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Jul 2019

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Evaluating Baseline and Sensitised Heat Nociception in Adult Drosophila
评估成年果蝇的基线和致敏热伤害感受   

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Abstract

Chronic pain is a complex disease that affects a large proportion of the population. With little to no effective treatments currently available for patients, this malady presents a large burden to society. Drosophila melanogaster has been previously used to describe conserved molecular components of nociception in larvae and adults. However, adult assays tend to rely on avoidance behaviours, and whilst larval acute thermal avoidance assays exist, larvae are not best suited to a chronic pain scenario as the condition must be long-term. Therefore, an adult thermal nociception response assay was required to study injury-evoked changes in heat nociception threshold (allodynia and hyperalgesia) over time, and we describe such a protocol here. Following leg amputation, flies display increased thermal sensitivity (allodynia) to innocuous temperatures but not an increase in magnitude of response (hyperalgesia) to noxious heat. Our method allows for individualised analysis of both allodynia and hyperalgesia.

Keywords: Drosophila (果蝇), Nociception (伤害性感受), Hyperalgesia (痛觉过敏), Allodynia (异常性疼痛), Nerve-injury (神经损伤), Heat (热), Hot-plate (加热板), Chronic-pain (慢性疼痛)

Background

Chronic pain represents a substantial burden on society. Patients with this malady often suffer and experience a reduced quality of life (Campbell and Meyer, 2006; Pfau et al., 2012). Importantly, available treatment options are ineffective for the majority of chronic pain patients (Turk et al., 2011); however, an understanding of the basic underlying biology will lead to effective therapeutics (Grosser et al., 2017).


Extensive modelling of chronic pain has been performed in rodents (Costigan et al., 2009); however, these systems are expensive, the genetics of evaluating mouse pain responses are slow, and inducing chronic pain in a significant numbers of animals is required, which can have ethical implications. In contrast, Drosophila are inexpensive to maintain, quick to raise, and have an extensive molecular toolbox that allows rapid genetic manipulation.


Much work has been done to define the underlying nociceptive machinery required for invertebrate “pain” perception (Tracey et al., 2003; Kang et al., 2010; Neely et al., 2010 and 2011; Hamoudi et al., 2018). These systems are primarily dependent on investigating nociception reflexes in the transient larval stage (Babcock et al., 2009 and 2011; Turner et al., 2016; Patel and Cox, 2017; Lopez-Bellido and Galko, 2020) or thermal avoidance assays in adult flies. The first adult Drosophila avoidance assay described involved a sealed tube, noxiously heated, and a light source at one end, which flies were prevented from reaching because of a noxious heat barrier (Manev and Dimitrijevic, 2004). An alternate acute heat nociception system relied on floating a sealed “heat” chamber on hot water, where one surface of the chamber was then rapidly heated to 46°C, while the other reached 31°C during a 4-min trial. Flies with intact heat nociception avoided the hot surface, whereas flies that could not sense heat, or those that had heat-related motor issues or other confounding phenotypes, would fail to avoid the hot surface and rapidly become incapacitated (Neely et al., 2010).


While these assays are useful for assessing a loss of acute heat nociception, they are ineffective for evaluating sensitisation of nociceptive pathways. Here, we present an adaptation of our previously described method (Khuong et al., 2019) for analysis of acute thermal nociception responses, which allows for individualised assessment of both allodynia and hyperalgesia following injury.

Materials and Reagents

  1. Sigmacote (Sigma Aldrich, catalog number: SL2)

  2. Falcon bacteriological Petri dish lids, sizes 35-60 mm (Falcon, Corning, catalog number: 351008)

  3. Sandpaper

  4. Heat Sink Compound (RS, RS Pro, catalog number: 554-311)

  5. Organic Fine Corn Flour (HBC Trading Australia, TUN number: 19339337303842)

  6. Molasses (Whole Body Health Company, catalog number: MOL 600)

  7. Yeast torula type B (H.J. Langdon Co., catalog number: 45014)

  8. Agar 750 g (H.J. Langdon Co., catalog number: 44305)

  9. Dulux Metalshield Flat Vivid White Epoxy Enamel Spray Paint (or similar) (Dulux, catalog number: 32C04912)

  10. Diets (see Recipes)

Equipment

  1. Bottle 6 oz square bottom PP (Pathtech, Genesee Scientific, catalog number: 076-32-130F)

  2. Drosophila vials, Narrow, PS (Pathtech, Genesee Scientific, catalog number: 076-32-109)

  3. Benchtop Flowbuddy Complete (Pathtech, Genesee Scientific, catalog number: 076-59-122BC)

  4. FitoClima 600/1200 PLH Insect Research Chamber (Aralab)

  5. Superfine Vannas Scissors, 8 cm (World Precision Instruments, catalog number: 501778)

  6. Viltrox LL-126VB LED light (Viltrox, catalog number: LL-126VB)

  7. Hot/Cold plate NG (Ugo Basile, catalog number: 35150)

  8. Testo 925 K input handheld digital thermometer (RS, testo, 0560 9250)

  9. C920 HD pro webcam (Logitech, catalog number: 960-000764)

  10. Aluminium plate (200 × 200 × 3 mm), manufactured in-house

Software

  1. BORIS (Friard and Gamba, 2016, https://www.boris.unito.it/)

  2. Ctrax (Branson et al., 2009, http://ctrax.sourceforge.net/)

  3. any2ufmf (Branson et al., 2009, http://ctrax.sourceforge.net/any2ufmf.html)

