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. Here, we demonstrate 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.     

Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity

As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating.     

ppk23-Dependent chemosensory functions contribute to courtship behavior in Drosophila melanogaster

Insects utilize diverse families of ion channels to respond to environmental cues and control mating, feeding, and the response to threats. Although degenerin/epithelial sodium channels (DEG/ENaC) represent one of the largest families of ion channels in Drosophila melanogaster, the physiological functions of these proteins are still poorly understood. We found that the DEG/ENaC channel ppk23 is expressed in a subpopulation of sexually dimorphic gustatory-like chemosensory bristles that are distinct from those expressing feeding-related gustatory receptors. Disrupting ppk23 or inhibiting activity of ppk23-expressing neurons did not alter gustatory responses. Instead, blocking ppk23-positive neurons or mutating the ppk23 gene delayed the initiation and reduced the intensity of male courtship. Furthermore, mutations in ppk23 altered the behavioral response of males to the female-specific aphrodisiac pheromone 7(Z), 11(Z)-Heptacosadiene. Together, these data indicate that ppk23 and the cells expressing it play an important role in the peripheral sensory system that determines sexual behavior in Drosophila.     

The receptor channel formed by ppk25, ppk29 and ppk23 can sense the Drosophila female pheromone 7,11‐heptacosadiene

In Drosophila, pheromones play a crucial role in regulating courtship behaviors. In males, female aphrodisiac pheromones promote male‐female courtship, and male antiaphrodisiac pheromones inhibit male‐male courtship. Previous studies have reported that receptor proteins belonging to the pickpocket (ppk) family, ionotropic receptor family and gustatory receptor family are required for pheromone detection and normal courtship. However, none of them has been shown to be sufficient for sensing pheromones after ectopic expression in originally unresponsive cells. “M” cells are activated by male antiaphrodisiac pheromones but not female aphrodisiac pheromones, and the activated cells inhibit male‐male courtship. In our study, male flies with ectopic expression of ppk25, ppk29 and ppk23 in “M” cells showed decreased male‐female courtship. Using an in vivo calcium imaging approach, we found that the “M” cells expressing these three ppks were significantly activated by the female aphrodisiac pheromone 7,11‐heptacosadiene (7,11‐HD). Our results indicate that a sodium channel consisting, at minimum, of ppk25, ppk29 and ppk23, can sense 7,11‐HD, most likely as a receptor. Our findings may help us gain insights into the molecular mechanisms of pheromonal functions.     

Pickpocket1 Is an Ionotropic Molecular Sensory Transducer

The molecular transformation of an external stimulus into changes in sensory neuron activity is incompletely described. Although a number of molecules have been identified that can respond to stimuli, evidence that these molecules can transduce stimulation into useful neural activity is lacking. Here we demonstrate that pickpocket1 (ppk1), a Drosophila homolog of mammalian Degenerin/epithelial sodium channels, encodes an acid-sensing sodium channel that conducts a transient depolarizing current in multidendritic sensory neurons of Drosophila melanogaster. Stimulation of Ppk1 is sufficient to bring these sensory neurons to threshold, eliciting a burst of action potentials. The transient nature of the neural activity produced by Ppk1 activation is the result of Ppk1 channel gating properties. This model is supported by the observation of enhanced bursting activity in neurons expressing a gain of function ppk1 mutant harboring the degenerin mutation. These findings demonstrate that Ppk1 can function as an ionotropic molecular sensory transducer capable of transforming the perception of a stimulus into phasic neuronal activity in sensory neurons.     

Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1

Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders.     

The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females

Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless ( pain ) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. In this study, we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNA interference (RNAi) in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. However, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity.     

