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Assaying Mechanonociceptive Behavior in Drosophila Larvae
果蝇幼虫机械伤害感受行为分析   

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Nature Neuroscience
Aug 2017

Abstract

Drosophila melanogaster larvae have been extensively used as a model to study the molecular and cellular basis of nociception. The larval nociceptors, class IV dendritic arborization (C4da) neurons, line the body wall of the animal and respond to various stimuli including noxious heat and touch. Activation of C4da neurons results in a stereotyped escape behavior, characterized by a 360° rolling response along the body axis followed by locomotion speedup. The genetic accessibility of Drosophila has allowed the identification of mechanosensory channels and circuit elements required for nociceptive responses, making it a useful and straightforward readout to understand the cellular and molecular basis of nociceptive function and behavior. We have optimized the protocol to assay mechanonociceptive behavior in Drosophila larvae.

Keywords: Nociception (痛觉感受), Noxious touch (有害触摸), Drosophila melanogaster (黑腹果蝇), Somatosensory network (躯体感觉网络), Mechanosensory (机械应力)

Background

Nociception, the innate ability to detect and avoid noxious stimuli, is highly conserved across the animal kingdom. Drosophila melanogaster larvae are capable to detect and avoid a variety of noxious stimuli including noxious touch, heat and light (Tracey et al., 2003; Hwang et al., 2007; Xiang et al., 2010). Mechanical stimulation above a certain threshold (> 30 mN) elicits a stereotyped rolling escape response at all larval stages (Almeida-Carvalho et al., 2017), which is thought to have evolved to avoid ovipositor injection by parasitic wasps such as L. boulardi (Hwang et al., 2007). This escape response is mediated by activation of nociceptive C4da neurons, which possess sensory dendrites covering the entire body wall allowing the animal to detect noxious cues. C4da neurons express several mechanosensory channels belonging to the DEG/ENaC family (pickpocket [ppk], ppk26/balboa) (Zhong et al., 2010; Gorczyca et al., 2014; Guo et al., 2014; Mauthner et al., 2014), a mechanosensitive TrpA1 isoform (Zhong et al., 2012), piezo (Kim et al., 2012) and the Trp channel painless (Tracey et al., 2003), all of which are required for normal mechanonociceptive responses.

The escape response can be assayed by using a von Frey filament exerting a force between 30-120 mN, which activates mechanosensory channels in C4da neurons (Hwang et al., 2007; Kim et al., 2012). Recent work has also shed light on circuit mechanisms required for mechanonociceptive responses. Mechanically induced escape responses require co-activation of class II da (C2da) and class III da (C3da) sensory neurons, as silencing of either subset impaired rolling behavior (Hu et al., 2017). Moreover, this sensory integration is specific for mechanonociception and in addition requires neuropeptide-mediated feedback. We provide a detailed protocol from our recent work (Hu et al., 2017), which employed a mechanonociception assay based on previously described methods (Hwang et al., 2007; Caldwell and Tracey, 2010; Zhong et al., 2010). We typically use a mechanical force of 45-50 mN, which elicits weak responses after the first stimulus, but enhanced responses after a second subsequent stimulus. This approach allows assaying changes in sensitivity and sensitization of mechanonociceptive responses, which can be coupled with genetic approaches to identify molecular and network components required for normal escape behavior.