  4. R (R foundation, https://www.r-project.org)

  5. R Studio (https://www.rstudio.com)

  6. Github Repository (https://github.sydney.edu.au/jmas3890/Hot-Plate-Assay.git)

Procedure

  1. Fly preparation and injury

    Note: Aside from the physical leg amputation, control flies should be treated exactly the same as experimental flies.

    1. Prepare fly crosses at a standard density of 20 female and 5 male flies for two days at 25°C, 65% humidity, and a 12 h light/dark cycle before removing parents.

    2. Two days following initial pupae eclosion, place F1 progeny into containers and allow to mate for two days.

    3. Select healthy, intact male flies on light CO2 (less than 5 L/min CO2 using a Flowbuddy, delivered through a Flypad) at a standard density in standard Drosophila vials and allow to age until flies are between 7-9 days old, flipping onto new food every 2 days.

    4. Under light CO2, amputate the right middle leg femur using surgical scissors and also expose control uninjured animals to CO2 in parallel (Figure 1).

    5. Place one fly per vial and allow to recover for a further 7 days, flipping onto new food every 2 days.



      Figure 1. Leg injury model showing the location of femur amputation


  2. Hot plate preparation

    1. Obtain an aluminium square plate with dimensions matching the diameter of the hot plate (ensure this plate is between 1-5 mm thick to ensure efficient heat transfer).

    2. Using thin layers, paint the aluminium plate with white matte paint.

      Note: Spray paint works best for this.

      1. Painting white is to ensure good tracking, as dark flies on a white background works best; however, if this is not a concern, skip this step.

      2. Once the paint has dried, place the aluminium plate over the top of the Ugo Basil Hot/Cold plate (Figure 2).



      Figure 2. Arrangement of the equipment required for the hot plate assay. A. Arrangement of the Ugo Basile Hot/cold plate, Digital thermometer, Aluminium plate, Arena, Camera, and External fan. B. Simplified overhead view showing the location of the aluminium plate above the Ugo Basile hot/cold plate and arena.


  3. Arena preparation

    1. Take the 35-60-mm diameter Petri dish lid and using sandpaper, remove most of the walls until they are between 1-2 mm high.

    2. Treat with Sigmacote to help stop the flies from climbing on the walls and ceiling.

      Note: 1-2 coats may be necessary.


  4. Generating a standard curve

    1. Set the hot plate to ramp from 25°C to 50°C over 3 min.

    2. Using a heat-transferring medium, place the tip of a digital thermal probe in the centre of the plate (or roughly where the fly will be).

    3. Allow the plate to run and cool several times and record the temperature every 2 s for 3 min and 15 s.

      Notes:

      1. This is best done by recording the readout with a camera, C920 HD pro webcam, or similar.

      2. This will be used later to correlate the time of the behaviour to the temperature at that time.


  5. Hot plate assay

    1. Set the hot plate to ramp from 25°C to 50°C over 3 min.

    2. Place an individual fly onto the plate and quickly cover/capture with the premade arena.

      Notes:

      1. This can be done by simply flipping an individual fly onto the plate from its vial and rapidly covering with the arena (less than 30 s recovery is required for this method). Alternatively, aspirators or light anaesthesia with ice can be used (between 1-2 min and then allow double that time for recovery).

      2. CO2 should not be used to anaesthetise flies on the day of the nociception experiment as this may affect behaviour.

    3. Start the camera (1080 p and 30 fps) and the hot plate at the same time and record for 3 min and 15 s (195 s) before allowing the plate to cool again.

      Note: Multiple external fans can be used to aid cooling.

    4. Before beginning the next experimental replicate, ensure the plate has returned to 25°C using a thermal probe.

    5. Repeat five times per experimental group (i.e., 5 control flies and 5 injured flies) for one technical replicate.

      Technical replicates should be performed on separate days to avoid same-day experimental error.

Data analysis

  1. Escape Response (Jump) Analysis using the BORIS behavioural Tracking Software. Detailed user manuals for the BORIS software can be found on their website (https://www.boris.unito.it/).

    1. Create a new project and set the time format to seconds.

    2. Create the ethogram with two behaviours. Assign these to any Key you wish; however, it is often easiest to use numbers such as 1 and 2. The behaviours we assign are:

      1. Jump behaviour (Figure 3).

      2. Death (this can also be useful in assessing thermal sensitivity).

        Note: Here death is defined by fly curling and cessation of further movement.