Impairment of proprioceptive movement and mechanical nociception in Drosophila melanogaster larvae lacking Ppk30, a Drosophila member of the Degenerin/Epithelial Sodium Channel family

The mechanosensory neurons of Drosophila larvae are demonstrably activated by diverse mechanical stimuli, but the mechanisms underlying this function are not completely understood. Here we report a genetic, immunohistochemical, and electrophysiological analysis of the Ppk30 ion channel, a member of the Drosophila pickpocket (ppk) family, counterpart of the mammalian Degenerin/Epithelial Na⁺ Channel family. Ppk30 mutant larvae displayed deficits in proprioceptive movement and mechanical nociception, which are detected by class IV sensory (mdIV) neurons. The same neurons also detect heat nociception, which was not impaired in ppk30 mutant larvae. Similarly, Ppk30 mutation did not alter gentle touch mechanosensation, a distinct mechanosensation detected by other neurons, suggesting that Ppk30 has a functional role in mechanosensation in mdIV neurons. Consistently, Ppk30 was expressed in class IV neurons, but was not detectable in other larval skin sensory neurons. Mutant phenotypes were rescued by expressing Ppk30 in mdIV neurons. Electrophysiological analysis of heterologous cells expressing Ppk30 did not detect mechanosensitive channel activities, but did detect acid‐induced currents. These data show that Ppk30 has a role in mechanosensation, but not in thermosensation, in class IV neurons, and possibly has other functions related to acid response.     

The mushroom body D1 dopamine receptor controls innate courtship drive

Mating is critical for species survival and is profoundly regulated by neuromodulators and neurohormones to accommodate internal states and external factors. To identify the underlying neuromodulatory mechanisms, we investigated the roles of dopamine receptors in various aspects of courtship behavior in Drosophila. Here, we report that the D1 dopamine receptor dDA1 regulates courtship drive in naïve males. The wild‐type naïve males actively courted females regardless their appearance or mating status. On the contrary, the dDA1 mutant (dumb) males exhibited substantially reduced courtship toward less appealing females including decapitated, leg‐less and mated females. The dumb male's reduced courtship activity was due to delay in courtship initiation and prolonged intervals between courtship bouts. The dampened courtship drive of dumb males was rescued by reinstated dDA1 expression in the mushroom body α/β and γ neurons but not α/β or γ neurons alone, which is distinct from the previously characterized dDA1 functions in experience‐dependent courtship or other learning and memory processes. We also found that the dopamine receptors dDA1, DAMB and dD2R are dispensable for associative memory formation and short‐term memory of conditioned courtship, thus courtship motivation and associative courtship learning and memory are regulated by distinct neuromodulatory mechanisms. Taken together, our study narrows the gap in the knowledge of the mechanism that dopamine regulates male courtship behavior.     

Courtship initiation is stimulated by acoustic signals in Drosophila melanogaster

Finding a mating partner is a critical task for many organisms. It is in the interest of males to employ multiple sensory modalities to search for females. In Drosophila melanogaster, vision is thought to be the most important courtship stimulating cue at long distance, while chemosensory cues are used at relatively short distance. In this report, we show that when visual cues are not available, sounds produced by the female allow the male to detect her presence in a large arena. When the target female was artificially immobilized, the male spent a prolonged time searching before starting courtship. This delay in courtship initiation was completely rescued by playing either white noise or recorded fly movement sounds to the male, indicating that the acoustic and/or seismic stimulus produced by movement stimulates courtship initiation, most likely by increasing the general arousal state of the male. Mutant males expressing tetanus toxin (TNT) under the control of Gr68a-GAL4 had a defect in finding active females and a delay in courtship initiation in a large arena, but not in a small arena. Gr68a-GAL4 was found to be expressed pleiotropically not only in putative gustatory pheromone receptor neurons but also in mechanosensory neurons, suggesting that Gr68a-positive mechanosensory neurons, not gustatory neurons, provide motion detection necessary for courtship initiation. TNT/Gr68a males were capable of discriminating the copulation status and age of target females in courtship conditioning, indicating that female discrimination and formation of olfactory courtship memory are independent of the Gr68a-expressing neurons that subserve gustation and mechanosensation. This study suggests for the first time that mechanical signals generated by a female fly have a prominent effect on males' courtship in the dark and leads the way to studying how multimodal sensory information and arousal are integrated in behavioral decision making.     