Materials and Reagents

  1. Drosophila vials (wide, K-Resin) (Dutscher, catalog number: 789002 )
  2. Flugs® fly plugs, plastic vials (wide) (Dutscher, catalog number: 789035 )
  3. Omniflex monofilament fishing line Shakespeare (6 lb test, diameter 0.23 mm) (Zebco, Tulsa, USA)
  4. Petri dishes (Ø 10 cm) (SARSTEDT, catalog number: 82.1473 )
  5. Fly stocks
    Chromsome , Bloomginton stock center No.:
    w1118 (X, BL 6326)
    w*; ppk-Gal4 (X, 3rd, BL 32079)
    w*;UAS-TNTE (X, 3rd, BL 28997)
    w*; TrpA11 (X, 2nd , BL 36342)
  6. Agar Kobe I ( Carl Roth, catalog number: 5210.4 )
  7. Agar plates (see Recipes)
  8. Fly food (see Recipes)
    1.  Agar (strings) (Gewürzmühle Brecht, Eggenstein, catalog number: 00262 )
    2. Corn flour (Davert, Newstartcenter, catalog number: 17080 )
    3. Soy flour (Davert, Newstartcenter, catalog number: 46985 )
    4. Brewer’s yeast (ground) (Gewürzmühle Brecht, Eggenstein, catalog number: 03462 )
    5. Malt syrup (MeisterMarken–Ulmer Spatz, Bingen am Rhein, catalog number: 728985 )
    6. Treacle (molasses) (Grafschafter Krautfabrik, Meckenheim, catalog number: 01939 )
    7. Nipagin (Methyl 4-hydroxybenzoate) (Sigma-Aldrich, catalog number: 54752-1KG-F )
    8. Propionic acid (Carl Roth , catalog number: 6026.3 )

Equipment

  1. Brush (Size 1, Boesner, model: Da Vinci Nova Serie 1570 , catalog number: D15701)
  2. Forceps (Dumont, #3) (Fine Science Tools, catalog number: 11231-30 )
  3. Light source (white light) LED Schott KL 1500 LCD (Pulch und Lorenz, catalog number: 150.200)
    Manufacturer: SCHOTT, model: KL 1500 LCD .
  4. SZX7 stereo microscope (Olympus, model: SZX7 )

Software

  1. Origin Pro 9.0 (OriginLab, Northampton, USA) or similar for statistical analysis

Procedure

  1. All fly stocks were maintained at 25 °C and 70% humidity with a 12 h dark/light cycle on standard fly food. All experiments were performed using 3rd instar larvae at 96 h (hours) after egg laying (AEL). In order to ensure that all larvae were about the same age, the egg laying was restricted to 4-6 h. Experimental crosses were raised on standard fly food at 25 °C with 70% humidity and a 12 h light/12 h dark cycle.
  2. Prepare your genetic crosses 2 days before staging with approx. 20-30 virgins and 10-15 males each (more if you are using weak genotypes). After 2 days, transfer flies to a fresh food vial for timed egg-laying for 4-6 h at 25 °C (Figure 1A). Transfer the adult flies to a fresh vial (for another round of staging on the same or next day). The original vial is maintained at 25 °C until 96 ± 3 h AEL. All larvae should be in the third instar (L3) foraging stage and not yet leaving the food (Figure 1B).
    Note: Precise staging is important, as nociceptive responses of larvae are reduced after 120 h AEL, likely due to the transition to the wandering stage and preparation for pupariation. The density of larvae in the food will also affect staging: too few animals cannot efficiently process the food, while having too many larvae will result in competition for food, both of which is affecting developmental progression and will broaden the developmental stage of the larval population. In case mutant animals with delayed development are used staging has to be adjusted accordingly (staging either by animal size or molting counting mouth hook teeth).


    Figure 1. Correct staging and larval density. A. A photograph showing appropriate embryo numbers for the used vial size (approx. 100-150 embryos laid within 4-6 h). B. At 96 h AEL, larvae should have processed the food well and still be in the foraging stage.

  3. Tool preparation
    1. Cut the omniflex monofilament fishing line (Shakespeare, 6 lb test, diameter 0.009 inch [0.23 mm]) to a length of 18 mm. 10 mm is attached to a toothpick such that 8 mm of the fiber protruded from the end of the toothpick (Figure 2A).
    2. Calibrate the force of the fiber by using it to depress a balance until the fishing line is seen to bend. Record the force (in grams) and convert to milli-Newtons (mN) by multiplying the measured grams by a factor of 9.81. Typically, this length of filament results in a force of 45-50 mN (Figures 2B-2D). Varying the filament length will change the mechanical force (the longer, the less the force), which might be desirable if different forces should be tested. The probability of nociceptive responses of larvae increases with the applied force from 30 to 120 mN (Kim et al., 2012; Almeida-Carvalho et al., 2017).
    3. Examine the filament under a stereoscope and make sure that no sharp edges remain, which might potentially injure the animal. Puncturing the body wall will result in altered behavioral responses.