      Figure 3. Consecutive frames from overhead video showing jump behaviour. Bottom left corner of each frame shows whole arena view.


    3. Save this project in the same location as the blinded videos.

    4. Create a new observation and name it the same as the video you will be scoring.

      Note: For ease of use, we randomly assign videos a sequential number.

    5. Limit the observation time between 0.000 s and 195.000 s (the length of the behavioural assay) and then load the video to score.

    6. Manually annotate jumps in blinded videos. Videos can be paused, slowed down, or skipped backwards to aid in behavioural annotation.

    7. Once all videos have been scored as their own observation, use the “export events” option under the “Observation” tab to export events as tabular events and select “.csv” as the file option. Be sure to export Death events and Jump events separately and into separate folders.


  2. Behavioural events analysis

    In general, this analysis script has three main components: standard curve creation, unblinding using the Key, and jump tabulation. Here, we provide a general description of the formatting required for the standard curve and key files and how these are then utilised within the script to perform analysis.

    To perform analysis, simply place all files (event .csv files, the standard curve file, and the Key) into the same folder, change the working directory to the path where this is stored (where to do this is indicated within the script), and execute the script. All scripts needed are in the GitHub Repository, as well as example files.


    1. Standard curve.

      The script will generate a curve from the average temperature at the time it was recorded. This curve is then utilised later in the script to correlate the time of the jump to the temperature at that time.

      Place temperatures recorded whilst generating the standard curve in a .csv file with columns labelled (Time, Rep1, Rep2, Rep3; see example provided in the GitHub Repository).

    2. Unblinding.

      This process will take the name of the event file exported from BORIS, which should correspond to the blinded video name (i.e., 1.csv, 2.csv, etc.), and correlate that to the original name of the video as listed in the Key. For this process to work properly, it is important that the key file be formatted correctly.

      The key should be formatted so that column 1 is the original name and column 2 is the blinded name (we use increasing numbers).

      Note: The unblinding process will convert the blinded names into a numeric value within the script. If numbers were not used in the blinding process, this section of the script may no longer work and will need to be rewritten for your analysis. The relevant section is marked in the script contained within the GitHub Repository.

    1. Tabulation of the jumps (Figure 4).

      1. Tabulation is done at 1-degree bin intervals (although this can be altered and is indicated where to do so in the script).

      2. Tabulation is started at 27.5°C and ends at 49.5°C; these temperatures can also be altered in the script. This process will include events at 49.5°C.

      3. This script will export a file where columns show individual flies and rows show the number of escape behaviours in that bin. Bins increase by 1°C and are labelled by the lower value and not the bin centre, which will be 0.5°C higher (i.e., a bin of 27.5-28.5°C will have a label of 27.5°C and a bin centre of 28°C).

      4. There is also a function that will tabulate total jumps, irrespective of temperature.

        Note: Multiple types of analysis can then be performed, including comparison of performance at individual temperature bins or peak analysis to understand the shape and distribution of the jumps.



    Figure 4. Jumps in control and injured Canton-S flies. 7-9-day-old male Canton-S flies underwent right femur amputation, and their escape response to thermal stimulation was recorded 7 days following initial injury. Data are presented as the mean ± SEM.


  1. Velocity analysis (Figure 5)

    Velocity analysis can be a useful tool to identify animals that may have motor issues arising from poor health (apart from the intended injury). Significant drops in velocity within experimental groups can be used to determine whether these animals should be removed from all analyses. Such animals can be identified using outlier removal assessment on the average velocity for the entire assay.

    1. Convert video files to .ufmf format (using “any2ufmf”).

    2. Track flies using Ctrax software or other appropriate tracking software (Detailed user guides for Ctrax and any2ufmf can be found on their website, http://ctrax.sourceforge.net/).

      Note: Due to the increased velocity at high temperatures, tracking is prone to errors. Increasing both frame rate and resolution will aid but not eliminate the possibility of errors occurring. Ctrax provides a MATLAB platform to manually annotate tracking errors.

    3. Following tracking, export as “.mat” file extension and use the R script to estimate velocities (velocities are estimated using the simple formula v = d/t). To perform analysis, place all relevant files into the same folder, change the working directory to this folder and then execute the R script.

      1. This R script will work in a very similar fashion to the escape response script; however, it will not perform unblinding, so videos must not be blinded. It will also require similar inputs (i.e., a standard curve file and all .mat files).

      2. Tracking errors can be removed at this step using a simple outlier removal test.

      3. This script will output two .csv files. One file will show columns as individual flies and rows as the velocity that occurred at the corresponding temperature. The other file will show the average velocity for each individual fly.



    Figure 5. Velocity (pixels.s-1) in control and injured Canton-S flies. 7-9-day-old male Canton-S flies underwent right femur amputation, and their escape response to thermal stimulation was recorded 7 days following initial injury. Data are presented as the mean ± SEM.

