 gene reduce or even abolish male courtship response to females in the dark, conditions under which detection of female pheromones is an essential courtship-activating sensory input. In contrast, the same mutations have no effect on other behaviors tested. Importantly, ppk25 mutant males that show no response to females in the dark execute all of the normal steps of courtship behavior in the presence of visible light, suggesting that ppk25 is required for activation of courtship behavior by chemosensory perception of female pheromones. A ppk25 mutant allele predicted to encode a truncated protein has dominant-negative properties, suggesting that the normal Ppk25 protein acts as part of a multiprotein complex. Together, these results indicate that ppk25 is necessary for response to female pheromones by D. melanogaster males, and suggest that members of the DEG/ENaC family of genes play a wider role in chemical senses than previously suspected (Lin, 2005).

As in most other animals, pheromones play key roles in the regulation of sexual behaviors of Drosophila. In particular, several pheromones modulate male courtship of the female, which involves a stereotyped series of behaviors. By analogy with olfactory and gustatory perception of organic molecules in both insects and vertebrates, perception of these pheromones most likely involves interactions with seven-transmembrane receptors and subsequent activation of a G protein-coupled signal transduction pathway. Indeed, a male-specific member of the seven-transmembrane gustatory receptor family, GR68a, has been identified as a putative receptor for female courtship-stimulating pheromones (Bray, 2003). In contrast, gustatory perception of hydrogen and sodium ions, perceived as sour and salty tastes, respectively, has been suggested to involve direct gating of sodium channels of the DEG/ENaC family (Lin, 1999; Lin, 2002). In support of this possibility, inactivation of either ppk11 or ppk19, two Drosophila DEG/ENaC subunit genes, results in loss of behavioral and electrophysiological responses to salt (Liu, 2003; Lin, 2005).

CheB42a, a member of a recently discovered family of Drosophila proteins, is only expressed in a small subset of gustatory sensilla on the front legs of males, suggesting that it may be involved in male-specific gustatory perception (Xu, 2002). Subsequently, Gr68a, a gustatory receptor gene, was found to be expressed in a similar pattern (Bray, 2003). The loss of male response to female pheromones upon either inactivation of Gr68a-expressing neurons or knock-down of Gr68a expression suggests that Gr68a may be a receptor for female pheromones that activate male courtship behavior. CheB42a and Gr68a are expressed in the same subset of gustatory sensilla on male front legs, suggesting that CheB42a also plays a role in this process. Intriguingly, ppk25, a gene predicted to encode another protein with a function in chemical senses, is found only 103 nt downstream of the 3' end of CheB42a (Lin, 2005).

Two deletions were generated that inactivate both CheB42a and ppk25: Delta5-2 and Delta5-22. Males homozygous for either deletion display a much reduced response to females but no similar decrease in other behaviors. In contrast, another deletion that results in complete loss of CheB42a expression but has no effect on ppk25 does not reduce male courtship behavior. A genomic fragment that includes both CheB42a and ppk25 rescues the response of Delta5-22 homozygous males to females, whereas an almost identical fragment lacking ppk25 does not. ppk25^PB, an independent mutation resulting from insertion of a transposable element into the second intron of ppk25, affects male response to females even more severely than Delta5-22, even though this allele has no detectable effect on CheB42a expression. Indeed, ppk25^PB has dominant-negative effects on male response to females, observable both in the presence or absence of a wild-type copy of ppk25. The dominant-negative properties of ppk25^PB are readily interpreted in light of the predicted generation in this mutant of a truncated Ppk25 protein retaining the N-terminal cytoplasmic domain, the first transmembrane domain, and part of the extracellular domain of the normal Ppk25. Similarly truncated variants of various members of the DEG/ENaC family, including several Drosophila ppks, also have dominant-negative properties (Lin, 2005).

The discovery of a role for ppk25 in male response to female pheromones was the unexpected result of an interest in the neighboring CheB42a. The data in this report show that deletion of CheB42a does not decrease overall male response to courtship-activating pheromones. However, the restricted expression of CheB42a in the same subset of gustatory sensilla that express Gr68a and are required for response to female courtship-activating pheromones (Bray, 2003) suggests that CheB42a's requirement may be obscured by functional redundancy with one or more of the other 11 Drosophila CheB genes or, alternatively, that CheB42a has a different role in male-specific chemical senses (Lin, 2005).

Is it a coincidence that two genes implicated in male-specific chemical senses are within ~103 nt of each other? These two genes produce mRNAs of different sizes with related, albeit different, expression patterns. Both are preferentially expressed in male gustatory appendages starting late in pupal development and remaining through at least sexual maturity of the adult males. However, whereas CheB42a is only expressed in male front legs (Xu, 2002), ppk25 mRNA is present at similar levels in legs and in the third antennal segment, and at much lower but detectable levels in heads and bodies. The proximity of these two genes may therefore reflect a shared dependence on regulatory elements important for overlapping spatial and/or temporal characteristics of their expression. Indeed, the lack of detectable ppk25 mRNA in males homozygous for Delta5-2 suggests the presence of a regulatory element essential for ppk25 expression within or immediately downstream of the 3' half of CheB42a. Alternatively, the proximity between these two genes may be more a reflection of their involvement in evolutionarily important and related aspects of sexual behavior (Lin, 2005).

Why can't ppk25 mutant males respond to females normally? Vision and pheromone detection have both been implicated in the response of Drosophila males to females. Absence of visible light or mutations that cause partial or complete blindness reduce, but do not eliminate, male response to females. In addition, a number of studies suggest that males detect courtship-stimulating female pheromones by using either gustation, olfaction, or both chemical senses. Although both vision and olfactory detection of pheromones are important for initiation of courtship behavior, gustatory perception of the same or other pheromones may be required for efficient progression to later steps in the courtship sequence. Because both initiation and maintenance of courtship bouts are affected in dominant-negative as well as null mutations in ppk25, this gene may be required for detection of pheromones by both sensory modalities, a possibility supported by the expression of ppk25 in both olfactory (antennae) and gustatory (wings and legs) appendages (Lin, 2005).

