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. Author manuscript; available in PMC: 2014 May 12.
Published in final edited form as: Curr Biol. 2009 Apr 16;19(10):799–806. doi: 10.1016/j.cub.2009.03.062

Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae

Daniel T Babcock 1,3, Christian Landry 4,#, Michael J Galko 1,2,*
PMCID: PMC4017352  NIHMSID: NIHMS578787  PMID: 19375319

Summary

Background

Heightened nociceptive (pain) sensitivity is an adaptive response to tissue damage that serves to protect the site of injury. Multiple mediators of nociceptive sensitization have been identified in vertebrates, but the complexity of the vertebrate nervous system and tissue repair responses has hindered identification of the precise roles of these factors.

Results

Here we establish a new model of nociceptive sensitization in Drosophila larvae, in which an aversive withdrawal behavior is altered after UV-induced tissue damage. We find that UV-treated larvae develop both thermal hyperalgesia, manifested as an exaggerated response to noxious thermal stimuli, as well as thermal allodynia, a responsiveness to sub-threshold thermal stimuli that are not normally perceived as noxious. Allodynia is dependent upon a Tumor Necrosis Factor (TNF) homolog, Eiger, released from apoptotic epidermal cells, and the TNF receptor, Wengen, expressed on nociceptive sensory neurons.

Conclusions

These results demonstrate that cytokine-mediated nociceptive sensitization is conserved across animal phyla and set the stage for a sophisticated genetic dissection of the cellular and molecular alterations in sensory neurons responsible for development of nociceptive sensitization.

Introduction

The ability to detect and respond to potentially damaging stimuli is crucial for the survival of many organisms. Nociceptors, the specialized sensory neurons that are activated in response to harmful stimuli, typically respond only to stimuli above a certain threshold [1]. However, tissue damage often alters the activation properties of these neurons, resulting in increased sensitivity. This sensitization, which can manifest as an exaggerated response to normally noxious stimuli (hyperalgesia) or an aversive response when presented with normally innocuous stimuli (allodynia) [2, 3], is adaptive as it fosters behavior that protects the damaged tissue while it heals. Unfortunately, chronic pain, a major health care burden, can ensue when the hypersensitivity remains after the damaged tissue is healed. To help alleviate this problem, it is important to understand the mechanisms responsible for the induction of this sensitivity. The astonishing complexity of the nervous and immune systems of vertebrates, where nociceptive sensitization has traditionally been studied, highlights the need for the establishment of simple model systems that could rapidly uncover the conserved genetic basis of damage-induced nociceptive sensitization.

Sensitization can be mediated by factors released from immune-responsive blood cells and likely from damaged epithelial cells as well. Some of these mediators are proteinaceous, such as the inflammatory cytokines Tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β), while others are lipids (prostaglandins), peptides (kinins and Substance P), or small molecule neurotransmitters such as ATP and serotonin [4-7]. In many of the current assays for sensitization it is difficult to separate the immune actions of these mediators from their neuromodulatory actions. However, UV-induced epidermal damage, a classical inducer of allodynia in humans, creates a sterile injury to the barrier epidermis by causing DNA damage [8]. UV-induced damage could thus be used to clarify the role of individual mediators. UV irradiation in rats induces both thermal and mechanical hyperalgesia and allodynia [9, 10], although the specific signaling pathways mediating this sensitization are not yet known. In Drosophila, UV irradiation of the developing retina induces cell death [11, 12], and acute doses of UV radiation induce a withdrawal behavior in larvae[13] but the effects of UV on the barrier epidermis and underlying nociceptive sensory neurons have not been investigated.

Recent studies on invertebrate nociception have revealed that many of the basic mechanisms of nociception are evolutionarily conserved [14-16]. Repeated exposure to mild noxious stimuli results in nociceptive sensitization in several invertebrates, including Aplysia [17], Manduca sexta [18], and the medicinal leech [19]. At least in Aplysia, inflammation and tissue damage also play a role in nociceptive sensitization [20, 21]. However, there is no work so far on damage-induced nociceptive hypersensitivity in a genetically tractable invertebrate model.

