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.     

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.     

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.     

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.     

The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice.

Cation channels in the DEG/ENaC family are proposed to detect cutaneous stimuli in mammals. We localized one such channel, DRASIC, in several different specialized sensory nerve endings of skin, suggesting it might participate in mechanosensation and/or acid-evoked nociception. Disrupting the mouse DRASIC gene altered sensory transduction in specific and distinct ways. Loss of DRASIC increased the sensitivity of mechanoreceptors detecting light touch, but it reduced the sensitivity of a mechanoreceptor responding to noxious pinch and decreased the response of acid- and noxious heat-sensitive nociceptors. The data suggest that DRASIC subunits participate in heteromultimeric channel complexes in sensory neurons. Moreover, in different cellular contexts, DRASIC may respond to mechanical stimuli or to low pH to mediate normal touch and pain sensation.     

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.     

painless,a Drosophila gene essential for nociception

We describe a paradigm for nociception in Drosophila. In response to the touch of a probe heated above 38 degrees C, Drosophila larvae produce a stereotypical rolling behavior, unlike the response to an unheated probe. In a genetic screen for mutants defective in this noxious heat response, we identified the painless gene. Recordings from wild-type larval nerves identified neurons that initiated strong spiking above 38 degrees C, and this activity was absent in the painless mutant. The painless mRNA encodes a protein of the transient receptor potential ion channel family. Painless is required for both thermal and mechanical nociception, but not for sensing light touch. painless is expressed in peripheral neurons that extend multiple branched dendrites beneath the larval epidermis, similar to vertebrate pain receptors. An antibody to Painless binds to localized dendritic structures that we hypothesize are involved in nociceptive signaling.     

TrpA1 regulates thermal nociception in Drosophila

Pain is a significant medical concern and represents a major unmet clinical need. The ability to perceive and react to tissue-damaging stimuli is essential in order to maintain bodily integrity in the face of environmental danger. To prevent damage the systems that detect noxious stimuli are therefore under strict evolutionary pressure. We developed a high-throughput behavioral method to identify genes contributing to thermal nociception in the fruit fly and have reported a large-scale screen that identified the Ca²⁺ channel straightjacket (stj) as a conserved regulator of thermal nociception. Here we present the minimal anatomical and neuronal requirements for Drosophila to avoid noxious heat in our novel behavioral paradigm. Bioinformatics analysis of our whole genome data set revealed 23 genes implicated in Ca²⁺ signaling that are required for noxious heat avoidance. One of these genes, the conserved thermoreceptor TrpA1, was confirmed as a bona fide “pain” gene in both adult and larval fly nociception paradigms. The nociceptive function of TrpA1 required expression within the Drosophila nervous system, specifically within nociceptive multi-dendritic (MD) sensory neurons. Therefore, our analysis identifies the channel TRPA1 as a conserved regulator of nociception.     

Mutant molecular motors disrupt neural circuits in Drosophila

A dominant negative mutation, Glued¹, that codes for a component of the dynactin complex, disrupted the axonal anatomy of leg sensory neurons in Drosophila. To examine neuron structure in mutant animals, a P[Gal4] enhancer trap targeted expression of lacZ to the sensory neurons and thereby labeled neurons in the femoral chordotonal organ and their axons within the central nervous system. When these sensory axons were examined in the Glued¹ mutant specimens, they were observed to arborize abnormally. This anatomical disruption of the sensory axons was associated with a corresponding disruption in a reflex. Normally, the tibial extensor motor neurons were excited when the femoral-tibial joint was flexed, but this resistance reflex was nearly absent in mutant animals. We used the P[Gal4] insertion strains to target expression of tetanus toxin light chain to these sensory neurons in wild-type animals and showed that this blocked the resistance reflex and produced a phenocopy of the Glued result. We conclude that disruption of the dynein-dynactin complex disrupts sensory axon path finding during metamorphosis, and this in turn disrupts synaptic connectivity. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 711–723, 1997     

Trigeminal-Rostral Ventromedial Medulla circuitry is involved in orofacial hyperalgesia contralateral to tissue injury

Background

Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. ?? and ??ENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

Results

Here we show that deleting ?? and ??ENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.

Conclusion

Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.     