Is a Ppk25-containing sodium channel involved in the peripheral detection of female pheromones? Here the data strongly support the requirement for ppk25 in the male's ability to respond to female courtship-activating pheromones. In addition, mutations in ppk25 do not similarly impair other behaviors that are either largely independent of sensory inputs, such as walking and preening, or sensory-driven such as geotaxis and chemosensory response to sugars. Most importantly, these mutations have no effect on the initiation of courtship behavior in the presence of visible light. Therefore, ppk25's requirement for male response to pheromones likely reflects a specific role in the sensory detection of pheromones or subsequent processing within the central nervous system rather than a more general requirement for neural function or even for performance of courtship behavior. Finally, ppk25 expression is first detectable during late pupal stages, after determination of all of the various types of chemosensory cells and as they undergo the final stages of differentiation, suggesting that ppk25 is required for the function, rather than the development of chemosensory organs (Lin, 2005).

Is ppk25 required in peripheral olfactory or gustatory neurons that sense and respond to female pheromones in the environment or in central nervous system neurons that receive and process the information coming from the periphery? Although these alternatives remain to be tested, the former hypothesis is supported by ppk25's preferential expression in male chemosensory appendages as well as by the established roles of other DEG/ENaC subunits in peripheral sensory responses to mechanical stimuli and salt. ppk25's putative role in pheromone detection may not involve direct participation in the primary molecular response to pheromones. However, recent imaging of the electrophysiological response in mechanosensory neurons indicate that the C. elegans DEG/ENaC gene mec-4 is specifically required for the mechanosensory function (O'Hagan, 2005) rather than the general physiology of the neurons in which it is expressed. Similar questions arise regarding the role ppk25 plays in male detection of female pheromones and in particular, whether it interacts, directly or indirectly, with the G protein-coupled signal transduction pathways that underlie chemical senses in Drosophila as in other animals (Lin, 2005).

Finally, the dominant-negative properties of the ppk25^PB allele most likely reflect the participation of the Ppk25 protein in a multisubunit protein complex. Proteins of the DEG/ENaC family are thought to interact in the formation of heteromeric sodium channels. Several truncated versions of DEG/ENaC proteins have dominant-negative properties that most likely result from their ability to form partial and inactive complexes with other DEG/ENaC subunits. By analogy, the results suggest that one or more of the ~30 other Ppk proteins encoded in the Drosophila genome interacts with Ppk25 within a heteromeric sodium channel. In conclusion, the data demonstrate a role for a member of the DEG/ENaC family of sodium channel subunits in the peripheral detection or central processing of a pheromonal signal. This finding opens the door to the dissection of ppk25's role in pheromone response and its relationship with other proteins involved in pheromone response. Finally, this work suggests that members of the Drosophila ppk family, as well as DEG/ENaC subunits in other organisms, play more complex roles in chemical senses than previously suspected (Lin, 2005).

Two Drosophila DEG/ENaC channel subunits have distinct functions in gustatory neurons that activate male courtship.

Trimeric sodium channels of the DEG/ENaC family have important roles in neurons, but the specific functions of different subunits present in heteromeric channels are poorly understood. The Drosophila DEG/ENaC subunit Ppk25 has been shown to be essential in a small subset of gustatory neurons for activation of male courtship behavior, likely through detection of female pheromones. This study shows that, like mutations in ppk25, mutations in another Drosophila DEG/ENaC subunit gene, nope (ppk29), specifically impair male courtship of females. nope regulatory sequences drive reporter gene expression in gustatory neurons of the labellum wings, and legs, including all gustatory neurons in which ppk25 function is required for male courtship of females. In addition, gustatory-specific knockdown of nope impairs male courtship. Further, the impaired courtship response of nope mutant males to females is rescued by targeted expression of nope in the subset of gustatory neurons in which ppk25 functions. However, nope and ppk25 have nonredundant functions, as targeted expression ofppk25 does not compensate for the lack of nope and vice versa. Moreover, Nope and Ppk25 form specific complexes when coexpressed in cultured cells. Together, these data indicate that the Nope and Ppk25 polypeptides have specific, nonredundant functions in a subset of gustatory neurons required for activation of male courtship in response to females, and suggest the hypothesis that Nope and Ppk25 function as subunits of a heteromeric DEG/ENaC channel required for gustatory detection of female pheromones (Liu, 2012).

Recently, it was reported that expression of ppk25 in a small subset of taste neurons on the legs and wings of males is sufficient for a normal male courtship response to females (Starostina, 2012). This study describes a second DEG/ENaC subunit gene, nope, that is also specifically required for male response to females. Several lines of evidence indicate that both genes are expressed in, and are required for the function of a common subset of gustatory neurons that activate courtship behavior. First, mutations in ppk25 and nope have indistinguishable effects on male courtship. Like mutations in ppk25, mutations in nope specifically impair the courtship response of males to females but have no general effect on other behaviors, such as walking or preening. Furthermore, like mutations in ppk25 (Starostina, 2012), but in contrast to mutations in fru or in the Gr32a gustatory receptor gene, mutations in nope do not increase homosexual behavior, suggesting that neither ppk25 nor nope is required for gustatory detection of inhibitory pheromones, such as the male-enriched 7-tricosene. Finally, consistent with gustatory function being required not only to initiate courtship but also to progress to late steps, mutations in either ppk25 (Starostina, 2012) or nope result in significant decreases in both the fraction of males that initiate courtship and the total time spent courting. Second, expression of ppk25-Gal4 and nope-Gal4 transgenes overlaps in a small subset of taste neurons on the legs and wings of males. In the front legs in particular, both transgenes are expressed in fru-expressing taste neurons that make sexually dimorphic projections onto the thoracic ganglia (Mellert, 2010), and all neurons that express ppk25-Gal4 also express nope-Gal4. Third, as in the case of ppk25 (Starostina, 2012), targeted RNAi-mediated knockdown of nope in gustatory neurons specifically impairs the male response to females, indicating that both genes are required for normal gustatory response to females. Finally, and most importantly, using the same ppk25-Gal4 driver, targeted expression of nope and ppk25 rescues the courtship of nope and ppk25 mutant males, respectively, indicating that for both DEG/ENaC subunits, expression in a common subset of gustatory neurons on the legs and wings of males is sufficient for normal response to females. Coupled with previous work (Starostina, 2012), these findings further demonstrate that this specific subset of gustatory neurons on the legs and wings has an essential role in activating male courtship, starting from the earliest steps (Liu, 2012).