In Drosophila larvae, a set of well-characterized multidendritic (md) sensory neurons underlie the body wall [22, 23] and the dendritic arbors of these neurons contact almost all barrier epidermal cells. A recent study demonstrated that a distinct subset of these sensory neurons, class IV md neurons, function as nociceptors that are involved in the sensation of noxious thermal and mechanical stimuli [24]. When presented with such stimuli, Drosophila larvae exhibit a unique aversive withdrawal behavior involving a “corkscrew” rolling motion that is distinct from the usual locomotive behavior. A genetic screen using this behavioral paradigm uncovered a novel Transient Receptor Potential (TRP) channel that is necessary for thermal and mechanical nociception [15]. TRP channels are also important in mammalian nociception, including transient receptor potential vanilloid receptor-1 (TRPV1), which is involved in thermal nociception and hyperalgesia [25, 26].

Here, with the goal of establishing a genetically tractable system to study nociceptive sensitization, we investigated whether UV-induced tissue damage could cause hypersensitization of nociceptive sensory neurons in Drosophila larvae and identified the conserved inflammatory cytokine that mediates this sensitization.

Results

Thermal probe and nociceptive threshold

To establish the thermal nociceptive threshold, third-instar larvae of our control (w1118) strain were stimulated with a custom-built heat probe (Figures 1A and 1B) capable of contacting a single body segment and maintaining a constant set-point temperature +/− 0.1 °C. Larvae were tested over a 10 °C range spanning the nociceptive threshold previously reported [15], with each individual stimulated only once to avoid the possibility of experience-dependent changes in the withdrawal behavior. Responses were categorized by withdrawal latency: fast responders withdrew in less than 5 seconds, slow responders between 5 and 20 seconds, and non-responders did not withdraw within our 20 second cutoff (Figure 1C). No larvae responded below 39 °C. At 44 °C nearly all larvae exhibited the aversive response, with an average withdrawal latency of 9.0 +/− 1.5 seconds. At 48 °C and above all of the larvae responded within 5 seconds, with an average withdrawal latency of 1.5 +/− 0.8 seconds (Figure 1D). These findings define temperatures appropriate for testing development of allodynia and hyperalgesia following tissue damage.

Figure 1. Larval responses to thermal stimuli and UV radiation.

Figure 1

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(A) Diagram of thermal probe illustrating the thermal control unit, feedback design (arrows) and brass tip. Box shows area of interest in B. (B) Schematic illustrating the major components of the heat probe. (C) Aversive response of third instar w1118 larvae to stimulation at various temperatures. Behaviors were classified as “no response” (white, > 20 s), “slow withdrawal” (gray, between 5 and 20 s) or “fast withdrawal” (black, < 5 s). n = 50 for each temperature tested. (D) Average withdrawal latency at different temperatures. 44 °C = 9.6 s +/− 1.6 s and 48 °C = 1.5 s +/− 0.6 s. n = 50 for each temperature. Error bars indicate standard error of the mean. The median withdrawal time was significantly different between groups (p < 0.001, by Log-rank test). (E) Survival rate of third instar larvae to adulthood after treatment with increasing doses of ultraviolet radiation. n = 75 for each group.

UV-induced epidermal tissue damage

Acute doses of UV radiation damage DNA through the production of thymine dimers. If the radiation causes sufficient genetic damage within barrier epidermal cells, they undergo apoptosis [8]. We found that third-instar control larvae can withstand a UV dosage of up to 20 mJ/cm2 without a decrease in survival to adulthood (Figure 1E). To determine the extent of tissue damage after this exposure, we visualized the epidermis and the underlying sensory neurons. Untreated barrier epidermal cells are highly regular in size and shape, while the sensory neurons that innervate them possess extensively branched dendrites (Figures 2A and 2B). Up to 16 hours after UV exposure, epidermal cell morphology appeared normal, but by 24 hours there were clear alterations in epidermal cell morphology that indicate tissue damage centered along the dorsal midline (Figure 2I and Figure S1 in the Supplemental Data available online). This morphological deterioration was accompanied by activation of caspase 3, a marker of apoptosis (Figure 2L). Curiously, although neither activated caspase 3 nor morphological deterioration were observed 4 hr post-irradiation (Figures 2E, 2F, and 2H), we did find activation of other stress-responsive reporters such as misshapen (the MAP4K in the JNK signaling pathway), using a msn-lacZ reporter allele [27] at this earlier time-point and beyond (Figures 2G, 2K and 2O). Despite the extensive epidermal damage, the underlying sensory neurons maintained their normal morphology (Figure 2J, 2N, and Figure S2). These results indicate that UV radiation damages the epidermis but leaves the underlying sensory neurons intact and establish an appropriate UV dose for our subsequent sensitization experiments.

