1Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, 2Scholars Academy/MARC Scholar, University of Houston-Downtown, 3Genes and Development Graduate Program, University of Texas Graduate School of Biomedical Sciences, 4Neuroscience Graduate Program, University of Texas Graduate School of Biomedical Sciences
Chattopadhyay, A., Gilstrap, A. T. V., Galko, M. J. Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae. J. Vis. Exp. (63), e3837, doi:10.3791/3837 (2012).
In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. 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 species1,2. 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 biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. 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 stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. 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 cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 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.
A typical outline of the experimental steps for preparing normal or UV-sensitized larvae for the two thermal nociception assays is shown in Figure 1.
1. Preparation of Larvae
2. Etherization and UV Irradiation
Note: The next two steps should be performed in a fume hood as ether is a potentially explosive chemical and its fumes can anesthetize a human as well as a larva.
3. Local Heat Probe Assay
We deliver a noxious thermal stimulus to an individual larval body segment using a custom-built thermal probe manufactured by Pro-Dev Engineering (see Table of materials). Although this probe has optimal design features (a small metal tip of ~ .07 mm2 area and the ability to precisely maintain a set point temperature from 23 °C to 65 °C) in principle any tool with a small tip that can be heated to a defined temperature for a period of up to 20 seconds should suffice. The probe tip is used to stimulate early 3rd instar larvae precisely on the dorsal midline at abdominal segment A4 (see Figure 2). In response to this thermal stimulus, larvae will generally exhibit an aversive withdrawal behavior of rolling laterally by 360 degrees or more. This behavior is distinct from their light touch response to a non-noxious room temperature metal probe which generally involves a brief pause in their locomotory activity13.
Protocol for the heat probe assay:
4. Global Heat Plate Assay
The heat plate assay was designed to measure thermal nociception in Drosophila larvae when the whole animal is confronted with a noxious thermal stimulus. Individual mid 3rd instar larvae are placed in an 80 μl drop of water on a 60 x 15 mm Petri dish - see Figure 3 for schematic and photos of the assay setup. The Petri dish is then placed onto a solid heating block (referred to as the "heat plate"). As the temperature of the water drop rises, the larva exhibits a series of five stereotyped behaviors we have termed head thrash, roll, whip, seizure, and paralysis. As these behaviors are to some extent a function of the setpoint temperature of the heat plate and the volume of water in which the larva is immersed we present here what we have found to be the optimum conditions (95 °C heat plate, 80 μl drop of water) for observing all 5 of the behaviors with minimal overlap between them.
Protocol for heat plate assay:
5. Representative Results
Local Heat Probe Assay:
Upon contact with the thermal probe, a larva typically shows a preliminary behavior of lifting its head and tail. Typically the lifting of the head is seen first followed by lifting of the tail. A few seconds after this preliminary behavior the larva usually starts rolling laterally which we refer to as the "aversive withdrawal behavior." The time after which the preliminary behavior or the withdrawal behavior is shown may vary by temperature or genetic background. In our initial study we measured the percentage of larvae exhibiting aversive withdrawal at different probe set point temperatures and found that 48 °C was the lowest temperature at which all larvae responded fast (< 5 s). Here, we report that there is a ceiling to the larval thermal nociception response (Figure 4A). 100% fast responders are observed up to a probe temperature of 52 °C. However, at 54 °C and higher, 90 % or more of the larvae fail to respond even after 20 s of contact. These larvae do continue moving following the 20 s cutoff.
As noted previously1, the categories of withdrawal behavior at each temperature can be plotted and compared statistically. Given that this is a behavioral assay there is some variability from larva to larva and between individual users. To account for this we usually measure 3 sets of 30 larvae per test condition. In addition to the simple categorization of withdrawal latency that we have previously reported, we report here that the amplitude (number of rolls or time spent in aversive withdrawal behavior) can also be measured (See Figure 4). Surprisingly, there appears to be an inverse relationship between the input temperature and the robustness of the response because lower temperatures appear to provoke a higher number of rolls (Figure 4B) and more time spent in aversive withdrawal (data not shown). This may indicate that the duration of exposure to a noxious temperature may be the main determinant of robustness.