In addition to the subset of neurons defined by coexpression of ppk25-Gal4 and nope-Gal4, some chemosensory neurons express only one of the two drivers; ppk25-Gal4 is expressed in two subsets of olfactory neurons implicated in activating courtship (Starostina, 2012), and nope-Gal4 is expressed in gustatory neurons on the legs and labellum. Furthermore, targeted expression of nope using the ppk25-Gal4 driver is sufficient to rescue the courtship of nope mutant males. This result indicates that, under these experimental conditions at least, gustatory neurons that express nope-Gal4 but not ppk25-Gal4 are not essential for activation of courtship in response to females, and suggests that expression of nope-Gal4 may occur in functionally distinct subsets of gustatory neurons. In summary, our data indicate that nope and ppk25 function in a common subset of gustatory neurons on the front legs and wings of males that is required for activation of male courtship in the presence of females (Liu, 2012). ppk25 and nope function in the same subset of gustatory neurons, mutants lacking either subunit display severely reduced male response to females, indicating that the ppk25 and nope genes have nonredundant functions. Furthermore, the phenotype of nope mutant males is rescued by targeted expression of nope, but not of ppk25, and vice versa, indicating that the lack of redundancy is due to intrinsic properties of the proteins encoded by each gene rather than differences in their expression patterns. In addition, when coexpressed in cultured cells, Nope forms specific complexes with Ppk25 more efficiently than with human β-ENaC, another DEG/ENaC subunit. Together, these data support the hypothesis that Ppk25 and Nope assemble into a heteromeric DEG/ENaC channel with a specific and essential function in gustatory activation of courtship behavior. The crystal structure of the vertebrate DEG/ENaC acid-sensing channel, ASIC1a, reveals that it is a homotrimer, suggesting that most if not all DEG/ENaC channels are also trimers (Jasti, 2007). Furthermore, ENaC, the vertebrate epithelial sodium channel is a heterotrimer containing three types of subunits (α, β, and γ), all of which must be present for assembly of a functional channel. Finally, heteromeric assemblies of C. elegans Mec-4 and Mec-10 are required for response to gentle touch (Bianchi, 2007). By analogy, Ppk25 and Nope are likely to assemble into a heterotrimeric DEG/ENaC channel, perhaps together with a third subunit yet to be discovered. In addition, while ppk25-Gal4 and nope-Gal4 have overlapping expression patterns, each driver is also expressed in cells where the other is not; ppk25-Gal4 is expressed in two subsets of olfactory neurons involved in activation of male courtship (Root, 2008; Grosjean, 2011; Starostina, 2012), and, from this study, nope-Gal4 is expressed in gustatory neurons of unknown function on the front legs and labellum of males. Ppk25 may therefore also participate in trimeric channels lacking Nope, and vice versa. By analogy with human ASICs, for which channels of different subunit composition have different functional properties, combinatorial subunit composition may confer functional variety to DEG/ENaC channels in gustatory and olfactory neurons (Liu, 2012).

Finally, the specific phenotypes of ppk25 and nope mutants coupled with their expression in a small subset of gustatory neurons required for response to females suggest that both DEG/ENaC subunits function specifically in detection of courtship-stimulating pheromones. What is the molecular role of DEG/ENaC channels in pheromone response? In the same way that other DEG/ENaCs channels function as gustatory receptors for water (Cameron, 2010; Chen, 2010) or sodium ions (Chandrashekar, 2010), heteromeric channels containing Ppk25 and Nope may be directly gated by pheromones or by other courtship-activating compounds. Alternatively, heteromeric DEG/ENaC channels containing Nope and Ppk25 may have a less direct role in modulating courtship, similar to the function of C. elegans ASIC1 in learning modulation through presynaptic facilitation of dopamine release. This work lays the foundation for the dissection of the molecular roles of DEG/ENaC channels and the contribution of subunit composition in gustatory activation of male courtship, with relevance to the function of DEG/ENaC channels involved in a number of physiological and pathological processes including hypertension, cystic fibrosis, touch and pain, memory formation, evoked fear, and neuronal cell death in stroke (Liu, 2012).

Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila

Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, a behavioral assay was established in Drosophila melanogaster, and the relationship between sexual experience and aggression was investigated. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel Ppk29 and was mediated by male-specific GABAergic neurons acting on the GABAA receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. It is proposed that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression (Yuan, 2013).

Aggression is a complex behavior that is regulated by various internal and external stimuli. To date, however, studies have remained largely focused on the sensory pathways involved in regulating baseline aggression, with less examination of the central components of the underlying neural pathway. Moreover, the close relationship between sex and aggression has been a fascinating topic in both biology and literature, but their intertwined nature and the underlying neurobiological basis have remained elusive. Using a behavioral genetics approach, this study identified a previously unknown neural pathway that underlies the modulation of sex-related male-male aggression in Drosophila by prior contacts with females (Yuan, 2013).

These results suggest that prior female encounter through direct physical contacts activates the pheromone-sensing ppk29 neurons, resulting in inhibition of the central aggression circuit via GABAergic mechanisms involving the RDL GABA_A receptor, thereby suppressing the behavioral output for male-male aggression. The three levels of the neural pathway involved in this experience-dependent behavior modification all exhibited sexual dimorphism, consistent with the notion that morphological differences in male and female brains correlate with their distinct behavioral needs. It was possible to modify the aggressive behavior output by manipulating the circuit at each of these three steps, which possibly represented the sequence of the information relay involved in the native behaviors, the sensory input, the information processing and the execution of the behavior. However, it is recognized that the circuit components elucidated by these experiments are clearly only parts of the machinery responsible for aggression modulation. In addition, by identifying RDL as a molecular target for aggression regulation, this study provides an entry point for characterizing the missing link of aggression studies, namely the central neurons that respond to experience-dependent modulation and mediate the execution of aggressive behaviors. Thus, this work provides new insights regarding the intricate interactions between sexual experience and aggression and delineates the underlying mechanisms to inform potential means to suppress excessive aggression (Yuan, 2013).

The female contact-dependent suppression of male aggression may also be viewed as a form of learning-induced plasticity. The learning procedure in this case requires extended physical interactions between the male and the female (over 10 h), which could consist of repeated sessions of male courtship attempts and female rejection. As no obvious defects were observed in aggression suppression in genetic mutants with deficits in courtship conditioning, such as homer, eag, Shaker< and orb2 mutants, this experience-induced suppression of aggression is likely different from conventional courtship conditioning. Another interesting feature of this suppression is that it is long term, yet reversible, lasting up to 2 d after the female encounter. However, it also differs from well-studied long-term memory formation, as no defect was observed in amn mutants, which is required for long-term memory formation. The results implicate the fru⁺ d5-HT1B⁺ and GABA⁺ cluster of neurons in the central brain as the regulators of this suppression, but it remains to be determined whether these neurons are involved in the initiation, acquisition, execution or consolidation phase(s) of this behavior, what takes form as the underlying 'memory trace' and whether plasticity is manifested at the level of the number of neurons activated, neurite arborization, neuronal activity or some other aspect of neuronal signaling (Yuan, 2013).