Notes

This protocol will have a large amount of variability based on the individual experimenter. The scoring should be performed blind and by the same person for each project. Further, poor data quality will occur under poor fruit fly health, and food quality will contribute largely to this. Flies should be kept on fresh food and transferred to new food every 2-3 days. This experiment is best performed in triplicate, with three separate crosses and three separate days of recording to eliminate same-day experimental error.

Recipes

  1. Diets

    Corn flour (3.5% (w/v))

    Agar (2% (w/v))

    Molasses (8% (w/v))

    Yeast (1.2% (w/v)) and water

Acknowledgments

This method is an adaptation of the method described by Khuong et al. (2019), DOI: 10.1126/sciadv.aaw4099.

This work was supported in part through National Health and Medical Research Council (NHMRC) project grants APP1026310, APP1029672, APP1028887, APP1046090, APP1042416, and APP1086851. G.G. Neely was supported by an NHMRC career development fellowship II CDF1111940. Finally, we thank the generosity of John Chong and Anne Chong for their financial support of work in our laboratory.

Competing interests

The authors of this work have no conflicts of interest to declare.

References

  1. Babcock, D. T., Landry, C. and Galko, M. J. (2009). Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae. Curr Biol 19(10): 799-806.
  2. Babcock, D. T., Shi, S., Jo, J., Shaw, M., Gutstein, H. B. and Galko, M. J. (2011). Hedgehog signaling regulates nociceptive sensitization. Curr Biol 21(18): 1525-1533.
  3. Branson, K., Robie, A. A., Bender, J., Perona, P. and Dickinson, M. H. (2009). High-throughput ethomics in large groups of Drosophila. Nat Methods 6(6): 451-457.
  4. Campbell, J. N. and Meyer, R. A. (2006). Mechanisms of neuropathic pain. Neuron 52(1): 77-92.
  5. Costigan, M., Scholz, J. and Woolf, C. J. (2009). Neuropathic pain: a maladaptive response of the nervous system to damage. Annu Rev Neurosci 32: 1-32.
  6. Friard, O., Gamba, M. J. (2016). BORIS: a free, versatile open‐source event‐logging software for video/audio coding and live observations. Methods Ecol Evol 7(11): 1325-1330.
  7. Grosser, T., Woolf, C. J. and FitzGerald, G. A. (2017). Time for nonaddictive relief of pain. Science 355(6329): 1026-1027.
  8. Hamoudi, Z., Khuong, T. M., Cole, T. and Neely, G. G. (2018). A fruit fly model for studying paclitaxel-induced peripheral neuropathy and hyperalgesia. F1000Res 7: 99.
  9. Kang, K., Pulver, S. R., Panzano, V. C., Chang, E. C., Griffith, L. C., Theobald, D. L. and Garrity, P. A. (2010). Analysis of Drosophila TRPA1 reveals an ancient origin for human chemical nociception. Nature 464(7288): 597-600.
  10. Khuong, T. M., Wang, Q. P., Manion, J., Oyston, L. J., Lau, M. T., Towler, H., Lin, Y. Q. and Neely, G. G. (2019). Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila. Sci Adv 5(7): eaaw4099.
  11. Lopez-Bellido, R. and Galko, M. J. (2020). An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae. J Vis Exp (164).
  12. Manev, H. and Dimitrijevic, N. (2004). Drosophila model for in vivo pharmacological analgesia research. Eur J Pharmacol 491(2-3): 207-208.
  13. Neely, G.G., Hess, A., Costigan, M., Keene, A.C., Goulas, S., Langeslag, M., Griffin, R.S., Belfer, I., Dai, F., Smith, S.B., Diatchenko, L., Gupta, V., Xia, C. P., Amann, S., Kreitz, S., Heindl-Erdmann, C., Wolz, S., Ly, C. V., Arora, S., Sarangi, R., Dan, D., Novatchkova, M., Rosenzweig, M., Gibson, D. G., Truong, D., Schramek, D., Zoranovic, T., Cronin, S. J., Angjeli, B., Brune, K., Dietzl, G., Maixner, W., Meixner, A., Thomas, W., Pospisilik, J. A., Alenius, M., Kress, M., Subramaniam, S., Garrity, P. A., Bellen, H. J., Woolf, C. J. and Penninger, J. M. (2010). A genome-wide Drosophila screen for heat nociception identifies alpha2delta3 as an evolutionarily conserved pain gene. Cell 143(4): 628-638.
  14. Neely, G. G., Keene, A. C., Duchek, P., Chang, E. C., Wang, Q. P., Aksoy, Y. A., Rosenzweig, M., Costigan, M., Woolf, C. J., Garrity, P. A. and Penninger, J. M. (2011). TrpA1 regulates thermal nociception in Drosophila. PLoS One 6(8): e24343.
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简介

