The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.
Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.
Drosophila larvae exhibit a characteristic aversive rolling behavior when exposed to different noxious stimuli: thermal1, mechanical2, and chemical3. This behavior is clearly distinct from normal locomotion. Here we describe an improved mechanical assay that can be used to assess mechanical nociception and mechanical sensitization.
In a recent study, we fabricated von Frey-like filaments using nitinol wires4. Probes exerting different forces and pressures were made by varying the lengths and diameters of the nitinol wires forming each probe. Mechanical probes were calibrated and the measured force values (in millinewton, mN) were converted to pressure (kilopascal, kPa), based on the tip area of each probe4. Custom fabrication of mechanical probes allowed to us to generate subthreshold (≤200 kPa) to suprathreshold (225 kPa to 5318 kPa) pressures, which could, in principle, be beneficial for studying mechanical hypersensitivity. Using these improved mechanical von Frey-like filaments, we showed that pressure4, as opposed to the previously examined force2,5,6 correlates more consistently with aversive behavioral responsiveness in Drosophila larvae. The improved mechanical assay described here also helped to identify a conserved Vascular Endothelial Growth Factor (VEGF)-related receptor tyrosine kinase signaling a pathway that regulates mechanical nociception in flies and rats4.
Mechanical allodynia and hyperalgesia, two modalities of hypersensitivity, are relatively understudied in Drosophila larvae, compared to the thermal (heat and cold) and chemical sensory modalities3,7,8,9,10. This is probably due to the lack of specific mechanical probes that span from innocuous stimuli to the high noxious range2,5,6. A normally innocuous stimulus that elicits the typical aversive rolling behavior after Drosophila larvae experience tissue damage3,7 is referred to as allodynia. An exaggerated rolling response to a typically noxious stimulus is known as hyperalgesia7. Noxious stimuli are defined as those that elicit tissue damage and can activate nociceptors11. Noxious stimuli delivered to Drosophila larvae damage either the barrier epidermis, the peripheral nociceptive sensory neurons3,4,7, or both.
In this article, we demonstrate how to custom fabricate and calibrate von Frey-like mechanical probes that are appropriate for Drosophila larvae. Further, we show how to use these probes to assay mechanical nociceptive responses in Drosophila larvae. Finally, we further demonstrate the utility of these probes by using them to demonstrate the presence of mechanical hypersensitivity, both allodynia and hyperalgesia, following tissue damage in Drosophila larvae (see Representative Results).
1. Mechanical probe construction
2. Preparation of larvae
3. Mechanical nociception assay
4. Confocal microscopy to assess neuronal morphology
5. Quantitation of tissue damage
We developed customized mechanical probes, using nitinol filaments (Figure 1A,N), to elicit mechanically-evoked behaviors and generated a full behavioral dose response curve using both innocuous and noxious mechanical probes of varying intensity (Figure 2D) demonstrating that these probes can be used to study baseline (in the absence of injury) mechanical nociception.
Our behavioral assay results determined that probes exerting pressures below 200 kPa (~1.57 mN) (Figure 1M), when applied to Drosophila larvae, do not provoke an aversive rolling response (Figure 2D and Video 3). As expected, these subthreshold or non-noxious mechanical probes (175 kPa or 200 kPa) did not elicit visible neuronal tissue damage (Figure 2E). Because they do not induce damage, such probes could be useful to assess mechanical allodynia (hypersensitivity to a normally non-noxious mechanical stimuli). Conversely, suprathreshold or noxious probes (from 462 kPa to 5,116 kPa), elicited an augmented behavioral response (Figure 2D) in a dose dependent manner—with the higher pressures eliciting stronger behavioral responses. As anticipated, suprathreshold mechanical pressure also induced dose-dependent tissue damage to the peripheral sensory neurons themselves (Figure 2E). The measured area of tissue damage (in µm2 ± standard deviation) taken from four larvae for each group were: 2,051.03 ± 703.81 (462 kPa), 5,102.29 ± 1,004.67 (2,283 kPa), and 12,238.83 ± 3,724.11 (5,116 kPa). Thus, pressures greater than or equal to 462 kPa (~63 mN), which evoke an aversive rolling response (in 25% or more of the larvae) and cause visible neuronal tissue damage (Figure 2E), could be appropriate to study mechanical hyperalgesia (hypersensitivity to normally noxious mechanical stimuli). Nociceptive mechanical probes (≥462 kPa) always induce tissue damage (n = 10, evaluated qualitatively) but do not always provoke an aversive rolling response.