      Figure 2. Tool preparation and calibration. A. 8 mm filament attached to toothpick prepared according to instructions above. B-D. Calibrating the filament: the filament should exert a force of 45-50 mN (4.59-5.10 g). B. Repeated depression of the filament should result in comparable forces (here 46 ± 5 mN). B’. Replicable forces are achieved by correct pressure and bending of the filament at the right angle as shown (approx. 45-60°). C’. Too little pressure and/or wrong angle of deflection result in lower forces. C’. Too much pressure and/or wrong angle of deflection result in too high forces.

  4. Distribute 2 ml of dH2O on a 2% agar plate to create a thin water film, which enables the animals to crawl freely and perform their rolling behavior. Without water, the larvae are not fully mobile and do not display consistent behavioral responses.
  5. Prepare 10-20 staged larvae by washing in dH2O to remove any residual food and place them gently on the agar plate using a brush (Figures 3A-3D).


    Figure 3. Preparation of mechanonociception assay. A and B. The assay is prepared by gently placing 10-20 staged 3rd instar larvae on a 10 cm 2% agar plate using a brush. C. A thin water film of 2 ml (colored magenta for illustration) is necessary for consistent behavioral responses. D. Agar plate with test animals is placed under a stereoscope with a light source. E. Illustration of a 3rd instar larvae. Boxed region indicates the dorsolateral region of abdominal segments A3-A6, which should be targeted with the filament calibrated to the chosen force (30-120 mN). F-H. Examples of correct and incorrect placement of the filament on the larva are shown. F. Correct placement on dorsolateral A3 region. G. Wrong placement with animal moving away from filament resulting in a bad angle for the stimulus. H. Filament placement at an anterior segment which generally does not elicit rolling behavior.

  6. Deliver the mechanical stimulus by rapidly depressing the larva with the filament on the dorsal side (abdominal segments four, five, or six) for approximately 1 sec. The quick release allows the larvae to perform escape behavior. Reapply the stimulus to the same larva after a pause of 2-3 sec.
  7. Score the response immediately on a scoring sheet according to Hwang et al. (2007): no response, stop, stop and turn (non-nociceptive responses), or rolling (nociceptive response); in addition, we introduced bending to score for an incomplete nociceptive response (C-shaped simultaneous convulsive head and tail movements) that did not result in rolling (response classification: 1 = no response, 2 = stop, 3 = stop and turn, 4 = bending, 5 = rolling). A positive rolling response is scored if at least one 360° rotation along the body axis occurred in response to the mechanical stimulus (Figures 4A-4D; Videos 1 and 2).
    Note: To develop and entrain precision in applying the correct force to the animal the experimenter should practice the correct motion on a balance. Hold the toothpick with the filament and depress the filament at a 45-60° angle until it bends, which should result in a consistent force every time. Practicing with control animals will further ensure consistent results. 50-70% of the animals should respond with rolling escape behavior after the 2nd stimulus.


    Figure 4. Behavioral responses to mechanonociceptive stimulation. A. Montage of nociceptive rolling response after mechanical stimulation. Note that the larva performs a full 360° roll along the body axis as visible following the main trachea. B. Montage of a partial nociceptive response (‘bending’) resulting in C-shaped body bending but no full 360° roll. C. Montage of stop and turn response (stop and at least 45° change in direction). D. Montage of stop response (no major change in direction of movement).

    Video 1. Behavioral responses to mechanical stimulation using a 50 mN von Frey filament (all 5 categories)

    Video 2. Side by side comparison rolling vs. bending

Data analysis

  1. Statistical differences in mechanonociceptive responses can be calculated using a χ2-test, which allows comparing categorical data between 2 genotypes (e.g., control vs. TrpA11). Distinguishing only nociceptive and non-nociceptive behaviors allows the use of a 2 x 2 contingency table providing the highest statistical power using a χ2-test with 1 degree of freedom. χ2 can be determined according to the following formula:

