Drosophila TRPA channel painless inhibits male-male courtship behavior through modulating olfactory sensation

The Drosophila melanogaster TRPA family member painless, expressed in a subset of multidendritic neurons embeding in the larval epidermis, is necessary for larval nociception of noxious heat or mechanical stimuli. However, the function of painless in adult flies remains largely unknown. Here we report that mutation of painless leads to a defect in male-male courtship behavior and alteration in olfaction sensitivity in adult flies. Specific downregulation of the expression of the Painless protein in the olfactory projection neurons (PNs) of the antennal lobes (ALs) resulted in a phenotype resembling that found in painless mutant flies, whereas overexpression of Painless in PNs of painless mutant males suppressed male-male courtship behavior. The downregulation of Painless exclusively during adulthood also resulted in male-male courtship behavior. In addition, mutation of the painless gene in flies caused changes in olfaction, suggesting a role for this gene in olfactory processing. These results indicate that functions of painless in the adult central nervous system of Drosophila include modulation of olfactory processing and inhibition of male-male courtship behavior.     

Thermal nociception in adult Drosophila: behavioral characterization and the role of the painless gene

Nociception, warning of injury that should be avoided, serves an important protective function in animals. In this study, we show that adult Drosophila avoids noxious heat by a jump response. To quantitatively analyze this nociceptive behavior, we developed two assays. In the CO2 laser beam assay, flies exhibit this behavior when a laser beam heats their abdomens. The consistency of the jump latency in this assay meets an important criterion for a good nociceptive assay. In the hot plate assay, flies jump quickly to escape from a hot copper plate (>45 degrees C). Our results demonstrate that, as in mammals, the latency of the jump response is inversely related to stimulus intensity, and innoxious thermosensation does not elicit this nociceptive behavior. To explore the genetic mechanisms of nociception, we examined several mutants in both assays. Abnormal nociceptive behavior of a mutant, painless, indicates that painless, a gene essential for nociception in Drosophila larvae, is also required for thermal nociception in adult flies. painless is expressed in certain neurons of the peripheral nervous system and thoracic ganglia, as well as in the definite brain structures, the mushroom bodies. However, chemical or genetic insults to the mushroom bodies do not influence the nociceptive behavior, suggesting that different painless-expressing neurons play diverse roles in thermal nociception. Additionally, no-bridge(KS49), a mutant that has a structural defect in the protocerebral bridge, shows defective response to noxious heat. Thus, our results validate adult Drosophila as a useful model to study the genetic mechanisms of thermal nociception.     

The ankyrin repeat domain of the TRPA protein painless is important for thermal nociception but not mechanical nociception

The Drosophila TRPA channel Painless is required for the function of polymodal nociceptors which detect noxious heat and noxious mechanical stimuli. These functions of Painless are reminiscent of mammalian TRPA channels that have also been implicated in thermal and mechanical nociception. A popular hypothesis to explain the mechanosensory functions of certain TRP channels proposes that a string of ankyrin repeats at the amino termini of these channels acts as an intracellular spring that senses force. Here, we describe the identification of two previously unknown Painless protein isoforms which have fewer ankyrin repeats than the canonical Painless protein. We show that one of these Painless isoforms, that essentially lacks ankyrin repeats, is sufficient to rescue mechanical nociception phenotypes of painless mutant animals but does not rescue thermal nociception phenotypes. In contrast, canonical Painless, which contains Ankyrin repeats, is sufficient to largely rescue thermal nociception but is not capable of rescuing mechanical nociception. Thus, we propose that in the case of Painless, ankryin repeats are important for thermal nociception but not for mechanical nociception.     