To evaluate mechanical hypersensitivity (allodynia and hyperalgesia), we used a well-established Drosophila larval model of nociceptive sensitization that uses ultraviolet light (UV) irradiation to induce tissue damage7,12. This assay has helped to dissect the genetic and cellular mechanisms of thermal nociceptive hypersensitivity8,9,10,13,14,15. To determine whether UV treatment causes mechanical allodynia, mid third-instar control (w1118) larvae were mock-irradiated or UV-irradiated (15–20 mJ/cm2) (Figure 3A). Then, the larvae were tested behaviorally at 2 h, 4 h, 8 h, 16 h, and 24 h post-treatment with a normally subthreshold mechanical probe (200 kPa, 1.57 mN). Approximately 20% of larvae responded as early as 2 h after UV treatment while 50% responded at 4 h, compared to 6.6% and 8.3% mock UV-irradiated animals, respectively (Figure 3B). This indicates that UV-induced tissue damage causes mechanical allodynia at 4 h post-irradiation. At later time points (8 h, 16 h, and 24 h) the behavioral response of the UV-treated larvae was in the range of 16%–20% responders (average mean of n = 3–6 sets of 10 larvae each), slightly increased (but not statistically significant) compared to the mock-irradiated control group (in the range of 3%–6% of responders, average mean of n = 3–6 sets of 10 larvae each) (Figure 3B).
To investigate mechanical hyperalgesia, a suprathreshold pressure (462 kPa, 3.63 mN), that normally induces an aversive rolling response in ~20% of larvae (Figure 2D) and causes neuronal tissue damage (Figure 2E), was used. We applied the 462 kPa probe onto the dorsal side of larvae with or without UV-induced tissue damage (Figure 3A). We found that larvae probed at 4 h, 8 h, and 16 h following UV treatment showed a significant increase in the aversive rolling response, with 4 h being the peak of the behavioral hypersensitivity (~60% responsive); mock UV-irradiated animals showed an ~27% of aversive response (Figure 3C). Similar to mechanical allodynia, the behavioral response at 8 h, 16 h, and 24 h of UV-treated animals (in the range of 36%–42%) was statistically indistinguishable from the non-treated larvae (in the range of 20%–26%). Larvae at the late third instar stage did show a slight decrease of the baseline behavioral response when compared with the middle third instar stage. We hypothesize this could be either by the increased size of the larvae (Figure 2A) or the increased thickness of the cuticle covering the body. This fact could explain why at a later stage of development the UV treatment does not induce greater mechanical sensitization, as observed 4 h post UV treatment.
Taken together, our results indicate that Drosophila larvae develop both mechanical allodynia and mechanical hyperalgesia following UV-induced tissue damage. The peak time of mechanical allodynia and hyperalgesia is the same, 4 h after UV treatment; however, mechanical hyperalgesia has a more pronounced temporal tail as it returns to baseline more slowly compared to mechanical allodynia.
Figure 1: Development of a Von Frey-like tool to evaluate mechanical nociception in Drosophila larvae. (A) Picture of a mechanical probe used to study mechanical nociception in Drosophila larvae. (B) Nitinol filaments and their relative diameters are shown to relative scale. (C) Picture of the diagonal wire cutter used to cut the nitinol filaments. (D) Smoothing the sharp edges of the cut nitinol filament with a sharpening stone. (E) Hypodermic needle used to make a hole into the wooden popsicle stick handle of the probe. The tip of the needle needs to reach at least half the height of the handle stick for secure filament insertion. (F–G) Attachment of the nitinol filament by gluing into a wooden popsicle stick handle with insertion hole. (H–L) Calibration of mechanical probes by pressing them against a scale. (M) Values of force (in mN) and pressure (in kPa) generated by different mechanical probes. The length of each nitinol filament used to construct the probes (P1–P10; P: probe) is detailed in centimeters (cm). (N) A picture of a complete set of mechanical probes, ranging from 174 kPa to 5,116 kPa. Please click here to view a larger version of this figure.