    O: observed values, E: expected value.
    The χ2-test requires the actual number of animals, as it cannot compute ratios, percentages or frequencies. Most statistics programs (Origin Pro, SigmaPlot, Prism, SAS, Statistica, R) can be used to calculate χ2 and compute statistical significances.
    Note: In cases where more than two genotypes should be compared, appropriate correction for multiple comparisons of statistical significances should be performed, e.g., a Bonferroni correction factor (significant if p < a/n, typically with a = 0.05 and n being the number of compared hypotheses).
  2. At least 60 animals per genotype should be tested to ensure that an effect of 20% or greater is resulting in statistical significance (P < 0.05) with sufficient power of the χ2-test (> 0.9).
    Note: All behavioral experiments should be blinded and randomized to avoid unwanted bias. The genotypes for the mechanonociception assay should be coded (e.g., numbers or letters), with the experimenter being unaware of the genotypes being tested.

Expected results

  1. The functionality of the mechanonociception assay can be assessed by inactivation of C4da neurons, either by genetic silencing using Tetanus toxin light chain (UAS-TnT), the inward rectifying potassium channel Kir2.1, or genetic mutants of mechano-sensitive channels (e.g., TrpA11) (Hwang et al., 2007; Zhong et al., 2012; Hu et al., 2017).
  2. Performing the mechanonociception assay with TrpA11 mutant larvae showed a decrease in nociceptive responses compared to control w1118 larvae (Video 3). After the first stimulation already 76% of control larvae displayed nociceptive responses (bending + rolling behavior) (Figure 5A). In contrast, none of TrpA11 larvae showed nociceptive responses to the mechanical force. Treating the larvae a second time resulted in 85% nociceptive response (50% rolling), whereas only 10% of TrpA11 animals reacted with nociceptive bending but no rolling (Figure 5B).

    Video 3. Exemplary behavioral response of TrpA11 animals to mechanical stimulation using a 50 mN von Frey filament


    Figure 5. TrpA11 mutant larvae exhibited defects in mechanonociceptive behavior. Response of 3rd instar larvae to a mechanonociceptive stimulus (50 mN). The behavioral response was categorized into nociceptive (rolling, bending) and non-nociceptive responses (stop and turn, stop, no response). Behavioral differences between nociceptive and non-nociceptive behavior of 3rd instar control and TrpA11 larvae after the 2nd stimulation were compared using a χ2-test. P < 0.0001 (χ2-test, 1 degree of freedom, n = 60/genotype).

  3. Alternatively, cell type specific genetic manipulation or silencing can be employed to explore circuit function by employing expression of the tetanus toxin light chain (UAS-TnT). C4da specific expression of TnT (ppk-Gal4/UAS-TnT) resulted in mechanonociceptive defects, displayed by a decrease in nociceptive behavior and an increase in non-nociceptive responses compared to control animals (Figure 6).
  4. These results confirmed the experimental design and C4da neuron function in mechanonociception. This approach should allow the identification of genes required for mechanonociception in C4da or downstream neurons of the nociceptive network.


    Figure 6. C4da neuron silencing with TnT impairs mechanonociceptive behavior. Percentage of 3rd instar larvae showing categorized responses after mechanonociceptive stimulation (50 mN). Behavioral responses of animals with C4da neuron-specific overexpression of TnT (ppk-Gal4/UAS-tnt) and controls were compared. Responses were categorized into nociceptive (rolling, bending) and non-nociceptive responses (stop and turn, stop, no response). Blocking synaptic transmission in C4da neurons by TnT expression resulted in strong defects in mechanonociceptive responses after the 2nd stimulation. P < 0.0001(***) (χ2-test, 1 degree of freedom, n = 63, 62, 64).

Notes

Under optimal conditions (proper staging, same filament, etc.) results are highly reproducible between experiments performed on different days and by different trained experimenters. From our experience, tight staging, density-controlled vials and gentle handling of the animals are critical for reproducible results. Behavioral response rates are highly dependent on applying the stimulus consistently and appropriately. Due to the manual procedure it takes practice to be able to consistently stimulate larvae. We recommend practicing with a balance first to ensure that the applied force is constant. Next, practicing with wildtype and mutant larvae displaying impaired mechanonociception (e.g., TrpA11 mutant larvae) will ensure consistent and reproducible behavioral responses.