Heat Plate Assay:
Five stereotyped locomotory behaviors are observed upon transfer of the larva immersed in water to the heat plate. These are described below and shown in the video along with the typical locomotory behavior of an unheated larva in water. The average latencies at which these behaviors are observed are shown in Figure 5A, as are the average water drop temperatures measured for each latency. Under the optimal assay conditions presented here the percentage of larvae showing each distinct behavior ranged from 77 to 100 % (Figure 5B) although occasionally the rolling and whipping behaviors are omitted, overlap, or occur in reverse order. The observed behaviors, in chronological order, are described as follows and can be watched in the video at the times indicated below:
These data suggest that the temperature of the water drop and the latency to onset of the five characteristic behaviors observed upon global heating of the larva/water drop are highly correlated.
Figure 1. Outline of experimental steps for preparation and assay of larvae. Prior to assaying nociception with the heat probe or the heat plate, larvae derived from a particular stock or genetic cross are harvested and cleaned of food. If nociceptive sensitization (as opposed to baseline nociception) is to be assessed, harvesting is followed by etherization (exposure to ether), mounting, UV irradiation, and a period of recovery on fly food. Harvested or recovered larvae are then subjected to noxious test temperatures using either the heat probe (local) or the heat plate (global) assay. Numbers refer to sections of the methods text that describe the step(s) shown.
Figure 2. Experimental set up for local heat probe assay. The heat probe is controlled by a thermal control unit which is used to set and maintain the temperature of the probe. The probe is held perpendicular to the anteroposterior axis and used to stimulate the larva at an angle of 45° to the horizontal. Probe contact is made specifically at abdominal segment A4 as shown. The user must maintain this contact with gentle pressure up until the 20 second cutoff or until the rolling behavior commences. If the temperature is perceived as noxious, the larva will show an aversive withdrawal behavior characterized by at least one 360° roll. The number of rolls can be single or multiple (See Figure 4B).
Figure 3. Experimental set-up for heat plate assay. (A) Cartoon of a horizontal view of the 60 x 15 mm Petri dish containing one mid 3rd instar larva in a 80 μl water drop. (B) Cartoon of a top view of the larva placed in the middle of the water drop as viewed through the microscope. (C) Photograph of a horizontal view of the workstation for this assay. (D) Photograph of the larva as viewed through the microscope.
Figure 4. Quantification of behavioral response using the heat probe assay. (A) Plot of the percent of responders belonging to each category (fast-, slow-, and non-responders) versus temperature. (B) Plot of the latency to aversive withdrawal behavior versus the number of rolls each larva exhibited at four different test temperatures of increasing noxiousness (42 °C, 44 °C, 46 °C, 48 °C, 50 °C, and 52 °C).
Figure 5. Heat plate assay: Latency and Temperature vs. Behavior. (A) Latency and average water drop temperature to each behavioral response under the optimal conditions of 95 °C (hot plate surface temperature) and 80 μl drop of water (n = 150). Note that each behavior is observed within a particular time interval. Water drop temperatures were measured using a thermocouple inserted into either the top or bottom of the drop (n = 10 drops for each location) and these measurements were averaged. (B) Percent of larvae exhibiting each behavioral response under optimal assay conditions. Thrashing, seizing, and paralysis are observed almost 100 % of the time while rolling and whipping are observed 77 and 80 % of the time, respectively. At lower surface temperature set points seizure and paralysis are not observed within 200 s and at higher set points rolling and whipping can be skipped or may occur simultaneously. n = 150. (C) Percentage of larvae that survive following initiation of the paralysis behavior. Larvae were heated until beginning paralysis and then removed to standard culture conditions to recover. Mock-treated larvae received equivalent treatment except for heat exposure. Formation of pupae and viable adults were quantified on days 7-13. n = 120.