[摘要]慢性疼痛是一种复杂的疾病,影响着很大一部分人口。由于目前对患者几乎没有有效的治疗方法,这种疾病给社会带来了巨大的负担。黑腹果蝇以前曾被用来描述幼虫和成虫伤害感受的保守分子成分。然而,成人试验往往依靠避税行为,同时存在幼虫急性热回避试验中,幼虫都没有最适合于慢性疼痛的情况作为条件必须是长- 学期。因此,需要成人热痛觉反应试验来研究热痛觉阈值(异常性疼痛和痛觉过敏)随时间的损伤诱发的变化,我们在这里描述了这样的协议。在腿部截肢后,果蝇对无害温度表现出增加的热敏感性(异常性疼痛),但对有害热量的反应(痛觉过敏)幅度没有增加。我们的方法允许对异常性疼痛和痛觉过敏进行个性化分析。

[背景]慢性疼痛是社会的沉重负担。患有这种疾病经常遭受体验一个生活质量下降(Campbell和迈耶,2006年;普福等,2012) 。重要的是,现有的治疗方案对大多数慢性疼痛患者无效(Turk等,2011);然而,一个Ñ理解的基本基础生物学的将导致有效的治疗(哥洛莎等人,2017) 。

已经在啮齿类动物中进行了广泛的慢性疼痛建模(Costigan等,2009);然而,这些系统是昂贵的,评估小鼠疼痛反应的基因的慢,而且诱导的慢性疼痛一动物显著数量需要,它可以有伦理问题。相比之下,果蝇的维护成本低,饲养速度快,并且拥有广泛的分子工具箱,可以进行快速的基因操作。

已经做了很多工作来定义无脊椎动物“疼痛”感知所需的潜在伤害性机制(Tracey等,2003;Kang等,2010;Neely等,2010 和 2011;Hamoudi等,2018)。这些系统主要依赖于研究瞬态幼虫阶段的伤害感受反射(Babcock等人,2009 年和 2011 年;Turner等人,2016 年;Patel 和 Cox,2017 年;Lopez-Bellido 和 Galko,2020 年)或在成年苍蝇。描述的第一个成年果蝇回避试验涉及一个密封管,有害加热,一端有光源,由于有害的热屏障,苍蝇无法到达(Manev 和 Dimitrijevic,2004)。一个替代急性热伤害感受系统依靠浮动密封的“热”室上热水,其中,所述腔室的一个表面上,然后迅速加热至46℃,而另一个为4时达到31°C -分钟试验。具有完整热伤害感受的果蝇会避开热表面,而无法感知热的果蝇,或那些有与热量相关的运动问题或其他混淆表型的果蝇,将无法避开热表面并迅速丧失行动能力(Neely等,2010 ) 。

虽然这些测定可用于评估急性热伤害感受的损失是有用的,它们是在有效用于评估伤害感受通路的致敏。在这里,我们提出了对我们先前描述的方法(Khuong等人,2019 年)的改编,用于分析急性热伤害反应,这允许对损伤后的异常性疼痛和痛觉过敏进行个性化评估。

关键字:果蝇, 伤害性感受, 痛觉过敏, 异常性疼痛, 神经损伤, 热, 加热板, 慢性疼痛



材料和试剂


Sigmacote (Sigma Aldrich,目录号:SL2)
鹘b acteriological的Petri d ISH盖子,尺寸35-60毫米中(Falcon,康宁,目录号:351008)
砂纸
散热器化合物(RS,RS Pro,目录号:554-311)
有机细玉米粉(HBC Trading Australia,TUN 编号:19339337303842)
糖蜜(w ^孔乙ODY ħ ealth Ç ompany ,目录号码:MOL 600)
B型酵母圆环(HJ Langdon Co.,目录号:45014)
琼脂 750 g(HJ Langdon Co.,目录号:44305)
多乐士Metalshield Flat Vivid 白色环氧搪瓷喷漆(或类似产品)(多乐士,目录号:32C04912)
饮食(见食谱)


设备


瓶子6盎司方底PP(Pathtech ,Genesee Scientific,目录号:076-32-130F)
果蝇v IALS,精细,PS(Pathtech ,杰纳西科学,目录号码:076-32-109)
Benchtop F lowbuddy Complete(Pathtech ,Genesee Scientific,目录号:076-59-122BC)
FitoClima 600/1200 PLH 昆虫研究室 ( Aralab )
Superfine Vannas Scissors,8 cm(World Precision Instruments,目录号:501778)
Viltrox LL-126VB LED 灯(Viltrox ,目录号:LL-126VB)
热/冷板NG(Ugo Basile,目录号:35150)
Testo 925 K 输入手持式数字温度计(RS,testo,0560 9250)
C920 HD pro 网络摄像头(罗技,目录号:960-000764)
铝板(200 × 200 × 3 mm),内部制造