Figure 2. Epidermal damage induced by UV radiation.

Figure 2

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(A-P) Wholemount staining showing the cellular effects of mock treatment (A-D) or UV treatment on larval tissues at 4 hours (E-H) 24 hours (I-L) or 48 hours (M-P) post-exposure. (A, E, I, M) Epidermal cell membranes of w1118;ppk1.9-Gal4, UAS-eGFP larvae labeled with anti-Fasciclin III. (B, F, J, N) Class IV dendritic arborization sensory neurons labeled in the same ppk1.9-Gal4, UAS-eGFP larvae as in A, E, and I. (C, G, K, O) X-Gal staining of msn-lacZ larvae. (D, H, L, P) Apoptotic cells of w1118 larvae labeled with an activated caspase-3 antibody. Scale bar in P, 100 μm for all panels.

UV damage induces thermal allodynia

To assess whether UV-induced damage causes changes in nociceptive sensitivity, third-instar control larvae were exposed to UV, stimulated with the highest normally subthreshold temperature (38 °C) and their withdrawal responses recorded. We found that some larvae responded as early as 4 hours after UV (average withdrawal latency = 13.0 +/− 1.9 seconds) while 62% responded at 24 hours (average withdrawal latency = 6.4 +/− 5.3 seconds) (Figure 3A), indicating that UV-induced tissue damage lowers the behavioral response threshold of treated larvae. At 48 hours after UV exposure the sensitization is diminished, coinciding with the healing of the dorsal epidermis and diminishment of overt epidermal cell apoptosis (Figure 2M-2P). To determine how far the nociceptive threshold was lowered, we stimulated groups of larvae 24 hours after UV treatment with progressively lower temperatures (Figure 3B). After tissue damage, more larvae withdrew from slightly supra-threshold temperatures (42 or 40 °C), 60 % of the larvae responded at 34 °C, and some even responded as low as 30 °C. Since the aversive withdrawal was not observed below 30 °C we conclude that it is primarily the temperature of the probe and not the light touch of the probe that is responsible for the withdrawal behavior. These results demonstrate that Drosophila larvae, like mammals, exhibit a lowering of their nociceptive threshold, or allodynia, following UV-induced tissue damage.

Figure 3. Thermal allodynia and hyperalgesia after tissue damage.

Figure 3

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(A-B) Comparison of response thresholds between UV-treated and mock treated w1118 larvae. (A), Response to the highest normally innocuous temperature (38 °C, see Figure 1B) at specified times after UV-induced tissue damage. White = no response, gray = response between 5 and 20 s, black = response in less than 5 s. Nociceptive threshold distributions are significantly different (p < 0.001, by Fisher’s exact test), at 8, 16, and 24 hours post-UV as compared to mock-treated larvae (asterisks). Mock-treated larvae were assessed 24 hours after mock irradiation, to compare with the greatest response seen in UV-treated larvae. (B) Responses to decreasing temperatures below the normal nociceptive threshold 24 hours after tissue damage. Gray = mock treated, hatched = UV treated. (C-D) Comparison of withdrawal latencies between UV-treated larvae and mock treated w1118 larvae. (C) Response to a normally noxious temperature (45 °C) at specified times after UV-induced tissue damage. White = no response, gray = response between 5 and 20 s, black = response in less than 5 s. Nociceptive threshold classification is significantly different at 8 (p < 0.001) and 16 (p = 0.039) hours post-UV as compared to mock-treated larvae (asterisks). Mock-treated larvae were assessed 8 hours after mock irradiation, to compare with the greatest response seen in UV-treated larvae. (D) Average withdrawal latency to 45 °C stimulation at various times after tissue damage. Error bars indicate standard error of the mean. Mean withdrawal was significantly different (p < 0.001, by Student’s t-test) at 8 hours, as compared to mock-treated larvae (asterisks). n = 50 for all conditions.