Recipes

  1. Agar plates
    Note: For mechanonociception assays, 2% agar plates were used.
    Dissolve Kobe agar I in dH2O and fill Petri dishes (Ø 10 cm) with a defined volume of 12 ml
  2. Fly food
    Use the following ingredients for 1 L of standard fly food:
    Ingredients for 1 L of standard fly food:

    Dissolved in 1 L dH2O

Acknowledgments

This work was supported by the Landesforschungsförderung LFF-FV27 (to P.S.) and the Deutsche Forschungsgemeinschaft priority program SPP1926 (project SO1337/2-1 to P.S.). The authors would like to acknowledge the previous work and effort of several groups developing and applying mechanonociceptive assays in Drosophila, in particular the labs of D. Tracey, A. Patapoutian, Y.N. Jan and Z. Wang. The authors declare that no conflicts of interest or competing interests exist.

References

  1. Almeida-Carvalho, M. J., Berh, D., Braun, A., Chen, Y. C., Eichler, K., Eschbach, C., Fritsch, P. M. J., Gerber, B., Hoyer, N., Jiang, X., Kleber, J., Klambt, C., Konig, C., Louis, M., Michels, B., Miroschnikow, A., Mirth, C., Miura, D., Niewalda, T., Otto, N., Paisios, E., Pankratz, M. J., Petersen, M., Ramsperger, N., Randel, N., Risse, B., Saumweber, T., Schlegel, P., Schleyer, M., Soba, P., Sprecher, S. G., Tanimura, T., Thum, A. S., Toshima, N., Truman, J. W., Yarali, A. and Zlatic, M. (2017). The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3 Drosophila larvae. J Exp Biol 220(Pt 13): 2452-2475.
  2. Caldwell, J. C. and Tracey, W. D. (2010). Alternatives to mammalian pain models 2: Using Drosophila to identify novel genes involved in nociception. Methods Mol Biol 617: 19-29.
  3. Gorczyca, D. A., Younger, S., Meltzer, S., Kim, S. E., Cheng, L., Song, W., Lee, H. Y., Jan, L. Y. and Jan, Y. N. (2014). Identification of ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila. Cell Rep 9(4): 1446-1458.
  4. Guo, Y., Wang, Y., Wang, Q. and Wang, Z. (2014). The role of PPK26 in Drosophila larval mechanical nociception. Cell Rep 9(4): 1183-1190.
  5. Hu, C., Petersen, M., Hoyer, N., Spitzweck, B., Tenedini, F., Wang, D., Gruschka, A., Burchardt, L. S., Szpotowicz, E., Schweizer, M., Guntur, A. R., Yang, C. H. and Soba, P. (2017). Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior. Nat Neurosci 20(8): 1085-1095.
  6. Hwang, R. Y., Zhong, L., Xu, Y., Johnson, T., Zhang, F., Deisseroth, K. and Tracey, W. D. (2007). Nociceptive neurons protect Drosophila larvae from parasitoid wasps. Curr Biol 17(24): 2105-2116.
  7. Kim, S. E., Coste, B., Chadha, A., Cook, B. and Patapoutian, A. (2012). The role of Drosophila Piezo in mechanical nociception. Nature 483(7388): 209-212.
  8. Mauthner, S. E., Hwang, R. Y., Lewis, A. H., Xiao, Q., Tsubouchi, A., Wang, Y., Honjo, K., Skene, J. H., Grandl, J. and Tracey, W. D., Jr. (2014). Balboa binds to pickpocket in vivo and is required for mechanical nociception in Drosophila larvae. Curr Biol 24(24): 2920-2925.
  9. Tracey, W. D., Jr., Wilson, R. I., Laurent, G. and Benzer, S. (2003). Painless, a Drosophila gene essential for nociception. Cell 113(2): 261-273.
  10. Xiang, Y., Yuan, Q., Vogt, N., Looger, L. L., Jan, L. Y. and Jan, Y. N. (2010). Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature 468(7326): 921-926.
  11. Zhong, L., Bellemer, A., Yan, H., Ken, H., Jessica, R., Hwang, R. Y., Pitt, G. S. and Tracey, W. D. (2012). Thermosensory and nonthermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP Channel. Cell Rep 1(1): 43-55.
  12. Zhong, L., Hwang, R. Y. and Tracey, W. D. (2010). Pickpocket is a DEG/ENaC protein required for mechanical nociception in Drosophila larvae. Curr Biol 20(5): 429-434.