Figure 6. Inactivation of larval nociceptive sensory neurons increases behavioral response latencies. Plot of the latency to each behavior for the indicated genotypes: w1118, Gal4109(2)80 = md-Gal4/+, UAS-Ork1.Δ-C/+, UAS-Ork1.Δ-NC /+, md-Gal4/UAS-Ork1.Δ-C, and md-Gal4/UAS-Ork1.Δ-NC. md-Gal4 drives expression of UAS-regulated transgenes in all four classes of multidendritic peripheral sensory neurons; UAS-Ork1.Δ-C14 expresses a modified version of the Drosophila open rectifier K+ channel required for synaptic transmission, and UAS-Ork1.Δ-NC14 expresses a further modified version of this same channel that does not interfere with synaptic transmission. Note that only larvae bearing both the md-Gal4 driver6 and the UAS-Ork1.Δ-C transgenes show increased latencies for four of the five observed behaviors (thrash, roll, seizure, and paralysis). Note also that because of the increased latency to rolling these animals whip before they roll. Asterisk denotes p < 0.05 by Student's t-test. n = 30 larvae per genotype.
Supplemental Video 1. Click here to watch video.
Supplemental Video 2. Click here to watch video.
Supplemental Video 3. Click here to watch video.
Supplemental Video 4. Click here to watch video.
Supplemental Video 5. Click here to watch video.
Supplemental Video 6. Click here to watch video.
The assays described here can be used to qualitatively and quantitatively assess larvae of different genotypes for responsiveness to noxious thermal stimuli. A main feature of the heat probe assay is that the stimulus is given only at a single locus. This presumably leads to firing of only a small subset of class IV neurons- the ones in the segment contacted by the probe and perhaps those in immediately adjacent segments11. Because of the local stimulation, the heat probe assay mimics the common sensory experience of detecting a noxious stimulus that is localized to a particular body region- such as a hand contacting a hot stove. A disadvantage of the heat probe assay is that it has some user-to-user variability that can likely be attributed to three factors: i) the pressure with which the user applies the probe to the larva, ii) the precise location of the probe on the larva relative to the underlying nociceptive neurons, and, iii) the precise angle at which the probe contacts the surface of the larva.
We previously reported a quantitative strategy of categorizing larvae into non-responders, slow responders, and fast responders based on their withdrawal latency to a given temperature1. Here we report on larval responsiveness to even higher temperatures. Interestingly, we find that there is a ceiling to larval thermal nociceptive responses and that this ceiling lies between 52 and 54 °C. This may indicate that larvae do not possess a transient receptor potential (TRP) channel capable of gating at temperatures higher than 52 °C. Alternatively, it could suggest that the neurons or muscles used to initiate or carry out the motor response become damaged before they can even function in aversive withdrawal. We also report a different analysis of the amplitude of the withdrawal response- using either the number of rolls as an indicator of the "robustness" of the response. Naively, one would expect that these parameters would increase with increasing temperature or time of stimulation. Surprisingly, we find that this is not the case. Larvae stimulated for a longer time at a temperature at the low end of the noxious range (42 °C) show more rolls and more time spent rolling than larvae probed at higher temperatures (48-52 °C). This suggests that within the noxious temperature window it is primarily the duration of exposure that determines the amplitude of the response. Since larvae exposed to highly noxious temperatures (48-52 °C) respond on average very quickly, they do not exhibit as many rolls as larvae exposed to a less noxious temperature for a longer length of time. The analysis of response amplitude reported here adds another quantitative dimension along which different genotypes or environmental manipulations can be compared.