Notwithstanding the emergence of Drosophila as a successful genetic model for aggression studies and the extensive characterization of its stereotypical motor display of aggression, the strong influence of genetic background over baseline aggression and locomotor activity often complicates Drosophila aggression studies. The current assay avoids such difficulties by consistently eliciting aggression in naive male flies in the presence of females and by inducing a strong suppression of aggression in males with prior female encounter. The small variations among different genetic backgrounds in the behavioral assay make it possible to identify critical cellular and molecular components involved in the regulation of aggression by experience (Yuan, 2013).

One purpose of studying aggression regulation in animal models is to eventually understand the basis of human violence and establish venues to reduce or prevent it. Psychophysiological studies suggest that the failure to maintain an appropriate level of aggression in humans is associated with impaired executive cognitive processes or emotion registration. As an innate behavior built largely on predetermined neural pathways, aggression in Drosophila males can be modulated by prior exposure to females through GABAergic inhibition. This study raises the possibility that an ancient and basic machinery of the central neuronal circuitry, GABAergic inhibition, could be part of a conserved mechanism to modulate the level of aggression in males and ensure proper balance between reproductive competition and individual survival (Yuan, 2013).

Amplification of Drosophila olfactory responses by a DEG/ENaC Channel

Insect olfactory receptors operate as ligand-gated ion channels that directly transduce odor stimuli into electrical signals. However, in the absence of any known intermediate transduction steps, it remains unclear whether and how these ionotropic inputs are amplified in olfactory receptor neurons (ORNs). This study finds that amplification occurs in the Drosophila courtship-promoting ORNs through Pickpocket (PPK25), a member of the degenerin/epithelial sodium channel family (DEG/ENaC). Pharmacological and genetic manipulations indicate that, in Or47b and Ir84a ORNs, PPK mediates Ca(2+)-dependent signal amplification via an intracellular calmodulin-binding motif. Additionally, hormonal signaling upregulates PPK expression to determine the degree of amplification, with striking effects on male courtship. Together, these findings advance understanding of sensory neurobiology by identifying an amplification mechanism compatible with ionotropic signaling. Moreover, this study offers new insights into DEG/ENaC activation by highlighting a novel means of regulation that is likely conserved across species (Ng, 2019).

Vertebrates detect odorants with G-protein-coupled receptors (GPCRs), the activation of which triggers subsequent metabotropic signaling cascades in the olfactory receptor neurons (ORNs) to transduce chemical stimuli into electrical signals. These series of transduction events also provide opportunities to amplify input signals. In contrast, insect olfaction is initiated by ligand-gated receptor channels that lack canonical G protein interacting domains. Although various G proteins and effectors have been implicated in the function of insect ORNs, it remains an open question whether those molecules play a specific role in olfactory transduction or a regulatory role in neuronal development, maintenance, and neuromodulation. Given that ligand-gated receptor channels can directly convert sensory stimuli to neuronal depolarization, it is unclear whether and how ionotropic inputs can be amplified in the absence of any known intermediate transduction steps (Ng, 2019).

In earlier work, it was found that the responses of Or47b ORNs, which detect aphrodisiac fly odors in D. melanogaster, increase with age in male flies (Lin, 2016), pointing to the possibility of signal amplification downstream of insect olfactory receptors. This age-dependent plasticity therefore presents an opportunity to investigate the mechanisms by which ionotropic sensory inputs can be amplified. Intriguingly, Or47b ORNs express a degenerin/epithelial sodium channel (DEG/ENaC) subunit named Pickpocket (PPK25). Within invertebrate genomes, DEG/ENaCs constitute one of the largest ion channel families. In mechanosensory and gustatory neurons, PPK subunits are involved in touch, proprioception, nociception, salt taste, water sensation, and recognition of contact pheromones. However, the functional role of PPK in olfaction remains unknown (Ng, 2019).

If PPK amplifies olfactory signals, how then is its activity regulated? Can it function as a transduction channel activated by intracellular second messengers downstream of receptor activation? Members of the DEG/ENaC superfamily, including the mammalian nonvoltage-gated sodium channels (SCNNs) and acid-sensing sodium ion channels (ASICs), are known to open in response to mechanical stimuli, extracellular ligands, or are otherwise constitutively active. In cultured cell lines, various intracellular signaling mechanisms can influence DEG/ENaC currents by regulating channel transcription, endocytosis, degradation, or translocation. Post-translational modification is also known to modulate DEG/ENaC function; for example, constitutive channel activity can be regulated by CaMKII-mediated phosphorylation or by protease-mediated cleavage of the extracellular domain. However, the possibility of DEG/ENaC activation by direct interaction with an intracellular ligand has not been explored (Ng, 2019).

This study shows that signal amplification can occur downstream of ligand-gated receptor channels. The age-dependent response plasticity of Or47b ORNs arises through PPK25-mediated amplification. Additionally, this mechanism is employed in another type of courtship-promoting ORN expressing the Ir84a receptor. Interestingly, the degree of amplification is determined by PPK expression levels, which are in turn upregulated by a reproductive hormone. Thus, a common hormone regulates these two parallel olfactory pathways to coordinate courtship behavior. Mechanistically, PPK operates as a transduction channel: its activation requires odor-induced Ca2+ influx and a calmodulin binding motif (CBM) in the N-terminal intracellular domain. This result therefore highlights a novel mechanism whereby DEG/ENaCs can be activated by second messengers, a critical feature common to all transduction channels. Moreover, similar intracellular CBMs are predicted in multiple DEG/ENaCs across animal species, suggesting an evolutionarily conserved regulatory mechanism for channels in this superfamily (Ng, 2019).

This study has demonstrated that ionotropic sensory inputs can be amplified in select Drosophila ORNs whose receptors are ligand-gated cation channels. Pharmacological and genetic experiments reveal a simple and elegant mechanism for this amplification. Upon odor stimulation, receptor excitation allows for direct influx of Ca2+, which serves as a second messenger to activate a DEG/ENaC channel, PPK25, and thereby amplify ORN responses (Ng, 2019).