Thermal hyperalgesia after tissue damage

A second type of nociceptive sensitization, hyperalgesia, involves an exaggerated response to supra-threshold stimuli. To assess thermal hyperalgesia after tissue damage, we stimulated larvae after UV treatment with a 45 °C probe that normally elicits a withdrawal response in the absence of tissue damage. Although all mock treated larvae withdraw from this temperature, the fast responders (< 5 s) increased from 26% in the mock treated group to 95% at 8 hours post-irradiation (Figure 3C). We found that the average withdrawal latencies of responsive larvae decreased from 7.8 +/− 0.6 seconds in the mock-treated group to 3.2 +/− 0.2 seconds 8 hours after UV treatment (Figure 3D). As with allodynia the response returned towards baseline, although the reduction in hyperalgesia occurs more rapidly with response latencies returning close to baseline 24 hours after UV treatment. Thus, both modes of nociceptive sensitization, allodynia and hyperalgesia, are observed in Drosophila larvae following tissue damage.

Allodynia requires apical apoptotic caspase activity within epidermal cells

24 hours after UV treatment, dorsal epidermal cells normally undergo apoptosis (Figures 2L and 4A-4F). To determine whether UV-induced nociceptive sensitization is mediated by signals produced by dying epidermal cells, we genetically blocked apoptosis in these cells and assessed nociceptive sensitivity after UV exposure. To block apoptosis in larval epidermal cells we used the GAL4/UAS system [28] and RNA interference (RNAi). Specifically, we combined a larval epidermal Gal4 driver (A58-Gal4) [27] with a UASRNAi transgene targeting the apical caspase Dronc [29] (UAS-droncIR), to knock down expression of Dronc only in epidermal cells. Epidermal expression of UAS-droncIR effectively blocked UV-induced apoptosis, as seen by persistent normal epidermal cell morphology and lack of caspase activation (Figures 4G-4I) 24 hours after irradiation. We then assessed withdrawal behaviors in these irradiated larvae that lacked epidermal apoptosis. While control larvae bearing either the Gal4 or UAS insert alone became sensitive to the normally subthreshold stimulus of 38 °C, this sensitization was almost completely absent in larvae where Dronc expression was blocked (Figure 4J). The lack of residual sensitization suggests that there is little if any contribution to sensitization from blood cells that might be recruited to the damaged dorsal epidermis. Consistent with this, genetic ablation of blood cells [30] did not affect the onset of allodynia following UV treatment (Figure 4L). Interestingly, larvae lacking epidermal Dronc activity did exhibit thermal hyperalgesia (Figure 4K), as the withdrawal latency to a normally noxious stimulus (45 °C) was not significantly altered, suggesting possible mechanistic differences mediating the onset of thermal allodynia and hyperalgesia. As with allodynia, hyperalgesia was also not dependent on the presence of blood cells (Figure 4M).

Figure 4. Apoptosis in epidermal cells is required for thermal allodynia.

Figure 4

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(A-I) Wholemount staining of w1118; A58-Gal4/+ (A-C), w1118;UAS-droncIR/+ (D-F) and w1118;UAS-droncIR/+;A58-Gal4/+ larvae (G-I). (A,D,G) Epidermal cell membranes (green) labeled with anti-fasciclin III. (B,E,H) Apoptotic cells (red) labeled with an activated caspase-3 antibody. (C,F,I) Merged panels. Bar in I, 100 μm for A-I. (J) Response to the highest normally innocuous temperature (38 °C) 24 hours post-UV. Larvae bearing both the A58-Gal4 and UAS-DroncIR inserts are significantly less sensitive (p < 0.001, by Fisher’s exact test) than the parental control strains when tested for thermal allodynia. (K) Response to a normally noxious temperature (45 °C, see figure 1C) 8 hours post-UV. Larvae bearing both the A58-Gal4 and UAS-DroncIR inserts displayed no statistically significant differences from the parental control lines or w1118 control line (Figure 3D) when tested for thermal hyperalgesia (p = 0.659, by one-way ANOVA). (L-M) Response to the highest normally innocuous temperature (38 °C) 24 hours post-UV (L) or a normally noxious temperature (45 °C) 8 hours post-UV (M). Larvae lacking hemocytes (Pxn-Gal4>UAS-hid) displayed no differences from the parental control lines when tested for thermal allodynia or hyperalgesia. n = 30 for each group in J through M. Error bars indicate standard error of the mean.