Figure 2: Mechanical nociception assay: Von Frey-like filaments generate a dose-response curve of aversive rolling behavior and cause tissue damage to sensory neurons. (A) Pictures of the different stages (second and third instar) of Drosophila larvae. Scale bar: 2 mm. (B) Cartoon of the dorsal view of the third instar Drosophila larvae. The red dot indicates the abdominal segment where the mechanical probe is applied. T: thoracic segment; A: abdominal segment. Other anatomical landmarks are labeled. (C) Cartoon of the assay: A mechanical probe is applied to the dorsal side of the larva until it bends against the surface below and is then held for 2 s. If the pressure is sufficiently high, this elicits an aversive rolling response upon release. (D) Behavioral dose response; each blue dot represents the percent of larvae that responded, with aversive rolling, to the mechanical stimulation within a set of 10 animals. Violin plot of the percent of aversive rolling behavior induced by different mechanical probes. kPa: kilopascals. Box plots represent median (green), whiskers (red) represent the 10th and 90th percentiles. (E) Tissue damage: Third instar larvae (of genotype ppk-Gal4>UAS-mCD8-GFP to label nociceptive sensory neurons) were probed at dorsal segment A8 with the indicated pressures. Fluorescently labeled paired ddaC class IV sensory neurons (across the dorsal midline) were then examined (see sections 4 and 5). White areas (red asterisks) represent gaps or tissue damage. Scale bar: 100 μm. In panel B, the larva is shown in the dorsal view, while in C it is the lateral view. Mechanical probes pressed against the dorsal cuticle-epidermis side of the larva produce a depression like-pocket at the point of contact of the tip of the probe and the surrounding areas. The solid black line curved toward the ventral side is the top of the pocket, while the dashed gray lateral line represents the lateral side and the bottom of the pocket. Please click here to view a larger version of this figure.
Figure 3: Mechanical hypersensitivity after UV damage. (A) Schematic of the experimental design to test sensitization. Mid third instar were mock treated (non-UV) or UV irradiated. The mechanical nociception assay was then performed at different time points (2 h, 4 h, 8 h, 16 h, and 24 h) following mock treatment or irradiation. (B) Mechanical allodynia: The percentage of larvae exhibiting aversive rolling after probing with a normally subthreshold or non-noxious mechanical stimulus (200 kPa, 1.57 mN) at the indicated time points after mock-treatment or UV irradiation. (C) Mechanical hyperalgesia: The percentage of larvae exhibiting aversive rolling after probing with a normally suprathreshold or noxious mechanical stimulus (462 kPa, 3.63 mN) at the indicated time points after mock-treatment or UV irradiation. Error bars indicate mean +/- SEM. Two-tailed unpaired t-test was used for statistical analysis: *p < 0.05, **p < 0.01; ns: not significant. Each red dot, in panels B and C, represents the mean proportion of 10 larvae, n = 3–6 sets per timepoint/condition. Please click here to view a larger version of this figure.
Video 1: Normal locomotion of Drosophila larvae. Please click here to download this video.
Video 2: Noxious mechanical stimulation of Drosophila larvae. Please click here to download this video.
Video 3: Subthreshold mechanical stimulation of Drosophila larvae. Please click here to download this video.
We modified an established mechanical assay1,2,16 using customized mechanical probes fabricated from nitinol filaments. This metal alloy allows us to use smaller diameter filaments that are appropriate to the size of the Drosophila larvae. Fishing line-based monofilaments have dominated the field of fly mechanical nociception to date2,5,6,16. Our nitinol filaments maintain their shape and measured pressure for approximately ~3–5 months (in our experience). By varying the length and diameter of the nitinol filaments, the user can generate a wide range of pressures spanning from subthreshold to a nearly complete rolling response. In particular, making subthreshold probes is simpler with the smaller diameter nitinol filaments. Using these probes, we found that pressure, rather than force, elicits more consistent nocifensive behavioral responses4. We demonstrate here, using a well-established UV-induced nociceptive sensitization model7,10,13, that these filaments are also a useful tool for studying mechanical hypersensitivity—allodynia and hyperalgesia.