Ionotropic signal amplification, as described, affords remarkable versatility in sensory signaling when compared against G-protein-mediated metabotropic mechanisms. In vertebrate olfaction, separate families of metabotropic receptors typically couple to different G proteins, each engaging a unique downstream signaling cascade. In contrast, activation of specific G proteins is not required for Ca2+-mediated amplification, making it compatible with a wide variety of ionotropic receptor channels, so long as these receptors are permeable to Ca2+. As evidenced in this study, PPK can function downstream of Or47b, Ir84a, and ChR2, despite their low sequence similarity and distinct topologies because these receptors can flux Ca2+ (Ng, 2019).

These findings highlight striking differences and commonalities between insect and vertebrate olfactory transduction. This study observed surprising heterogeneity within insect olfactory transduction: PPK is neither expressed nor functional in another ORN type expressing Or22a, which belongs to the same receptor family as Or47b. It is unclear whether input signals in Or22a ORNs are amplified. If so, it is likely through a different mechanism. This finding indicates that insect ORNs expressing the same family of receptors do not necessarily employ the same mechanism for amplification, in contrast with vertebrate olfaction where receptors from the same family share a common signaling pathway. Despite this difference, the activation mechanism and function of PPK are remarkably similar to those of Anoctamin (ANO2), a key transduction channel in vertebrate ORNs. Specifically, both PPK and ANO are activated by calcium to amplify olfactory inputs. Although the sources of Ca2+may differ-ligand-gated ion channels in insects or cyclic nucleotide-gated cation channels in vertebrates-Ca2+-mediated amplification may represent a shared signaling motif between the two olfactory systems (Ng, 2019).

Interestingly, the impact of signal amplification on spike output differs between insect and vertebrate ORNs. Consistent with its role as a transduction channel, Ano mutation markedly reduces odor-evoked currents. However, the spike output of Ano knockout ORNs is higher than that of wild-type neurons. Vertebrate ORN spike number peaks when the transduction current is largely carried by ANO at intermediate odor concentrations, suggesting a strong depolarization block whereby amplification provides negative feedback to clamp total spike output. In contrast, the local field potential and spike responses of insect ORNs both peak at saturating odor concentrations. As such, blocking PPK25-mediated amplification not only reduces LFP response but also total spike number. Therefore, signal amplification in insect ORNs may predominantly serve to modulate the gain of neuronal output (Ng, 2019).

What is the functional significance of PPK in the Or47b and Ir84a ORNs? Notably, these are the only ORN types known to promote courtship in D. melanogaster males, whose fertility and courtship drive increase and peak at about days of age. Male mating drive is highly influenced by external olfactory cues, including the availability of mates and food signaled by Or47b and Ir84a ORNs, respectively. Remarkably, the responses of both ORN types exhibit age-dependent plasticity, which is coordinated by the same reproductive hormone-juvenile hormone-through upregulation of PPK expression in older males. The expression level of PPK in turn determines the ORN response magnitude, with striking impacts on courtship. Therefore, flexibility over this biologically salient behavior is afforded by the dynamic regulation of PPK expression, which heightens males' sensitivity to food and mate odors at their age of peak fertility. This upregulation of PPK provides a molecular mechanism for how sex-specific refinements of olfactory circuits are achieved via hormonal signaling (Ng, 2019).

The critical role of the intracellular CBM in PPK function argues that Ca2+/CaM activates the channel by directly interacting with this motif. Such regulation contrasts sharply with previously reported mechanisms, in which Ca2+/CaM indirectly modulates ENaC activity through intermediate proteins. For example, in cultured Xenopus cells, Ca2+/CaM can inhibit ENaC currents by interacting with MARCKS (myristoylated alanine-rich C kinase substrate) to modulate channel open probability and also by activating CaMKII to regulate ENaC apical trafficking. Together, these findings highlight the complexity of interactions between Ca2+/CaM and neuronal DEG/ENaC (Ng, 2019).

The results described in this study further advance understanding of DEG/ENaC activation. The gating mechanisms for this family of sodium channels are known to be highly diverse: some open in response to mechanical stimuli; others to extracellular ligands; and still others are constitutively active. In light of these findings, it is possible that other members of the DEG/ENaC superfamily may also be directly activated by intracellular second messengers, allowing them to function as transduction channels to amplify sensory inputs. In support of this notion, similar N-terminal intracellular CBMs were bioinformatically identified in multiple members of the DEG/ENaC superfamily across species-including worm, fruit fly, mosquito, mouse, and human-suggesting that those channels have the potential to function as Ca2+-activated transduction channels (Ng, 2019).

Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship

The elaborate courtship ritual of Drosophila males is dictated by neural circuitry established by the transcription factor Fruitless and triggered by sex-specific sensory cues. Deciphering the role of different stimuli in driving courtship behavior has been limited by the inability to selectively target appropriate sensory classes. This study identified two ion channel genes belonging to the degenerin/epithelial sodium channel/pickpocket (ppk) family, ppk23 and ppk29, which are expressed in Fruitless-positive neurons on the legs and are essential for courtship. Gene loss-of-function, cell-inactivation, and cell-activation experiments demonstrate that these genes and neurons are necessary and sufficient to inhibit courtship toward males and promote courtship toward females. Moreover, these cells respond to cuticular hydrocarbons, with different cells selectively responding to male or female pheromones. These studies identify a large population of pheromone-sensing neurons and demonstrate the essential role of contact chemosensation in the early courtship steps of mate selection and courtship initiation (Thistle, 2012).

Evolved differences in larval social behavior mediated by novel pheromones

Pheromones, chemical signals that convey social information, mediate many insect social behaviors, including navigation and aggregation. Several studies have suggested that behavior during the immature larval stages of Drosophila development is influenced by pheromones, but none of these compounds or the pheromone-receptor neurons that sense them have been identified. This study report sa larval pheromone-signaling pathway. Larvae were found to produce two novel long-chain fatty acids that are attractive to other larvae. A single larval chemosensory neuron was identified that detects these molecules. Two members of the pickpocket family of DEG/ENaC channel subunits (ppk23 and ppk29) are required to respond to these pheromones. This pheromone system is evolving quickly, since the larval exudates of D. simulans, the sister species of D. melanogaster, are not attractive to other larvae. These results define a new pheromone signaling system in Drosophila that shares characteristics with pheromone systems in a wide diversity of insects (Mast, 2014).