软件


BORIS (Friard和岗巴,2016年,https://www.boris.unito.it/)
Ctrax (布兰森等人,2009,http://ctrax.sourceforge.net/)
any2ufmf (布兰森等人,2009 年,http: //ctrax.sourceforge.net/any2ufmf.html )
R(R 基金会,https://www.r-project.org)
R Studio ( https://rstudio.com )
Github上ř epository(https://github.sydney.edu.au/jmas3890/Hot-Plate-Assay.git)


程序


飞准备和我njury
注意:除了物理腿截肢,对照苍蝇应与实验苍蝇完全一样对待。


在 2 5 的两天内以 20 只雌性和 5 只雄性苍蝇的标准密度准备飞交叉° Ç ,65%湿度,以及一个去除父母之前12小时光照/黑暗周期。
在最初的蛹羽化两天后,将 F1 后代放入容器中并允许交配两天。
上的光CO选择健康的,完整的雄蝇2 (升ESS大于5升/分的CO 2使用Flowbuddy ,通过一个递送Flypad )在标准标准密度果蝇小瓶中并允许其老化直到苍蝇7-9天龄之间,每 2 天换一次新食物。
在轻度 CO 2 下,使用手术剪截断右中腿股骨,同时将对照未受伤动物暴露于 CO 2中(图 1 )。
每瓶放一只苍蝇,并允许再恢复 7 天,每 2 天换一次新食物。


图片包含,动物,水,房间描述已自动生成


图1 。腿部受伤的模型显示了股骨截肢的位置


热板准备
得到铝方板与尺寸小号热板的直径相匹配(这确保板为1-5毫米厚之间,以确保有效的热传递)。
使用薄层,在铝板上涂上白色哑光漆。 
注:小号祈祷涂料最适合这个。


画白色是为了确保良好的跟踪,因为白色背景上的黑色苍蝇效果最好;但是,如果这不是问题,请跳过此步骤。
油漆干燥后,将铝板放在Ugo Basil 热/冷板的顶部(图 2 )。


图形用户界面 描述已自动生成


图2 。的布置的设备所需的热板试验。一个。的排列的羽后巴西莱热/冷板,数字吨hermometer,铝板,竞技场,照相机,和外部风扇。乙。简化示出俯视图的位置的铝板的上方的羽后巴西莱热/冷板和舞台。


竞技场准备
取35-60 -毫米直径的Petri培养皿盖,并使用砂纸,除去大部分的壁直到它们之间1-2毫米高。
治疗与Sigmacote帮助制止了苍蝇从攀爬墙壁和天花板上。
注意:可能需要 1-2 层。


生成一个标准曲线
将热板设置为在3 分钟内从 25 °C升至 50° C 。
使用热-转印介质,放置所述的尖端一个数字热探针在板的中心(或大致其中蝇会)。
让板运行并冷却几次,并每 2 秒记录一次温度,持续 3 分钟和 15 秒。
注意小号:


这是最好的拍摄用相机,C920高清网络摄像头亲读出完成,或类似的。
这将在稍后用于将行为的时间与当时的温度相关联。


热p后期分析
将热板设置为在3 分钟内从 25°C 升温至 50° C 。
将一只苍蝇放在盘子上,然后用预制的舞台快速覆盖/捕获。
笔记:


这可以通过简单地将单个苍蝇从其小瓶翻转到板上并快速覆盖竞技场来完成(此方法需要不到 30 秒的恢复时间)。或者,可以使用抽吸器或轻度冰麻醉(在 1-2 分钟之间,然后让恢复时间加倍)。
CO 2不应在伤害感受实验当天用于麻醉苍蝇,因为这可能会影响行为。
同时启动相机(1080 p 和 30 fps)和热板,并在让板再次冷却之前记录 3 分 15 秒(195 秒)。
注意:可以使用多个外部风扇来帮助冷却。


开始前的下一个实验重复,保证了板已经返回至25℃使用一个热探针。
每个实验组重复5次(即,5只对照果蝇和5只人受伤苍蝇)为一个技术重复。
技术重复应在不同的日子进行,以避免同-天实验误差。


数据一nalysis


逃生使用响应(跳转)分析了BORIS行为跟踪软件。有关详细的用户手册中的BORIS软件可以在他们的网站上找到(https://www.boris.unito.it/)。
创建一个新项目并将时间格式设置为秒。
创建具有两种行为的 ethogram 。一个SSIGN这些任何ķ你想EY ; 然而,使用 1 和 2 等数字通常是最简单的。我们分配的行为是:
跳转行为(图 3 )。
死亡(这也可用于评估热敏感性)。
注意:这里的死亡是由飞卷曲和停止进一步运动来定义的。