Response of Drosophila to wasabi is mediated by painless, the fly homolog of mammalian TRPA1/ANKTM1

A number of repellent compounds produced by plants elicit a spicy or pungent sensation in mammals . In several cases, this has been found to occur through activation of ion channels in the transient receptor potential (TRP) family . We report that isothiocyanate (ITC), the pungent ingredient of wasabi, is a repellent to the insect Drosophila melanogaster, and that the painless gene, previously known to be required for larval nociception, is required for this avoidance behavior. A painless reporter gene is expressed in gustatory receptor neurons of the labial palpus, tarsus, and wing anterior margin, but not in olfactory receptor neurons, suggesting a gustatory role. Indeed, painless expression overlaps with a variety of gustatory-receptor gene reporters. Some, such as Gr66a, are known to be expressed in neurons that mediate gustatory repulsion . painless mutants are not taste blind; they show normal aversive gustatory behavior with salt and quinine and attractive responses to sugars and capsaicin. The painless gene is an evolutionary homolog of the mammalian "wasabi receptor" TRPA1/ANKTM1 , also thought to be involved in nociception. Our results suggest that the stinging sensation of isothiocyanate is caused by activation of an evolutionarily conserved molecular pathway that is also used for nociception.     

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.     

Local and global methods of assessing thermal nociception in Drosophila larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors while the second involves a wholesale (global) activation of most or all such neurons. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons. The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli in addition to their recently discovered role as photoreceptors. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system where they may connect to second-order neurons that project to the brain. Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.     

Fruit flies for anti-pain drug discovery

Recent work has indicated that fruit flies (Drosophila melanogaster) can be used in nociception research. Genetic screening identified a gene, painless, that is required for thermal and mechanical nociception in Drosophila larvae. On the other hand, pharmacological techniques and noxious heat were used to assay antinocieceptive behavior in intact adult Drosophila. In general, animal models for pain research are bound by ethical concerns. Since no serious ethical controversies have been raised regarding experiments in insects, Drosophila may be, for the time being an ethically acceptable animal model for combined genetic and pharmacological analgesia research.     

Growing pains: development of the larval nocifensive response in Drosophila

The ability to perceive and avoid harmful substances or stimuli is key to an organism's survival. The neuronal cognate of the perception of pain is known as nociception, and the reflexive motion to avoid pain is termed the nocifensive response. As the nocifensive response is an ancient and evolutionarily conserved behavioral response to nociceptive stimuli, it is amenable to study in relatively simple and genetically tractable model systems such as Drosophila. Recent studies have taken advantage of the useful properties of Drosophila larvae to begin elucidating the neuronal connectivity and molecular machinery underlying the nocifensive response. However, these studies have primarily utilized the third-instar larval stage, and many mutations that potentially influence nociception survive only until earlier larval stages. Here we characterize the nocifensive responses of Drosophila throughout larval development and find dramatic changes in the nature of the behavior. Notably, we find that prior to the third instar, larvae are unable to perform the characteristic "corkscrew-like roll" behavior. Also, we identify an avoidance behavior consistent with a nocifensive response that is present immediately after larval hatching, representing a paradigm that may be useful in examining mutations with an early lethal phenotype.     

Optogenetic manipulation of neural circuits and behavior in Drosophila larvae

Optogenetics is a powerful tool that enables the spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third-larval instar. We have previously shown that, using spatially defined GAL4 drivers and potent UAS (upstream activation sequence)-channelrhodopsin-2∷YFP transgenic strains developed in our laboratory, it is possible to manipulate neuronal populations in response to illumination by blue light and to test whether the activation of defined neural circuits is sufficient to shape behaviors of interest. Although we have only used the protocol described here in larval stages, the procedure can be adapted to study neurons in adult flies--with the caveat that blue light may not sufficiently penetrate the adult cuticle to stimulate neurons deep in the brain. This procedure takes 1 week to culture optogenetic flies and ~1 h per group for the behavioral assays.     

Thermosensation: hot findings make TRPNs very cool

TRPV and TRPM proteins have been shown to form temperature-responsive cation channels that act in nociception. Recent work on the mouse ANKTM1 and Drosophila Painless proteins shows that members of a third TRP subfamily, TRPN, also function in thermosensation.     