REGULATION

The Drosophila gene CheB42a is a novel modifier of Deg/ENaC channel function

Degenerin/epithelial Na(+) channels (DEG/ENaC) represent a diverse family of voltage-insensitive cation channels whose functions include Na(+) transport across epithelia, mechanosensation, nociception, salt sensing, modification of neurotransmission, and detecting the neurotransmitter FMRFamide. The Drosophila melanogaster Deg/ENaC gene lounge lizard (llz, ppk25) is co-transcribed in an operon-like locus with another gene of unknown function, CheB42a. Because operons often encode proteins in the same biochemical or physiological pathway, it is hypothesized that CHEB42A and LLZ might function together. Consistent with this hypothesis, both genes were found to be expressed in cells previously implicated in sensory functions during male courtship. Furthermore, when coexpressed, LLZ coprecipitated with CHEB42A, suggesting that the two proteins form a complex. Although LLZ expressed either alone or with CHEB42A did not generate ion channel currents, CHEB42A increased current amplitude of another DEG/ENaC protein whose ligand (protons) is known, acid-sensing ion channel 1a (ASIC1a). It was also found that CHEB42A is cleaved to generate a secreted protein, suggesting that CHEB42A may play an important role in the extracellular space. These data suggest that CHEB42A is a modulatory subunit for sensory-related Deg/ENaC signaling. These results are consistent with operon-like transcription of CheB42a and llz and explain the similar contributions of these genes to courtship behavior (Ben-Shahar, 2010).

The predicted protein structure of CHEB42A indicated it might interact with LLZ. First, the CHEB42A structure resembles that of the accessory subunits of other ion channels: an example is the human protein MiRP1, which associates with the HERG K+ channel. In addition, the C. elegans protein MEC-6 has a similar general predicted structure to the CHEB42A protein and associates with the DEG/ENaC channel subunits MEC-4 and MEC-10 (Chelur, 2002). Accessory subunits can alter the gating and regulation of ion channels and/or serve as a chaperone to regulate the level of channel presence on the cell surface. Thus, it was considered that CHEB42A might associate with LLZ and modulate its function. Second, the predicted CHEB42A structure is similar to that of some odorant binding proteins in which a transmembrane segment can act as a signal peptide, anchoring an extracellular odorant binding domain that is released from the membrane following protease cleavage. Thus, it was considered that CHEB42A might be proteolytically released from the membrane and interact with LLZ as a secreted protein (Shahar, 2010).

The current results support the conclusion that the CheB42a/llz locus has operon-like transcription. In addition, a recent report suggested that another chemosensory-related locus in the fly genome is transcribed as a polycistronic mRNA (Slone, 2007); the Gr64a-f locus encodes several sugar receptors. Finding that sensory-related loci are co-transcribed suggests an evolutionary solution for finely controlling the spatial, temporal and quantitative aspects of chemosensation. Tight control on the expression of membrane-bound complexes is probably essential in many eukaryotic systems. Hence, these results also raise the intriguing possibility that operon-like transcription may be more common in eukaryotes than has previously been appreciated, including organisms outside the Drosophila lineage. It is speculated that identification of other co-transcribed genes may reveal novel protein interactions and pathways (Shahar, 2010).

DEVELOPMENTAL BIOLOGY

To evaluate a possible involvement of ppk25 in male response to pheromones, ppk25 expression was tested in pooled adult appendages that are highly enriched for gustatory (legs and wings) and olfactory (third antennal segment sensory hairs, as well as in body parts that have much fewer chemosensory cells relative to their total mass: heads (without third antennal segment) and bodies (without heads or appendages). mRNA was isolated from all three types of body parts and analyzed by Northern blot using a full-length ppk25 cDNA probe. Remarkably, hybridization is by far the strongest to mRNAs from the appendages fraction, yielding a set of bands between 2.1 and 2.4 kb in size, consistent with the predicted ppk25 transcript. Upon longer exposure of the filter, mRNAs of identical sizes, but much lower abundance, are detected in both head and body fractions. Probing the same filter with CheB42a sequences reveals a much smaller mRNA of ~700 nt that is present only in the appendages fraction (Lin, 2005).

To determine which appendages express ppk25 mRNA, quantitative real-time RT-PCR was used on total RNA extracted from different types of male appendages. This analysis confirms that, in adults, ppk25 mRNA is most abundant in appendages and also shows that ppk25 expression is approximately three times higher in male than female appendages. Interestingly, however, ppk25 mRNA is present at equivalent levels in male legs and wings, appendages that carry many gustatory sensilla, and in the third antennal segment, the main olfactory organ of the fly. Finally, ppk25 mRNA is not detectable in larvae or at early pupal stages (light pupae), but first appears at late pupal stages (dark pupae), and persists for at least 3 days after eclosion, by which time males are sexually mature (Lin, 2005).

Together, these data show that ppk25 expression is highest in olfactory and gustatory appendages of sexually mature males, a distribution consistent with a role in response to female pheromones. In addition, two observations argue that, despite their proximity, CheB42a and ppk25 are indeed two separate genes that are independently transcribed into two separate mRNAs: (1) no evidence was found of any transcript containing both CheB42a and ppk25 sequences; (2) the two mRNAs have related, but not identical, tissue distributions. Both transcripts are present at highest levels in male appendages. However, whereas CheB42a expression is only detectable in male front legs, ppk25 mRNA is expressed equally in male legs and antennae and at lower, but significant levels in female appendages as well as bodies and heads of either sex (Lin, 2005).

The Drosophila female aphrodisiac pheromone activates ppk23(+) sensory neurons to elicit male courtship behavior

Females of many animal species emit chemical signals that attract and arouse males for mating. For example, the major aphrodisiac pheromone of Drosophila melanogaster females, 7,11-heptacosadiene (7,11-HD), is a potent inducer of male-specific courtship and copulatory behaviors. This study demonstrates that a set of gustatory sensory neurons on the male foreleg, defined by expression of the ppk23 marker, respond to 7,11-HD. Activity of these neurons is required for males to robustly court females or to court males perfumed with 7,11-HD. Artificial activation of these ppk23⁺ neurons stimulates male-male courtship even without 7,11-HD perfuming. These data identify the ppk23⁺ sensory neurons as the primary targets for female sex pheromones in Drosophila (Toda, 2012).