Thermal allodynia requires TNF signaling

The cytokine TNF-α and its receptors have been implicated in both cell death and nociceptive sensitization in mammals [31–34]. Indeed, exposure to UV light causes human keratinocytes to synthesize and release TNF [35, 36]. The Drosophila genome contains one clear homolog of the TNF ligand [37, 38] and TNF receptor [39] and we examined whether these genes are responsible for alterations in nociceptive sensitivity. Larvae with null mutations in eiger, the gene encoding the TNF-α homolog, were exposed to UV and stimulated with the highest normally subthreshold temperature, 38 °C, and their withdrawal responses recorded 24 hours later. We found that larvae transheterozygous for independent eiger null alleles showed a complete absence of thermal allodynia (Figure 5A). Larval epidermal specific expression of a UAS-eigerIR transgene also led to a complete absence of thermal allodynia (Figure 5A), suggesting that the epidermis is the source of the TNF that mediates sensitization. Expression of UAS-eigerIR in sensory neurons using the ppk1.9-Gal4 driver did not affect development of allodynia, indicating that sensory neuron-derived Eiger does not contribute to sensitization (Figure 5A).

Figure 5. Thermal allodynia is induced by TNF signaling.

Figure 5

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(A) Response to the highest normally innocuous temperature (38 °C) of eiger mutants or larvae lacking either Eiger, or Wengen in epidermal cells (A58-Gal4) or in nociceptive sensory neurons (ppk1.9-Gal4), 24 hours after UV treatment. (B) Response to a normally innocuous temperature (38 °C) of control larvae and of larvae with ectopic expression of Eiger in class IV sensory neurons in the absence of UV treatment. White = no response within 20 s, gray = response between 5 and 20 s, black = response in less than 5 s. n = 30 for each condition. Asterisk, p < 0.001 compared to wild type by Fisher’s exact test. (CE) Whole-mount staining of egr1/egr3 larvae. (C) Epidermal cell membranes (green) labeled with anti-fasciclin III. (D) Apoptotic cells (red) labeled with an activated caspase-3 antibody. (E) Merged panel. (F) Response to innocuous (38 °C) and noxious (48 °C) temperatures in the absence of tissue damage. Both eiger mutants and control larvae do not respond within 20 seconds to normally subthreshold temperatures (white bars). However, all larvae within all groups rapidly withdraw from noxious stimuli (black bars), suggesting that normal nociception is not impaired in eiger mutants. Epidermal knockdown of Eiger and nociceptive sensory neuron knockdown of Wengen also did not affect normal nociception. Error bars indicate standard error of the mean. n = 25 for each condition. Bar in E, 100 μm for C-E.

One possibility suggested by the epidermal requirement for Eiger in nociceptive sensitization is that the primary function of Eiger released from UV-treated epidermal cells is to initiate apoptosis [37, 38], which then leads to production of factors that mediate development of allodynia. Three lines of evidence argue against this model. First blocking TNF signaling through epidermal expression of an RNAi transgene targeting the Drosophila TNF receptor, Wengen [39], does not block allodynia (Figure 5A). This RNAi transgene has potent on-target effects as it efficiently blocks Eiger-induced cell death in the developing Drosophila eye disc [39]. Second, larvae transheterozygous for eiger null mutations have a normal epidermal apoptosis response to UV irradiation (Figures 5C-5E). Third, to test whether Eiger directly targets nociceptive sensory neurons after UV-induced tissue damage, we also knocked down expression of Wengen using a nociceptive sensory neuron-specific Gal4 driver (ppk1.9-Gal4) [40]. Larvae expressing UAS-wengenIR in the Class IV sensory neurons recently shown to be sufficient for nociception [24] displayed normal nociceptive responses (Figure 5F), but failed to develop thermal allodynia after UV treatment (Figure 5A), suggesting that Eiger does not act through the production of signaling intermediates such as prostaglandins or other cytokines. Importantly, neither eiger null mutants nor any combination of Gal4 and UAS transgenes affected developmental progression through the larval stages to pupariation (data not shown).