Cells expressing Desiccate are essential for morphogenesis of labial sensilla in Drosophila melanogaster adults

We recently discovered a new gene, Desiccate (Desi), that is expressed in the epidermis and protects larvae from desiccation stress in Drosophila melanogaster. In the present study, we found that taste organs express more Desi than the epidermis both in larvae and adults. Green fluorescent protein (GFP) expression in larvae under the direction of a Desi promoter‐Gal4 line containing the 1,010‐bp 5′ flanking region of Desi produced no signal in the epidermis but strong signals in cells of the larval gustatory sense organs, indicating that this driver works specifically in the gustatory organs. In adults, GFP expression was also observed in basal cells of sensilla on labella, tarsi and wings. More precise morphological analysis of GFP expression located its expression in the outer accessory cells rather than neurons of the labial sensilla. Although Desi knockdown or induction of cell death in Desi‐expressing cells did not change the morphological or physiological characters of the larvae, larvae lacking Desi‐expressing cells failed to metamorphose normally, and all of them died inside puparia. Dying pharate adults were found to lack all labial sensilla. The proneural genes Achaete and Scute, which are involved in the development of the adult central and peripheral nervous system, were normally expressed in the pupae lacking Desi‐expressing cells. These results suggested that the lack of Desi‐expressing cells makes it impossible to produce outer accessory cells for development of the sensilla, thereby signifying that cells expressing Desi are essential for normal morphogenesis of the labial sensilla in Drosophila adults.     

Role of Sensory Experience in Functional Development of Drosophila Motor Circuits

Neuronal circuits are formed according to a genetically predetermined program and then reconstructed in an experience-dependent manner. While the existence of experience-dependent plasticity has been demonstrated for the visual and other sensory systems, it remains unknown whether this is also the case for motor systems. Here we examined the effects of eliminating sensory inputs on the development of peristaltic movements in Drosophila embryos and larvae. The peristalsis is initially slow and uncoordinated, but gradually develops into a mature pattern during late embryonic stages. We tested whether inhibiting the transmission of specific sensory neurons during this period would have lasting effects on the properties of the sensorimotor circuits. We applied Shibire-mediated inhibition for six hours during embryonic development (15–21 h after egg laying [AEL]) and studied its effects on peristalsis in the mature second- and third-instar larvae. We found that inhibition of chordotonal organs, but not multidendritic neurons, led to a lasting decrease in the speed of larval locomotion. To narrow down the sensitive period, we applied shorter inhibition at various embryonic and larval stages and found that two-hour inhibition during 16–20 h AEL, but not at earlier or later stages, was sufficient to cause the effect. These results suggest that neural activity mediated by specific sensory neurons is involved in the maturation of sensorimotor circuits in Drosophila and that there is a critical period for this plastic change. Consistent with a role of chordotonal neurons in sensory feedback, these neurons were activated during larval peristalsis and acute inhibition of their activity decreased the speed of larval locomotion.     

Slit and Robo regulate dendrite branching and elongation of space-filling neurons in Drosophila

Space-filling neurons extensively sample their receptive fields with fine dendritic branches. In this study we show that a member of the conserved Robo receptor family, Robo, and its ligand Slit regulate the dendritic differentiation of space-filling neurons. Loss of Robo or Slit function leads to faster elongating and less branched dendrites of the complex and space-filling class IV multi-dendritic dendrite-arborization (md-da) neurons in the Drosophila embryonic peripheral nervous system, but not of the simpler class I neurons. The total dendrite length of Class IV neurons is not modified in robo or slit mutant embryos. Robo mediates this process cell-autonomously. Upon Robo over-expression in md-da neurons the dendritic tree is simplified and time-lapse analysis during larval stages indicates that this is due to reduction in the number of newly formed branches. We propose that Slit, through Robo, provides an extrinsic signal to coordinate the growth rate and the branching level of space-filling neurons, thus allowing them to appropriately cover their target field.     

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.     

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.     

Steroid Receptor Isoform Expression in Drosophila Nociceptor Neurons Is Required for Normal Dendritic Arbor and Sensitivity

Steroid hormones organize many aspects of development, including that of the nervous system. Steroids also play neuromodulatory and other activational roles, including regulation of sensitivity to painful stimuli in mammals. In Drosophila, ecdysteroids are the only steroid hormones, and therefore the fly represents a simplified model system in which to explore mechanisms of steroid neuromodulation of nociception. In this report, we present evidence that ecdysteroids, acting through two isoforms of their nuclear ecdysone receptor (EcR), modulate sensitivity to noxious thermal and mechanical stimuli in the fly larva. We show that EcRA and EcRB1 are expressed by third instar larvae in the primary nociceptor neurons, known as the class IV multidendritic neurons. Suppression of EcRA by RNA interference in these cells leads to hyposensitivity to noxious stimulation. Suppression of EcRB1 leads to reduction of dendritic branching and length of nociceptor neurons. We show that specific isoforms of the ecdysone receptor play critical cell autonomous roles in modulating the sensitivity of nociceptor neurons and may indicate human orthologs that represent targets for novel analgesic drugs.     

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.