Drosophila nociceptors mediate larval aversion to dry surface environments utilizing both the painless TRP channel and the DEG/ENaC subunit, PPK1

A subset of sensory neurons embedded within the Drosophila larval body wall have been characterized as high-threshold polymodal nociceptors capable of responding to noxious heat and noxious mechanical stimulation. They are also sensitized by UV-induced tissue damage leading to both thermal hyperalgesia and allodynia very similar to that observed in vertebrate nociceptors. We show that the class IV multiple-dendritic(mdIV) nociceptors are also required for a normal larval aversion to locomotion on to a dry surface environment. Drosophila melanogaster larvae are acutely susceptible to desiccation displaying a strong aversion to locomotion on dry surfaces severely limiting the distance of movement away from a moist food source. Transgenic inactivation of mdIV nociceptor neurons resulted in larvae moving inappropriately into regions of low humidity at the top of the vial reflected as an increased overall pupation height and larval desiccation. This larval lethal desiccation phenotype was not observed in wild-type controls and was completely suppressed by growth in conditions of high humidity. Transgenic hyperactivation of mdIV nociceptors caused a reciprocal hypersensitivity to dry surfaces resulting in drastically decreased pupation height but did not induce the writhing nocifensive response previously associated with mdIV nociceptor activation by noxious heat or harsh mechanical stimuli. Larvae carrying mutations in either the Drosophila TRP channel, Painless, or the degenerin/epithelial sodium channel subunit Pickpocket1(PPK1), both expressed in mdIV nociceptors, showed the same inappropriate increased pupation height and lethal desiccation observed with mdIV nociceptor inactivation. Larval aversion to dry surfaces appears to utilize the same or overlapping sensory transduction pathways activated by noxious heat and harsh mechanical stimulation but with strikingly different sensitivities and disparate physiological responses.     

A novel thermosensitive escape behavior in Drosophila larvae

We describe a novel thermosensitive escape behavior in Drosophila larvae and a simple assay to accurately define the response temperature. When a larva is placed in a droplet of water that is subsequently heated, a stereotypical escape response is robustly elicited at 29°C. Larvae defective for the painless TRP receptor, or blocked in the function of class IV multi-dendritic sensory dendrites respond to this stimulus at reproducibly higher temperature (34°C). The escape response has novel behavioral components and a lower temperature threshold in comparison with the responses to touch with a hot needle. Furthermore the assay minimizes operator bias that is present in current tests of thermosensitive nociception and generates a precise determination of temperature at the point of response. This response is highly reproducible and directly applicable to genetic and neural circuit analysis of a simple escape behavior.     

Larval Defense against Attack from Parasitoid Wasps Requires Nociceptive Neurons

Parasitoid wasps are a fierce predator of Drosophila larvae. Female Leptopilina boulardi (LB) wasps use a sharp ovipositor to inject eggs into the bodies of Drosophila melanogaster larvae. The wasp then eats the Drosophila larva alive from the inside, and an adult wasp ecloses from the Drosophila pupal case instead of a fly. However, the Drosophila larvae are not defenseless as they may resist the attack of the wasps through somatosensory-triggered behavioral responses. Here we describe the full range of behaviors performed by the larval prey in immediate response to attacks by the wasps. Our results suggest that Drosophila larvae primarily sense the wasps using their mechanosensory systems. The range of behavioral responses included both “gentle touch” like responses as well as nociceptive responses. We found that the precise larval response depended on both the somatotopic location of the attack, and whether or not the larval cuticle was successfully penetrated during the course of the attack. Interestingly, nociceptive responses are more likely to be triggered by attacks in which the cuticle had been successfully penetrated by the wasp. Finally, we found that the class IV neurons, which are necessary for mechanical nociception, were also necessary for a nociceptive response to wasp attacks. Thus, the class IV neurons allow for a nociceptive behavioral response to a naturally occurring predator of Drosophila.     