Previous studies using mechanical probes fabricated from fishing line have led to a certain variability in behavioral responsiveness2,6,16,17. Several factors may account for this. First, because pressure is the important variable, the buffing of the filament tip so that it is rounded and does not have any sharp edges is critical. Second, reporting pressure values rather than only force is important for the reproducibility of the experiments, because different mechanical probes that generate similar forces can elicit disparate pressures4. Third, it is critical to apply only one mechanical stimulation per larva using noxious probes, because such probes produce a dose-dependent tissue damage at the epidermal4 and sensory neuronal levels (Figure 2E). A second or subsequent noxious mechanical stimulus, after tissue damage has been induced, could conceivably impair the function of the affected peripheral sensory neurons and elicit an altered behavioral response. In another study, larvae stimulated twice with noxious mechanical probes mostly displayed an enhanced behavioral response5, suggesting development of an acute mechanical sensitization (hyperalgesia), which might result from the tissue damage provoked by the first noxious mechanical stimulus. Conversely, other authors6 reported a mixed (increased or decreased) behavioral response, indicating that the altered behavioral response could be due to damage/dysfunction of the neuronal tissue. Stimulating each larva only once eliminates possible variance in behavioral responses resulting either from sensitization or tissue damage. Fourth, we mechanically stimulated segment A8, which is more posterior than previous studies (preferred areas A3–A4)2,5,16. Probes between ~3,900 kPa and 5,300 kPa applied to either segment A2 or A8 did not show any behavioral differences4. In addition, A8, compared to A2–A4, is easier to stimulate with mechanical probes that generate lower pressures (<300 kPa) because the larva is thinner in this region and thus more easily compressed. Other studies showed that noxious mechanical stimulation of the posterior end of the larva (delivered by a rigid insect pin, held with forceps) mostly evoked forward locomotion, rather than an aversive or rolling response18. This different behavioral response could be due to differences in the properties of the used materials (bendable nitinol filament vs incompressible insect pin) or to different pressures delivered to the larvae (the pressure value of the insect pin was not reported).
The development of a mechanical nociception assay for Drosophila larvae has enabled the field to discover that different mechanical sensory ion channels and neural circuits mediate mechanical nociception5,6,16,17. However, the study of the mechanical hypersensitivity (allodynia and hyperalgesia) has lagged, compared to sensitization of the other sensory modalities—heat7,8,10,13,14, cold9, and chemical3. This lag may be due in part to the absence of suitable mechanical probes that can generate a full response range spanning subthreshold to suprathreshold pressures. Of particular importance, especially for assessing mechanical allodynia, are subthreshold probes that do not elicit an aversive rolling response from uninjured larvae. The significance of our improved mechanical probes is that they can be fabricated to span innocuous stimuli (subthreshold ~174 kPa–200 kPa) or the low to high noxious range (suprathreshold ~225 kPa to ~5,116 kPa). Here, we demonstrate using the nitinol von Frey-like filaments that Drosophila larvae develop both mechanical allodynia and mechanical hyperalgesia after UV irradiation. The mechanical sensitization shows some differences when compared to thermal sensitization. Both the onset and the peak of mechanical sensitization is earlier (~4 h) compared to thermal (heat) sensitization (~8 h for hyperalgesia and ~24 h for allodynia)7. In addition, the mechanical allodynia and hyperalgesia are concomitant (both peak at ~4 h). Furthermore, while heat sensitization (allodynia and hyperalgesia) resolves completely at later time points7, mechanical hypersensitivity exhibited a long tail that remained slightly above baseline. Cold sensitization in Drosophila involves a switch in cold-evoked behaviors9 and the emergence of new cold-evoked behaviors—a phenomenon that is not observed with mechanical stimulation. These differences in onset, duration, and observed behaviors suggest that each sensory modality may be controlled by different signaling pathways. Combining the sensitization assay described here with the powerful genetic tools available in Drosophila should allow a precise genetic dissection of the mechanical hypersensitivity (allodynia and hyperalgesia) observed.