Finally, we tested whether ectopic expression of Eiger was sufficient to induce allodynia even in the absence of UV irradiation. Pan-epidermal overexpression of Eiger is lethal (data not shown) suggesting that the ligand is either sequestered from its receptor in the untreated epidermis, or that it is produced and/or released locally in response to irradiation. We found, however, that we could obtain viable larvae when ectopically expressing Eiger from nociceptive sensory neurons via ppk1.9-Gal4. In these experiments, only larvae carrying both the Gal4 insert and the Eiger overexpression transgene (eigerGS9830) [37] developed allodynia even in the absence of UV irradiation (Figure 5B). Taken together, our results indicate that a conserved cytokine/receptor module directly mediates a subset of nociceptive sensitization responses in Drosophila larvae.

Discussion

We introduce a new model of nociceptive sensitization in Drosophila larvae (Figure 6). In this model, UV radiation activates the apical apoptotic caspase Dronc, resulting in the production and/or release of Eiger from epidermal cells. Secreted or released Eiger then binds to its receptor, Wengen, on nociceptive sensory neurons underlying the epidermal sheet, and lowers the threshold of the behavioral response. The complete absence of thermal allodynia upon tissue specific knockdown of epidermal TNF (Eiger) or sensory neuron TNFR (Wengen) presented here suggests that TNF is a crucial mediator of this sensitization in Drosophila larvae and that its effects are direct. TNF signaling has been implicated in nociceptive sensitization in vertebrates and there is evidence for both direct and indirect effects of this cytokine. Some studies suggest that the primary role of TNF is to mediate the production or release of secondary neuromodulatory factors (Interleukin-1β, prostaglandins) that then sensitize nearby nociceptive sensory neurons [41] while other studies have suggested that soluble TNF can directly alter the firing properties of these cells [42-45]. An important experimental test of the relevance of our proposed model to vertebrate systems would likely involve assessing development of thermal allodynia in mice harboring a skin-specific knockout of TNF-alpha or a sensory neuron-specific knockout of TNF receptors.

Figure 6. Model.

Figure 6

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Top-down illustration depicting the relationship between UV-induced epidermal damage and changes in underlying sensory neurons. Ultraviolet light is absorbed by epidermal cells (1), which eventually undergo apoptosis/activate Dronc (2). As a result, Eiger is produced (3) and released from epidermal cells (4) and ultimately activates Wengen on the nociceptive sensory neuron membrane (5). This activation leads to intracellular signaling that sensitizes the nociceptor (6) to normally subthreshold stimuli. VNC = Ventral Nerve Cord.

Does the TNF/TNFR signaling module represent an ancestral danger-signaling system? Vertebrate TNFs have been implicated in immune responses to pathogens and toxins [31] and in immune modulation of nervous system function [41] following tissue insult. TNF family ligands have been identified in several invertebrates including Drosophila [37, 38], molluscs [46], and the urochordate, Ciona savignyi [47]. In addition to their role in nociceptive sensitization described here, other functional data on the role of invertebrate TNFs show that Drosophila Eiger is required for combating extracellular pathogens [48] but can also, as in vertebrates, lead to infection-induced pathologies [49]. Thus, as in vertebrates, invertebrate TNF family ligands have both immune and neuromodulatory functions, which may be interconnected. We speculate that the TNF signaling system evolved as a general initiator of cell type-specific responses to a variety of pathogens and other noxious insults. In vertebrates, TNF can act both directly on sensory neurons and indirectly through stimulating production of other nociceptive mediators, but our data suggests that in organisms with simpler nervous and immune systems, direct effects may predominate. It will be interesting to test whether damage-induced nociceptive sensitization in urochordates (Ciona) or lower vertebrates employs a direct or indirect mode of TNF signaling and, conversely, whether damage-induced sensitization can occur in even simpler organisms, such as Cnidaria or Ctenophores, that have only a simple neural net but do possess TNF ligand and receptor homologs [50].