Visual sex discrimination in goldfish: seasonal, sexual, and androgenic influences

The olfactory signals used by goldfish for sexual and aggressive communication have been studied extensively, but little work has addressed the role of other sensory modalities in social communication in this species. We therefore investigated the role that visual stimuli play in sex discrimination and the ability of androgens, which masculinize courtship behavior, to affect behavioral responses toward female visual stimuli. We found that males selectively orient toward female visual stimuli during the breeding season but not outside it, whereas prostaglandin F2-alpha (PGF2alpha)-injected females do not differentially approach male and female visual stimuli, even during the breeding season. Implanting adult females with testosterone (T) and 11-ketotestosterone (KT), however, induced orientation responses toward female visual stimuli similar to those observed in males. These results indicate that visual sexual stimuli are likely important for reproductive signaling in goldfish, potentially helping males identify ovulating females from a distance in a shoal of fish, and that androgens can influence mechanisms associated with orientation responses toward such stimuli.     

Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in Drosophila melanogaster

A male-specific component, 11-cis-vaccenyl acetate (cVA) works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. In this study, we demonstrate that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABA_A receptors contributed to this olfactory habituation. We also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, we illustrate a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment.     

Drosophila retained/dead ringer is necessary for neuronal pathfinding, female receptivity and repression of fruitless independent male courtship behaviors

Mutations in the Drosophila retained/dead ringer (retn) gene lead to female behavioral defects and alter a limited set of neurons in the CNS. retn is implicated as a major repressor of male courtship behavior in the absence of the fruitless (fru) male protein. retn females show fru-independent male-like courtship of males and females, and are highly resistant to courtship by males. Males mutant for retn court with normal parameters, although feminization of retn cells in males induces bisexuality. Alternatively spliced RNAs appear in the larval and pupal CNS, but none shows sex specificity. Post-embryonically, retn RNAs are expressed in a limited set of neurons in the CNS and eyes. Neural defects of retn mutant cells include mushroom body beta-lobe fusion and pathfinding errors by photoreceptor and subesophageal neurons. We posit that some of these retn-expressing cells function to repress a male behavioral pathway activated by fruM.     

The hector G-protein coupled receptor is required in a subset of fruitless neurons for male courtship behavior

Male courtship behavior in Drosophila melanogaster is controlled by two main regulators, fruitless (fru) and doublesex (dsx). Their sex-specific expression in brain neurons has been characterized in detail, but little is known about the downstream targets of the sex-specific FRU and DSX proteins and how they specify the function of these neurons. While sexual dimorphism in the number and connections of fru and dsx expressing neurons has been observed, a majority of the neurons that express the two regulators are present in both sexes. This poses the question which molecules define the sex-specific function of these neurons. Signaling molecules are likely to play a significant role. We have identified a predicted G-protein coupled receptor (GPCR), CG4395, that is required for male courtship behavior. The courtship defect in the mutants can be rescued by expression of the wildtype protein in fru neurons of adult males. The GPCR is expressed in a subset of fru-positive antennal glomeruli that have previously been shown to be essential for male courtship. Expression of 4395-RNAi in GH146 projection neurons lowers courtship. This suggests that signaling through the CG4395 GPCR in this subset of fru neurons is critical for male courtship behavior.     

The neuropeptide SIFamide modulates sexual behavior in Drosophila

The expression of Drosophila neuropeptide AYRKPPFNGSIFamide (SIFamide) was shown by both immunohistology and in situ hybridization to be restricted to only four neurons of the pars intercerebralis. The role of SIFamide in adult courtship behavior in both sexes was studied using two different approaches to perturb the function of SIFamide; targeted cell ablation and RNA interference (RNAi). Elimination of SIFamide by either of these methods results in promiscuous flies; males perform vigorous and indiscriminant courtship directed at either sex, while females appear sexually hyper-receptive. These results demonstrate that SIFamide is responsible for these behavioral effects and that the four SIFamidergic neurons and arborizations play an important function in the neuronal circuitry controlling Drosophila sexual behavior.     