This study presents evidence that ppk23⁺ bitter-sensing gustatory sensory neurons (GSNs) are activated by the female aphrodisiac pheromone 7,11-HD and that the activity of these neurons is both necessary and sufficient for the courtship behavior that it elicits. These conclusions are consistent with data obtained independently in the Scott (Thistle, 2012) and Ben-Shahar (Lu, 2012) labs (Toda, 2012).

Silencing ppk23⁺ GSNs increases the latency and reduces the intensity with which males court either females or other males that have been perfumed with 7,11-HD. The effect of silencing these neurons is significantly more pronounced under dark conditions, suggesting that, at least in the small assay chambers used in the experiments, the inability to detect 7,11-HD can be compensated in part by visual cues. This result, which was also observed by Lu (2012), is consistent with the finding that males still court females lacking 7,11-HD and other cuticular pheromones, as well as data suggesting that males can flexibly use multiple cues to find and woo a mate. Nonetheless, insensitivity to the major female aphrodisiac pheromone is likely to significantly compromise a male's reproductive fitness in the competitive conditions of the natural environment (Toda, 2012).

Activation experiments further highlight the importance of context in the behavioral response to the 7,11-HD signal. Artificially activating ppk23⁺ GSNs is sufficient to stimulate courtship in the absence of 7,11-HD, but not in the absence of another fly. In contrast, activating specific brain neurons induces even isolated males to court. Thus, if the 7,11-HD signal ultimately feeds into these brain neurons, then it is likely to be only one of multiple signals required to activate them. Whatever these other contextual cues are, they are evidently not specific to females, and indeed may not even be specific to Drosophila (Toda, 2012).

Silencing ppk23⁺ GSNs not only impaired male-female courtship, but also resulted in a low but significant level of male-male courtship, as also observed by Thistle (2012). As 7,11-HD is produced only by females, this male-male courtship cannot be attributed to abnormal responses to 7,11-HD. Rather, it implies that ppk23⁺ GSNs are also involved in the detection of male inhibitory pheromones. The simplest scenario is that the ppk23⁺ neurons are functionally heterogeneous. Some ppk23⁺ GSNs, including those in the Tm4c sensilla that this study recorded from, respond to female pheromones, whereas others might respond to male pheromones. Indeed, recent imaging studies have shown that a subset of ppk23⁺ GSNs respond to male inhibitory pheromones 7-T and cVA (Thistle, 2012). Artificial activation of ppk23⁺ GSNs does not, however, mimic exposure to these inhibitory pheromones by suppressing male-female courtship, presumably because the 7,11-HD-responsive neurons are also activated in these experiments. This interpretation is supported by data from perfuming experiments demonstrating that the application of 7,11-HD overrides the inhibitory effects of 7-T and cVA. Further disentanglement of the functional diversity of ppk23⁺ GSNs awaits the development of more specific genetic reagents for the selective targeting of these distinct cell types (Toda, 2012).

What is the molecular nature of the receptor for 7,11-HD expressed in ppk23⁺ GSNs? Two members of the GR family of gustatory receptors, Gr68a and Gr39a, have previously been proposed to mediate male responses to female pheromones. The cells that express these GRs are, however, distinct from the ppk23⁺ neurons this study has described, and indeed, further study of Gr68a has failed to confirm its putative role in the chemosensory regulation of male courtship behavior. It is therefore unlikely that either Gr68a or Gr39a functions as a 7,11-HD receptor in ppk23⁺ neurons. An alternative possibility is that the Ppk23 protein itself is a component of the 7,11-HD receptor. Like the canonical families of insect chemoreceptors, some members of the Deg/ENaC family are also thought to form ligand-gated cation channels (Toda, 2012).

Deg/ENac proteins typically form heteromeric complexes of three to nine subunits, raising the possibility that Ppk23 might form a receptor complex together with other members of the Ppk family. In such a scenario, ligand specificity might be conferred by subunits other than Ppk23, explaining why ppk23 is required for the behavioral responses to both female and male pheromones. Indeed, at least two other members of the Ppk family, Ppk25 and Ppk29, are likely to be coexpressed with Ppk23 in some of the fru⁺ GSNs. Both Ppk25 and Ppk29 are required for robust male courtship toward females, but neither is required to prevent males from courting each other. Ppk23 might thus form heteromeric complexes with Ppk25 and/or Ppk29 to detect female pheromones and with other subunits to detect male pheromones. It is cautioned, however, that the data do not preclude a more general role for Ppk23 in the function of these pheromone-sensing cells. Additional data from heterologous systems will be required to test whether Ppk23 and other members of this family indeed act as pheromone receptors (Toda, 2012).

>Regardless of the molecular nature of the 7,11-HD receptor, identification of its cellular targets paves the way for investigating the neural mechanisms by which this pheromone ultimately induces males to court. It is now possible to begin to trace the neural pathways that further process the 7,11-HD signal, which thereby integrate it with other sensory inputs to elicit a robust courtship response and ensure that this response is directed only at the most appropriate targets (Toda, 2012).

REFERENCES

Search PubMed for articles about Drosophila Pickpocket 23, Pickpocket 25 and Pickpocket 29

Ben-Shahar, Y., et al. (2010). The Drosophila gene CheB42a is a novel modifier of Deg/ENaC channel function. PLoS ONE 5: e9395. PubMed Citation: 20195381

Chelur, D. S., et al. (2002). The mechanosensory protein MEC-6 is a subunit of the C. elegans touch- cell degenerin channel. Nature 420: 669--673. PubMed Citation: 12478294

Slone, J., Daniels, J. and Amrein, H. (2007). Sugar receptors in Drosophila. Curr. Biol. 17: 1809-1816. PubMed Citation: 17919910

Bray, S. and Amrein, H. (2003). A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship. Neuron 39: 1019-1029. 12971900

Bianchi, L. (2007) Mechanotransduction: touch and feel at the molecular level as modeled in Caenorhabditis elegans. Mol. Neurobiol. 36: 254-271. PubMed Citation: 17955200

Cameron, P., Hiroi, M., Ngai, J. and Scott, K. (2010). The molecular basis for water taste in Drosophila. Nature 465: 91-95. PubMed Citation: 20364123

Chandrashekar, J., et al. (2010). The cells and peripheral representation of sodium taste in mice. Nature 464: 297-301. PubMed Citation: 20107438

Chen, Z,, Wang, Q. and Wang, Z. (2010). The amiloride-sensitive epithelial Na+ channel PPK28 is essential for Drosophila gustatory water reception. J. Neurosci. 30: 6247-6252. PubMed Citation: 20445050