Thermosensation and pain

We feel a wide range of temperatures spanning from cold to heat. Within this range, temperatures over about 43 degrees C and below about 15 degrees C evoke not only a thermal sensation, but also a feeling of pain. In mammals, six thermosensitive ion channels have been reported, all of which belong to the TRP (transient receptor potential) superfamily. These include TRPV1 (VR1), TRPV2 (VRL-1), TRPV3, TRPV4, TRPM8 (CMR1), and TRPA1 (ANKTM1). These channels exhibit distinct thermal activation thresholds (>43 degrees C for TRPV1, >52 degrees C for TRPV2, > approximately 34-38 degrees C for TRPV3, > approximately 27-35 degrees C for TRPV4, < approximately 25-28 degrees C for TRPM8 and <17 degrees C for TRPA1), and are expressed in primary sensory neurons as well as other tissues. The involvement of TRPV1 in thermal nociception has been demonstrated by multiple methods, including the analysis of TRPV1-deficient mice. TRPV2, TRPM8, and TRPA1 are also very likely to be involved in thermal nociception, because their activation thresholds are within the noxious range of temperatures.     

A novel thermosensitive escape behavior in Drosophila larvae

We describe a novel thermosensitive escape behavior in Drosophila larvae and a simple assay to accurately define the response temperature. When a larva is placed in a droplet of water that is subsequently heated, a stereotypical escape response is robustly elicited at 29°C. Larvae defective for the painless TRP receptor, or blocked in the function of class IV multi-dendritic sensory dendrites respond to this stimulus at reproducibly higher temperature (34°C). The escape response has novel behavioral components and a lower temperature threshold in comparison with the responses to touch with a hot needle. Furthermore the assay minimizes operator bias that is present in current tests of thermosensitive nociception and generates a precise determination of temperature at the point of response. This response is highly reproducible and directly applicable to genetic and neural circuit analysis of a simple escape behavior.     

Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit

BACKGROUND: Drosophila larvae possess only 21 odorant-receptor neurons (ORNs), whereas adults have 1,300. Does this suggest that the larval olfactory system is built according to a different design than its adult counterpart, or is it just a miniature version thereof? RESULTS: By genetically labeling single neurons with FLP-out and MARCM techniques, we analyze the connectivity of the larval olfactory circuit. We show that each of the 21 ORNs is unique and projects to one of 21 morphologically identifiable antennal-lobe glomeruli. Each glomerulus seems to be innervated by a single projection neuron. Each projection neuron sends its axon to one or two of about 28 glomeruli in the mushroom-body calyx. We have discovered at least seven types of projection neurons that stereotypically link an identified antennal-lobe glomerulus with an identified calycal glomerulus and thus create an olfactory map in a higher brain center. CONCLUSIONS: The basic design of the larval olfactory system is similar to the adult one. However, ORNs and projection neurons lack cellular redundancy and do not exhibit any convergent or divergent connectivity; 21 ORNs confront essentially similar numbers of antennal-lobe glomeruli, projection neurons, and calycal glomeruli. Hence, we propose the Drosophila larva as an "elementary" olfactory model system.     