图片包含 文字, 墙壁, 白色 描述已自动生成


图3 。来自头顶视频的连续帧显示跳跃行为。每帧的左下角显示整个竞技场视图。


将此项目保存在与盲视频相同的位置。
创建一个新观察并将其命名为与您要评分的视频相同的名称。
注意:为了便于使用,我们随机为视频分配一个序列号。


将观察时间限制在 0.000 秒和 195.000 秒之间(行为al检测的长度),然后加载视频进行评分。
手动注释盲视频中的跳跃。影片可以暂停,放慢,或跳过向后,以帮助我ñ行为人的注释。
将所有视频作为自己的观察评分后,使用“观察”选项卡下的“导出事件”选项将事件导出为表格事件,并选择“.csv”作为文件选项。请务必将死亡事件和跳转事件分别导出到单独的文件夹中。


行为人的事件分析
一般情况下,这种分析的脚本有三个主要组件:小号TANDARD曲线创建,使用揭盲ķ EY ,和跳跃列表。在这里,我们提供了标准曲线和关键文件所需的格式的一般描述,以及如何在脚本中利用这些来执行分析。


  为了进行分析,只需将所有文件(事件的.csv文件,标准曲线文件,以及ķ EY)到同一个文件夹,更改工作目录的路径,这是存储在哪里(在哪里做,这是在脚本中表示) ,然后执行脚本。需要的所有脚本都在GitHub的[R epository以及示例文件。


1.标准曲线。     

该脚本将根据记录时的平均温度生成一条曲线。然后在脚本中稍后使用该曲线来将跳跃时间与当时的温度相关联。


将在生成标准曲线时记录的温度记录在 .csv 文件中,列标记为(时间、Rep1、Rep2、Rep3 ;参见GitHub 存储库中提供的示例)。


2.揭盲。     

这一过程将花费BORIS导出的事件文件的名称,这应该对应于盲视频名称(即,1.csv,2.csv,等等。),以及对视频的原始名称中列出的归属关系钥匙。要使此过程正常工作,正确格式化密钥文件很重要。


关键应格式化,使第1列是原来的名字和列2是盲名(w ^ E使用增加的数字)。


注意:解盲过程将在脚本中将盲名转换为数值。如果在盲法过程中未使用数字,则脚本的这一部分可能不再起作用,并且需要重新编写以进行分析。相关部分在 GitHub 存储库中包含的脚本中进行了标记。


跳跃的列表(图 4 )。
制表是以 1 度的 bin 间隔完成的(尽管这可以更改并且在脚本中指示了在哪里这样做)。
制表开始于 27.5°C,结束于 49.5°C ;这些温度也可以在脚本中更改。此过程将包括 49.5°C 下的事件。
此脚本将导出一个文件,其中列显示单个果蝇,行显示该 bin 中的逃逸行为数量。bins 增加 1°C,并用较低的值而不是 bin 中心标记,后者将高 0.5°C(即,27.5-28.5°C 的 bin 将具有 27.5°C 的标签和 bin 中心28°C)。
还有一个功能可以将总跳跃量制成表格,而与温度无关。
注意:然后可以执行多种类型的分析,包括比较各个温度区间的性能或峰值分析,以了解跳跃的形状和分布。






图4 。跳跃控制和受伤的 Canton-S 飞行。7 - 9天-老雄广-S飞行右股骨截肢和他们逃跑反应热刺激被记录7天后最初的损伤。数据表示为在平均值±SEM。


速度一个nalysis (图5 )
速度分析可识别的动物一个有用的工具是可能出现的,从健康状况不佳(除了预期损伤)电机的问题。在实验组内速度显著滴可用于确定是否这些动物应从所有analys去除ë秒。可以使用对整个测定的平均速度的异常值去除评估来识别此类动物。


将视频文件转换为 . ufmf格式(使用“any2ufmf”)。
轨道苍蝇使用Ctrax软件或其它适当的跟踪软件(详细的用户指南,Ctrax和any2ufmf可以Ø找到ñ他们的网站,http://ctrax.sourceforge.net/)。
注意:由于在高温下速度增加,跟踪容易出错。提高帧速率和分辨率将有助于但不能消除发生错误的可能性。Ctrax提供了一个 MATLAB 平台来手动注释跟踪错误。


跟踪后,导出为“.mat ”文件扩展名并使用 R 脚本估算速度(使用简单公式v = d/t估算速度)。要执行分析,请将所有相关文件放在同一文件夹中,将工作目录更改为该文件夹,然后执行 R 脚本。
这个 R 脚本的工作方式与转义响应脚本非常相似;^ h H但是,它不会进行揭盲,所以影片不能被蒙蔽。这也需要类似的输入(即,标准曲线文件和所有.MAT文件)。
在这一步可以使用简单的异常值去除测试来去除跟踪错误。
此脚本将输出两个 .csv 文件。一个文件将列显示为单独的苍蝇,行显示为在相应温度下发生的速度。另一个文件将显示每个单独飞行的平均速度。