Edwards, A. C., et al. (2009). Mutations in many genes affect aggressive behavior in Drosophila melanogaster. BMC Biol. 7: 29. PubMed Citation: 19519879

Grosjean, Y., et al. (2011). An olfactory receptor for food-derived odours promotes male courtship in Drosophila. Nature 478:236-240. PubMed Citation: 21964331

Jasti, J., Furukawa, H., Gonzales, E. B., and Gouaux, E. (2007). Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature 449: 316-323. PubMed Citation: 17882215

Krstic D., Boll, W. and Noll, M. (2009). Sensory integration regulating male courtship behavior in Drosophila. PLoS ONE 4: e4457. PubMed Citation: 19214231

Laughlin, J. D., Ha, T. S., Jones, D. N. and Smith, D. P. (2008). Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein. Cell 133: 1255-1265. PubMed Citation: 18585358

Lin, H., Mann, K. J., Starostina, E., Kinser, R. D. and Pikielny, C. W. (2005). A Drosophila DEG/ENaC channel subunit is required for male response to female pheromones. Proc. Natl. Acad. Sci. 102(36): 12831-6. 16129837

Lin, H. H., Cao, D. S., Sethi, S., Zeng, Z., Chin, J. S. R., Chakraborty, T. S., Shepherd, A. K., Nguyen, C. A., Yew, J. Y., Su, C. Y. and Wang, J. W. (2016). Hormonal modulation of pheromone detection enhances male courtship success. Neuron 90(6): 1272-1285. PubMed ID: 27263969

Lin, W., Finger, T. E., Rossier, B. C. and Kinnamon, S. C. (1999). Epithelial Na+ channel subunits in rat taste cells: localization and regulation by aldosterone. J. Comp. Neurol. 405: 406-420. 10076935

Lin, W., Ogura, T. & Kinnamon, S. C. (2002). Acid-activated cation currents in rat vallate taste receptor cells. J. Neurophysiol. 88: 133-141. 12091539

Liu, L., Leonard, A. S., Motto, D. G., Feller, M. A., Price, M. P., Johnson, W. A. and Welsh, M. J. (2003). Contribution of Drosophila DEG/ENaC genes to salt taste. Neuron 39: 133-146. 12848938

Liu, T., Starostina, E., Vijayan, V. and Pikielny, C. W. (2012). Two Drosophila DEG/ENaC channel subunits have distinct functions in gustatory neurons that activate male courtship. J. Neurosci. 32(34): 11879-89. PubMed Citation: 22915128

Lu, B., LaMora, A., Sun, Y., Welsh, M. J. and Ben-Shahar, Y. (2012). ppk23-Dependent chemosensory functions contribute to courtship behavior in Drosophila melanogaster. PLoS Genet. 8(3): e1002587. PubMed Citation: 22438833

Mano, I. and Driscoll, M. (1999). DEG/ENaC channels: a touchy superfamily that watches its salt. Bioessays 21(7): 568-78. 10472184

Mast, J. D., De Moraes, C. M., Alborn, H. T., Lavis, L. D. and Stern, D. L. (2014). Evolved differences in larval social behavior mediated by novel pheromones. Elife 3: e04205. PubMed ID: 25497433

Mellert, D. J., et al. (2010). Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors. Development 137: 323-332. PubMed Citation: 20040498

Ng, R., Salem, S. S., Wu, S. T., Wu, M., Lin, H. H., Shepherd, A. K., Joiner, W. J., Wang, J. W. and Su, C. Y. (2019). Amplification of Drosophila olfactory responses by a DEG/ENaC Channel. Neuron 104(5): 947-959. PubMed ID: 31629603

O'Hagan, R., Chalfie, M., Goodman, M. B. (2005). The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals. Nat. Neurosci. 8(1): 43-50. 15580270

Parks, A. L., et al. (2004). Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat. Genet. 36: 288-292. 14981519

Park, S. K., et al. (2006). A Drosophila protein specific to pheromone-sensing gustatory hairs delays males' copulation attempts. Curr. Biol. 16: 1154-1159. PubMed Citation: 16753571

Poët, M., et al. (2001). Exploration of the pore structure of a peptide-gated Na+ channel. EMBO J 20: 5595-5602. PubMed Citation: 11598003

Root C. M., et al. (2008) A presynaptic gain control mechanism fine-tunes olfactory behavior. Neuron 59: 311-321. PubMed Citation: 18667158

Silbering, A. F. and Benton, R. (2010). Ionotropic and metabotropic mechanisms in chemoreception: 'chance or design'? EMBO Rep 11: 173-179. PubMed Citation: 20111052

Starostina, E., et al. (2012). A Drosophila DEG/ENaC subunit functions specifically in gustatory neurons required for male courtship behavior. J Neurosci 32: 4665-4674. PubMed Citation: 22457513

Thibault, S. T., et al. (2004). A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat. Genet. 36: 283-287. 14981521

Thistle, R., Cameron, P., Ghorayshi, A., Dennison, L. and Scott, K. (2012). Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship. Cell 149(5): 1140-1151. PubMed ID: 22632976

Thistle, R., Cameron, P., Ghorayshi, A., Dennison, L. and Scott, K. (2012). Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship. Cell 149(5): 1140-51. PubMed Citation: 22632976

Toda, H., Zhao, X. and Dickson, B. J. (2012). The Drosophila female aphrodisiac pheromone activates ppk23(+) sensory neurons to elicit male courtship behavior. Cell Rep. 1(6): 599-607. PubMed Citation: 22813735

Voglis, G. and Tavernarakis, N. (2008). A synaptic DEG/ENaC ion channel mediates learning in C. elegans by facilitating dopamine signalling. EMBO J 27: 3288-3299. PubMed Citation: 19037257

Xu, A., Park, S. K., D'Mello, S., Kim, E., Wang, Q. and Pikielny, C. W. (2002). Novel genes expressed in subsets of chemosensory sensilla on the front legs of male Drosophila melanogaster. Cell Tissue Res. 307: 381-392. 11904775

Yu, Y., et al. (2010). A nonproton ligand sensor in the acid-sensing ion channel. Neuron 68: 61-72. PubMed Citation: 20920791

Yuan, Q., Song, Y., Yang, C. H., Jan, L. Y. and Jan, Y. N. (2013). Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila. Nat Neurosci 17(1): 81-8. PubMed ID: 24241395

date revised: 15 April 2020

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.