Nociceptive neurons protect Drosophila larvae from parasitoid wasps

BACKGROUND: Natural selection has resulted in a complex and fascinating repertoire of innate behaviors that are produced by insects. One puzzling example occurs in fruit fly larvae that have been subjected to a noxious mechanical or thermal sensory input. In response, the larvae "roll" with a motor pattern that is completely distinct from the style of locomotion that is used for foraging. RESULTS: We have precisely mapped the sensory neurons that are used by the Drosophila larvae to detect nociceptive stimuli. By using complementary optogenetic activation and targeted silencing of sensory neurons, we have demonstrated that a single class of neuron (class IV multidendritic neuron) is sufficient and necessary for triggering the unusual rolling behavior. In addition, we find that larvae have an innately encoded preference in the directionality of rolling. Surprisingly, the initial direction of rolling locomotion is toward the side of the body that has been stimulated. We propose that directional rolling might provide a selective advantage in escape from parasitoid wasps that are ubiquitously present in the natural environment of Drosophila. Consistent with this hypothesis, we have documented that larvae can escape the attack of Leptopilina boulardi parasitoid wasps by rolling, occasionally flipping the attacker onto its back. CONCLUSIONS: The class IV multidendritic neurons of Drosophila larvae are nociceptive. The nociception behavior of Drosophila melanagaster larvae includes an innately encoded directional preference. Nociception behavior is elicited by the ecologically relevant sensory stimulus of parasitoid wasp attack.     

Delta expression in post-mitotic neurons identifies distinct subsets of adult-specific lineages in Drosophila

The Drosophila ventral nerve cord is comprised of numerous neuronal lineages, each derived from a stereotypically positioned neuroblast (NB). At the embryonic stage the unique identities of each NB, and several of their neuronal progeny, are well characterized by spatial and temporal expression patterns of molecular markers. These patterns of expression are not preserved at the larval stage and thus the identity of adult-specific lineages remains obscure. Recent clonal analysis using MARCM has identified 24 adult-specific lineages arising from thoracic NBs at the larval stage. In this study, we have explored a role for the Delta protein in development of the post-embryonic Drosophila ventral nerve cord. We find that Delta expression identifies 7 of the 24 adult-specific lineages of the thoracic ganglia by being highly enriched in clusters of newly born post-mitotic neurons and their neurite bundles. The Delta lineages constitute the majority of bundles projecting to the ventral neuropil, consistent with a role in processing leg sensory information. Targeted knockdown of Delta in neurons using RNAi results in significantly decreased leg chemosensory response and a relatively unaffected leg mechanosensory response. Delta RNAi knockdown in Delta lineages also gives a more diffuse bundle terminal morphology while the overall path-finding of neurite bundles is unaffected. We also identify a male-specific Delta lineage in the terminal abdominal ganglia, implicating a role for Delta in development of sexually dimorphic neural networks. Examples of Delta-expressing neurites contacting Notch-expressing glia are also seen, but are not common to all Delta lineages. Altogether, these data reveal a fundamental pattern of Delta expression that is indicative of an underlying developmental program that confers identity to adult lineage neurons.     

Genetic dissection of trophic interactions in the larval optic neuropil of Drosophila melanogaster

The larval visual system of Drosophila melanogaster consists of two bilateral clusters of 12 photoreceptors, which express Rhodopsin 5 and 6 (Rh5 and Rh6) in a non-overlapping manner. These neurons send their axons in a fascicle, the larval optic nerve (LON), which terminates in the larval optic neuropil. The LON is required for the development of a serotonergic arborization originating in the central brain and for the development of the dendritic tree of the circadian pacemakers, the small ventral lateral neurons (LNv) [Malpel, S., Klarsfeld, A., Rouyer, F., 2002. Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development. Development 129, 1443-1453; Mukhopadhyay, M., Campos, A.R., 1995. The larval optic nerve is required for the development of an identified serotonergic arborization in Drosophila melanogaster. Dev. Biol., 169, 629-643]. Here, we show that both Rh5- and Rh6-expressing fibers overlap equally with the 5-HT arborization and that it, in turn, also contacts the dendritic tree of the LNv. The experiments described here aimed at determining whether Rh5- or Rh6-expressing fibers, as well as the LNv, influence the development of this serotonergic arborization. We conclude that Rh6-expressing fibers play a unique role in providing a signal required for the outgrowth and branching of the serotonergic arborization. Moreover, the innervation of the larval optic neuropil by the 5-HT arborization depends on intact Rac function. A possible role for these serotonergic processes in modulating the larval circadian rhythmicity and photoreceptor function is discussed.