A main feature of the heat plate assay is that it involves a global exposure to the noxious heat. As such, it is more akin to the animal sitting in a heating cauldron than touching a hot stove. Although it is not clear when a larva might experience a globally noxious stimulus in the wild, in the lab the behavioral responses to this global exposure are more complex than those observed upon local stimulation. A strength of the heat plate assay, also noted by others3, is that it has little user-to-user variability since touching the larva is not a component of the protocol. The only substantial variance seems to be in defining when each behavior commences and this can be minimized with repeated viewing/familiarity. An interesting difference between the assays is the temperatures at which the aversive behaviors commence. These are much lower in the heat plate assay than with the heat probe. The preliminary behavior exhibited by larvae contacted with the heat probe (head and tail raise) may be a correlate of the head thrash observed at ~27 °C in the heat plate assay. It is possible that this response reflects "discomfort" more than "pain". We have not observed a correlate of whipping, seizure, and paralysis even at high (up to 48°C) temperatures in the heat probe assay and it may be that a critical mass of sensory neuron firing from more than one region of the body is needed to bring on these behaviors. Interestingly, the seizure and paralysis behaviors are observed at temperatures (~34 – 37 °C) below the bottom end of the nociceptive threshold observed with the heat probe indicating that global stimulation may involve summing of neuronal responses that are insufficient to trigger behavior with local application of the heat probe. That these temperatures are actually perceived as noxious to the larvae is supported by the observation that larvae that begin the paralysis behavior and are subsequently allowed to recover on fly food do not in most cases survive (Figure 5C). Further supporting the argument that the heat plate assay is reading out nociceptive responses is the fact that blocking synaptic transmission in known nociceptive sensory neurons increases the latency of most of the observed behaviors (Figure 6). The observation that there is no increase in latency for the higher-temperature whipping behavior suggests that other sensory neurons that do not express md-Gal4 may be required for this behavior.
In sum, both assays involve exposing an individual larva to a noxious thermal stimulus of defined temperature - the hot tip of a small metal probe in the local assay and immersion in a drop of rapidly heating water in the global assay. Behavioral responses of Drosophila larvae of varying genetic backgrounds and/or exposed to varying environmental conditions (for instance plus or minus tissue damage), can be studied and quantified using these assays. Ultimately, results from these assays will help us better understand genetic networks controlling nociception in Drosophila and other related species.
We have nothing to disclose.
We thank Christian Landry for heat probe design, Daniel Babcock for developing the larval heat probe assay, Sean Sweeney for suggesting the heat plate assay, the Bloomington Drosophila Stock Center for fly stocks, and Galko lab members for critical reading of the manuscript. This work was supported by NIH R01 NS069828 to MJG and an NIH MARC U-STAR Training Grant (T34GM079088 to the University of Houston-Downtown Scholars Academy) for minority access to research careers (AVG).
|Thermal Probe||Pro-Dev Engineering||Custom-built on demand||Contact information can be provided on request|
|Dry Bath Incubator||Fisher Scientific||11-718||1 solid heating block and 1 heating block with 16mm wells|
|Leica DFC290 12v/400mA Color camera||Leica Microsystems||12730080||Any equivalent
camera will do.
|Leica MZ6 microscope||Leica Microsystems||Part number for MZ6 zoom body (optics carrier) is 10445614|
|Schott Ace Modulamp Unit||Schott AG||A20500|
|Schott Dual Gooseneck 23 inch Fiber Optic Light Guide||Schott AG||Schott A08575|
|Thermal Control Unit||TSCI corp.||Custom Built||Details can be provided on request|
|Zeiss Stemi 2000 microscope||Carl Zeiss, Inc.||NT55-605||Any equivalent
microscope will do.
|Forceps||Fine Science Tools||FS-1670|
|1mm mesh||Genesee Scientific||57-101|
|Paintbrush||Blick Art Materials||06762-1002|
|UV crosslinker||Fisher Scientific||1199289|
|Coplin Jars||Fisher Scientific||08-816|
|10ml beaker||Fisher Scientific||02-540C|
|Diethyl ether||Fisher Scientific||E138-500|
|35 X 10 mm Polystyrene Petri Dish||Falcon BD||351008||We have not tested
|Glass Microscope Slide||Corning||26003|
|Thermocouple||Omega Engineering, Inc.||HH802U|
|Piece of vinyl||Office Depot||480009|
|Microcentrifuge tube||Denville Scientific||C-2170|