The authors have nothing to disclose.
We thank Thomas Wang for developing the prototype von Frey filaments, Patrick J. Huang for improving the mechanical probe assay, the Bloomington Drosophila Stock Center for the control (w1118) and ppk-Gal4>UAS-mCD8-GFP fly stocks, and Galko lab members for critically reading the manuscript. This work was supported by R21NS087360 and R35GM126929 to MJG.
Beaker | Fisher Scientific | 02-540C | Beaker of 10 ml of capacity. Any similar container will do. |
Black (Arkansas) bench stone | Dan’s Whetstone | SKU: I200306B24b-HQ-BAB-622-C | Used to smoothe any irregularities of the nitinol wire tips. https://www.danswhetstone.com/product/special-extra-wide-black-bench-stone-6-x-2-1-2-x-1-2/ |
Confocal microscope | Olympus | FV1000 | Any equivalent confocal microscope will do |
Coplin Jar | Fisher Scientific | 08-816 | https://www.fishersci.com/shop/products/fisherbrand-glass-staining-dishes-10-slides-screw-cap/08816#?keyword=08-816 |
Diethyl ether | Fisher Scientific | E138-500 | For anesthetizing larvae. |
Etherization chamber | This is a homemade customized chamber. Please see details of its construction in our previous published paper12. The purpose of the etherization chamber is allow entry of diethyl ether fumes but prevent larval escape. | ||
Fiber Optic Light Guide | Schott AG | A08575 | Schott Dual Gooseneck 23 inch |
Forceps | Fine Science Tool | FS-1670 | For transferring larvae |
Glue | Aleene's | N/A | Aleene's® Wood Glue, formerly called (Aleene's All-Purpose Wood Glue) https://www.aleenes.com/aleenes-wood-glue |
Graspable holder | Loew Cornell | N/A | Loew-Cornell Simply Art Wood Colored Craft Sticks, 500 pieces. |
Halocarbon oil 700 | Sigma | H8898-100ML | |
Hypodermic needle 30G 1/2"L | Fisher Scientific | NC1471286 | BD Precisionglide® syringe needles, gauge 30, L 1/2 inches. Used to make a hole into the wooden holder for the nitinol wires |
Large Petridish | Falcon | 351007 | 60 mm x 10 mm Polystyrene Petridish |
Microscope (Zeiss) Stemi 2000 | Carl Zeiss, Inc. | NT55-605 | Any equivalent microscope will do |
Microscope Cover Glass 22×22 | Fisher | 12-545-B | |
Microscope Cover Glass 22×40 | Corning | 2980-224 | Tickness 1 1/2 |
Microscope Slides | Globe Scientific Inc. | 1358Y | |
Mini Diagonal Cutter | Fisher Scientific | S43981 | For cutting nitinol filaments |
Nitinol filaments, Diameters: 0.004”, 0.006”, 0.008” | Mailin Co | N/A | Fifteen pieces of each diameter of 12” length were ordered. https://malinco.com/ |
Piece of black vinyl | Office Depot | N/A | We use a small piece of vinyl cut from a binder. Dark color provides contrast. A small piece allows orientation of the larva |
Small Petridish | Falcon | 351008 | 35 mm x 10 mm Polystyrene Petridish |
Spatula | Fisher Scientific | 21-401-10 | Double-Ended Micro-Tapered Stainless Steel Spatula. Used to place the food in the petri dish |
Wipes | Fisher Scientific | 06-666A | Kimpes KMTECH, Science Brand. Used to dry larvae of excess moisture. |
W1118 | Bloomington Drosophila Stock Center | 3605 | Control strain for behavioral assays |
ppk-Gal4>UAS-mCD8-GFP | Bloomington Drosophila Stock Center | 8749 | Strain for fluorescent labeling of class IV md neurons |