The system established here provides a vehicle to apply a new and complementary set of experimental approaches- including the powerful toolkit of Drosophila genetics, to the problem of nociceptive sensitization. Genetic dissection of the sensitization response described here should lead to identification of the specific downstream events by which sensory neuron sensitivity is altered following engagement of the TNF receptor. The robustness of the sensitization response and the clear separation of allodynia (Dronc and TNF-dependent) and hyperalgesia (Dronc-independent) in this system suggest that there may be conserved mechanistic differences mediating the onset of these responses. Given the evolutionary conservation of genes mediating most aspects of neuronal development and function, we expect that this system will broadly inform our understanding of both the onset and potentially the aberrant persistence of nociceptive sensitization in chronic pain syndromes.

Experimental Procedures

Fly strains and genetics

w1118 served as a control strain for baseline nociceptive sensitivity and for most sensitization experiments. The GAL4/UAS system [28] was used to drive expression of UAS transgenes in class IV larval sensory neurons (ppk1.9-Gal4) [40], the larval epidermis (A58-Gal4) [27] and larval blood cells (Pxn-Gal4) [51]. UAS-eGFP [52] served to label sensory neurons, UAS-DroncIR (National Institute of Genetics, JAPAN: http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp) blocked apoptosis in the UV-treated larval epidermis, and UAS-hid [53] was used to ablate larval blood cells. msn-lacZ [54], a transcriptional reporter for the kinase encoded by the misshapen gene, was used as a stress-responsive reporter [27] for UV-treated epidermis. UAS-wengenIR, which blocks Eiger-induced apoptosis in the Drosophila eye [39], was used to knock down Wengen expression in nociceptors and epidermal cells. eiger1 and eiger3 [37] served to measure nociceptive sensitization in larvae lacking Eiger, and UAS-eigerIR [37] was used to knock down Eiger expression in epidermal cells and sensory neurons. eigerGS9830, which contains UAS elements inserted upstream of the eiger locus, was used to overexpress eiger in nociceptive sensory neurons.

UV treatment

Early third instar larvae were mounted dorsal-side-up using double-sided tape on glass slides and placed in a Spectrolinker XL-1000 ultraviolet crosslinker (Spectronics Corporation). UV treatment varied from 0 mJ/cm2 (mock) to 50 mJ/cm2 at a wavelength of 254 nm. For sensitization experiments we used 20 mJ/cm2, the highest dose at which all larvae survive to adulthood. The UV exposure lasted ~6 seconds. After treatment, larvae were returned to fly food at 25 °C before nociceptive sensitivity was assessed at various times post-UV.

Behavioral analysis

Nociceptive stimuli were delivered using a custom-built thermal probe consisting of a thermal control unit (Thermal Solutions, Inc), a thermocouple that measured tip temperature, and a 300 μm micro-fabricated brass tip that allowed stimulation within an individual body segment. The thermal probe, fabricated in the form of a pencil, has a cartridge heater (Watlow Electric Manufacturing Company, FIREROD® Cartridge Heater) embedded into the tip of the probe. A 0.25 inch brass tube covered the heater wires and allowed for the user to grip the probe with an acrylic handle that fitted over the brass tubing. Thermal conduction was controlled by a closed loop feedback configuration using a thermocouple and a temperature control unit with a PID configuration . The temperature control unit was programmed so the heater ramped to a desired set point so overshoot was non-existent and the probe maintained the desired temperature throughout its use. The probe was capable of maintaining a set-point temperature (23-70 °C) within one-tenth of a degree. Larvae were stimulated along the dorsal midline of segment A4 and the withdrawal response was viewed under a light microscope (Leica MZ6) and the time to initiation of aversive withdrawal (withdrawal latency) was measured up to a 20 second cutoff. As in previous studies, the withdrawal behavior was defined as at least one complete 360° roll. All stimulations were to separate individuals to avoid issues of habituation to the noxious stimulus.