图5 。控制和受伤的 Canton-S 苍蝇的速度 (pixels.s -1 )。7 - 9天-老雄广-S飞行右股骨截肢和他们逃跑反应热刺激被记录7天后最初的损伤。数据表示为在平均值±SEM。


笔记


该协议将具有基于个体实验者的大量可变性。每个项目的评分应由同一人盲进行。此外,在果蝇健康状况不佳的情况下,数据质量会很差,而食品质量将在很大程度上促成这一点。苍蝇应保持新鲜食物,并每 2-3 天转移到新食物。这个实验是表现最好的一式三份,具有三个独立的十字架和记录,以消除同三个独立的天-天实验误差。


食谱


饮食
玉米粉 (3.5% (w/v))


琼脂 (2% (w/v))


糖蜜 (8% (w/v))


酵母 (1.2% (w/v)) 和水


致谢


此方法是该方法的一个适配描述了ð通过Khuong等。,2019 年,DOI:10.1126/ sciadv.aaw 4099 。


  这项工作得到了国家卫生和医学研究委员会 (NHMRC) 项目资助 APP1026310、APP1029672、APP1028887、APP1046090、APP1042416 和 APP1086851 的部分支持。GG Neely 得到了 NHMRC 职业发展奖学金 II CDF1111940 的支持。最后,我们感谢 John Chong 和 Anne Chong 对我们实验室工作的财政支持。


竞争我nterests


这项工作的作者没有要声明的利益冲突。


参考


1. Babcock, DT, Landry, C. 和Galko , MJ (2009)。细胞因子信号在果蝇幼虫中介导紫外线诱导的伤害性致敏。Curr Biol 19(10):799-806。     

2. Babcock, DT, Shi, S., Jo, J., Shaw, M., Gutstein, HB 和Galko , MJ (2011)。Hedgehog 信号调节伤害性敏感性。Curr Biol 21(18):1525-1533。     

3. Branson, K., Robie , AA, Bender, J., Perona , P. 和 Dickinson, MH (2009)。大量果蝇中的高通量伦理学。Nat 方法6(6):451-457。     

4. Campbell, JN 和 Meyer, RA (2006)。神经性疼痛的机制。神经元52(1):77-92。     

5. Costigan, M.、Scholz, J. 和 Woolf, CJ (2009)。神经性疼痛:神经系统对损伤的适应不良反应。Annu Rev Neurosci 32:1-32。     

6. Friard , O., Gamba , MJ (2016)。BORIS:一个自由,开放的多功能-源事件-记录软件,视频/音频编码和实时观测。生态学与进化方法7(11): 1325-1330 。     

7. Grosser, T., Woolf, CJ 和 FitzGerald, GA (2017)。是时候非成瘾性地缓解疼痛了。科学355(6329):1026-1027。     

8. Hamoudi , Z., Khuong , TM, Cole, T. 和 Neely, GG (2018)。用于研究紫杉醇引起的周围神经病变和痛觉过敏的果蝇模型。F1000Res 7: 99。     

9. Kang, K., Pulver, SR, Panzano , VC, Chang, EC, Griffith, LC, Theobald, DL 和 Garrity, PA (2010)。果蝇 TRPA1 的分析揭示了人类化学伤害感受的古老起源。自然464(7288):597-600。     

10. Khuong , TM, Wang, QP, Manion, J., Oyston , LJ, Lau, MT, Towler , H., Lin, YQ 和 Neely, GG (2019)。神经损伤驱动果蝇高度警惕和神经性致敏状态。Sci Adv 5(7):eaaw4099。 

11.洛佩兹Bellido ,R。和Galko酒店,MJ(2020)。用于测量果蝇幼虫机械伤害感受的改进方法和工具。J Vis Exp (164)。 

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14. Neely, GG, Keene, AC, Duchek , P., Chang, EC, Wang, QP, Aksoy, YA, Rosenzweig, M., Costigan, M., Woolf, CJ, Garrity, PA 和Penninger , JM (2011) )。TrpA1 调节果蝇的热伤害感受。PLoS一6(8): e24343。 

15. Patel, AA 和 Cox, DN (2017)。用于研究果蝇幼虫有害寒冷检测和多模式感官处理机制的行为和功能分析。Bio - protoc ol 7(13) : e2388。 

16. Pfau, DB, Geber, C., Birklein , F. 和Treede , RD (2012)。神经性疼痛患者的定量感觉测试:潜在的机制和治疗意义。Curr疼痛头痛代表16(3):199-206。 

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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Massingham, J. N., Baron, O. and Neely, G. G. (2021). Evaluating Baseline and Sensitised Heat Nociception in Adult Drosophila. Bio-protocol 11(13): e4079. DOI: 10.21769/BioProtoc.4079.
  2. Khuong, T. M., Wang, Q. P., Manion, J., Oyston, L. J., Lau, M. T., Towler, H., Lin, Y. Q. and Neely, G. G. (2019). Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila.Sci Adv 5(7): eaaw4099.
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