The fragile X-related gene affects the crawling behavior of Drosophila larvae by regulating the mRNA level of the DEG/ENaC protein pickpocket1

BACKGROUND: Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 (FMR1) gene. How FMR1 affects the function of the central and peripheral nervous systems is still unclear. FMR1 is an RNA binding protein that associates with a small percentage of total mRNAs in vivo. It remains largely unknown what proteins encoded by mRNAs in the FMR1-messenger ribonuclear protein (mRNP) complex are most relevant to the affected physiological processes. RESULTS: Loss-of-function mutations in the Drosophila fragile X-related (dfmr1) gene, which is highly homologous to the human fmr1 gene, decrease the duration and percentage of time that crawling larvae spend on linear locomotion. Overexpression of DFMR1 in multiple dendritic (MD) sensory neurons increases the time percentage and duration of linear locomotion; this phenotype is similar to that caused by reduced expression of the MD neuron subtype-specific degenerin/epithelial sodium channel (DEG/ENaC) family protein Pickpocket1 (PPK1). Genetic analyses indicate that PPK1 is a key component downstream of DFMR1 in controlling the crawling behavior of Drosophila larvae. DFMR1 and ppk1 mRNA are present in the same mRNP complex in vivo and can directly bind to each other in vitro. DFMR1 downregulates the level of ppk1 mRNA in vivo, and this regulatory process also involves Argonaute2 (Ago2), a key component in the RNA interference pathway. CONCLUSIONS: These studies identify ppk1 mRNA as a physiologically relevant in vivo target of DFMR1. Our finding that the level of ppk1 mRNA is regulated by DFMR1 and Ago2 reveals a genetic pathway that controls sensory input-modulated locomotion behavior.     

Identification of guanylyl cyclases that function in thermosensory neurons of Caenorhabditis elegans

The nematode Caenorhabditis elegans senses temperature primarily via the AFD thermosensory neurons in the head. The response to temperature can be observed as a behavior called thermotaxis on thermal gradients. It has been shown that a cyclic nucleotide-gated ion channel (CNG channel) plays a critical role in thermosensation in AFD. To further identify the thermosensory mechanisms in AFD, we attempted to identify components that function upstream of the CNG channel by a reverse genetic approach. Genetic and behavioral analyses showed that three members of a subfamily of gcy genes (gcy-8, gcy-18, and gcy-23) encoding guanylyl cyclases were essential for thermotaxis in C. elegans. Promoters of each gene drove reporter gene expression exclusively in the AFD neurons and, moreover, tagged proteins were localized to the sensory endings of AFD. Single mutants of each gcy gene showed almost normal thermotaxis. However, animals carrying double and triple mutations in these genes showed defective thermotaxis behavior. The abnormal phenotype of the gcy triple mutants was rescued by expression of any one of the three GCY proteins in the AFD neurons. These results suggest that three guanylyl cyclases function redundantly in the AFD neurons to mediate thermosensation by C. elegans.     

Female contact activates male-specific interneurons that trigger stereotypic courtship behavior in Drosophila

We determined the cellular substrate for male courtship behavior by quasinatural and artificial stimulation of brain neurons. Activation of fruitless (fru)-expressing neurons via stimulation of thermosensitive dTrpA1 channels induced an entire series of courtship acts in male Drosophila placed alone without any courting target. By reducing the number of neurons expressing dTrpA1 by MARCM, we demonstrated that the initiation of courtship behavior is significantly correlated with the activation of the transmidline P1 interneurons, the descending P2b interneurons, or both, indicating that these interneurons trigger courtship. Using an experimental paradigm in which a tethered male can be stimulated to initiate courtship by touching his foreleg tarsus to a female's abdomen, we found that P1 neurites of tethered males showed a transient Ca(2+) rise after tarsal stimulation with the female-associated sensory cues. These observations strongly suggest that P1 neurons are the prime components of the neural circuitry that initiates male courtship.