For statistical analysis of the behavioral responses we used the Log-rank test to measure differences in median withdrawal time (seconds) between temperature groups. When comparing categorical data sets (no response, slow response, and fast response), we used Fisher’s exact test to measure response classification differences between time-points and between various genotypes. The Student’s t-test or one-way Analysis of Variance (ANOVA) were used in all other cases. All allodynia experiments comparing different genotypes were blinded and performed in triplicate.

Wholemount immunofluorescence and immunohistochemistry

Dissection and immunostaining of larval epidermal wholemount preparations were performed as described [27]. Primary antibodies were anti-Fasciclin III [55] (Developmental Studies Hybridoma Bank, 1:50) to label epidermal membranes, anti-GFP (Molecular Probes, 1:500) to label GFP-expressing sensory neurons, and anti-activated-Caspase-3 (Cell Signaling, 1:150) to label apoptotic cells. Secondary antibodies (Jackson ImmunoResearch) were goat-anti-mouse Cy3 (1:1,000) and goat-anti-rabbit-FITC (1:300). msn-lacZ bearing larvae were stained with X-Gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) as described [27] to reveal msn-lacZ-expressing cells.

Supplementary Material

01

Supplementary Figure 1. UV damage is localized to the exposed dorsal epidermis. (A) Orientation of a dissected wholemount preparation of a third instar larva. Dissections are carried out in a way that allows easy imaging of the dorsal surface. Orange line = dorsal midline. Red box = approximate area of images in subsequent panels. (B-C) UV damage localizes to the dorsal epidermis, as seen by msn-lacZ immunohistochemistry (B) and activated caspase (C) staining 24 hours after UV exposure. Orange line, dorsal midline; v, ventral body wall.

Supplementary Figure 2. Nociceptive sensory neuron morphology remains intact despite epidermal damage Epidermal wholemounts of w1118;ppk1.9-Gal4, UAS-eGFP larvae. (A-C) Epidermal and nociceptor morphology without UV treatment (A), 4 hours (B), and 24 hours (C) after UV exposure. Epidermal cell membranes are marked with anti-Fasciclin III (red) and nociceptors are marked with GFP (green). White boxes outline the approximate area magnified in subsequent panels. (A’-C’) Closer views of nociceptor dendritic arbors with and without epidermal damage. Bar in (C) is 100 μm.

Acknowledgements

We thank Hirotaka Kanuka, Andreas Bergmann, Michael Welch, and the Bloomington and NIG-Fly stock centers for fly stocks, the MD Anderson radiation physics shop for microfabrication of our heat probe, Shana Palla for assistance with statistical analysis, and Howard Gutstein, Terry Walters, Sid Strickland, and members of the Galko laboratory for comments on the manuscript. D.T.B. was supported by National Institutes of Health predoctoral training grant T32-HD07325-16 and an American Heart Association predoctoral fellowship (0815339F). M.J.G. was supported by University of Texas MD Anderson Cancer Center institutional startup funds.

Footnotes

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Associated Data

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Supplementary Materials

01

Supplementary Figure 1. UV damage is localized to the exposed dorsal epidermis. (A) Orientation of a dissected wholemount preparation of a third instar larva. Dissections are carried out in a way that allows easy imaging of the dorsal surface. Orange line = dorsal midline. Red box = approximate area of images in subsequent panels. (B-C) UV damage localizes to the dorsal epidermis, as seen by msn-lacZ immunohistochemistry (B) and activated caspase (C) staining 24 hours after UV exposure. Orange line, dorsal midline; v, ventral body wall.

Supplementary Figure 2. Nociceptive sensory neuron morphology remains intact despite epidermal damage Epidermal wholemounts of w1118;ppk1.9-Gal4, UAS-eGFP larvae. (A-C) Epidermal and nociceptor morphology without UV treatment (A), 4 hours (B), and 24 hours (C) after UV exposure. Epidermal cell membranes are marked with anti-Fasciclin III (red) and nociceptors are marked with GFP (green). White boxes outline the approximate area magnified in subsequent panels. (A’-C’) Closer views of nociceptor dendritic arbors with and without epidermal damage. Bar in (C) is 100 μm.

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