简介

果蝇幼虫已广泛用作研究伤害感受的分子和细胞基础的模型。 幼虫伤害感受器,IV类树突状树枝状(C4da)神经元,排列在动物体壁上,并对各种刺激作出反应,包括有害的热和触觉。 激活C4da神经元导致刻板脱逃行为,其特征在于沿着身体轴线的360°滚动响应,随后是运动加速。 果蝇的遗传可及性允许识别伤害性反应所需的机械感受通道和电路元件,使其成为理解伤害性功能和行为的细胞和分子基础的有用和直接的读数。 我们已经优化了该协议以检测果蝇幼虫中的机械伤害行为。


【背景】伤害感受,是检测和避免有害刺激的先天能力,在整个动物界高度保守。果蝇幼虫能够检测并避免各种有害刺激,包括有害触觉,热和光(Tracey等人,2003; Hwang等人, ,2007; Xiang et al。,2010)。高于特定阈值(> 30mN)的机械刺激在所有幼虫阶段(Almeida-Carvalho等人,2017)都引发了定型滚动逃避反应,其被认为已经进化以避免通过寄生蜂如L。 boulardi (Hwang et。,2007)。这种逃避反应是由伤害性C4da神经元的激活介导的,伤害性C4da神经元具有覆盖整个体壁的感觉树突,允许动物检测有害线索。 C4da神经元表达属于DEG / ENaC家族的几种机械感受通道(Pickpocket [ppk],ppk26 / balboa)(Zhong等人,2010; Gorczyca等人,, 2014; Guo等人,2014; Mauthner等人,2014),机械敏感的TrpA1同种型(Zhong等人,2012) ,压电(Kim等人,2012)和Trp通道无痛(Tracey等人,2003),所有这些都是正常机械伤害性反应所需的。

可以通过使用施加30-120mN之间的力的von Frey细丝来测定逃避反应,其激活C4da神经元中的机械感受通道(Hwang et al。,2007; Kim等人,2012年)。最近的工作也阐明了机械感受反应所需的电路机制。机械诱导的逃避反应需要II类da(C2da)和III类da(C3da)感觉神经元的共激活,因为任一子集的沉默都会使滚动行为受损(Hu等人,2017)。此外,这种感觉整合对于机械伤害感受是特异的,并且另外需要神经肽介导的反馈。我们从我们最近的工作(Hu等人,2017)提供了一个详细的方案,该方案采用了基于先前描述的方法的机械伤害性分析方法(Hwang et al。,2007; Caldwell和Tracey,2010; Zhong等人,2010)。我们通常使用45-50mN的机械力,在第一次刺激后引起弱反应,但在第二次刺激后增强反应。这种方法可以检测机械感受反应的敏感性和敏感性的变化,其可以与遗传方法结合以鉴定正常逃避行为所需的分子和网络组分。

关键字:痛觉感受, 有害触摸, 黑腹果蝇, 躯体感觉网络, 机械应力

材料和试剂

  1. 果蝇小瓶(宽,K-树脂)(Dutscher,目录号:789002)
  2. Flugs 蝇塞,塑料小瓶(宽)(Dutscher,目录号:789035)
  3. Omniflex单丝钓鱼线莎士比亚(6磅测试,直径0.23毫米)(Zebco,塔尔萨,美国)
  4. 培养皿(直径10厘米)(SARSTEDT,目录号:82.1473)
  5. 飞股票
    Chromsome,Bloomginton股票中心编号:
    1118 (X,BL 6326)
    W *; ppk-Gal4(X,3,BL 32079)
    w *; UAS-TNTE (X,3 ,BL 28997)
    W *; TrpA1 1 (X,2 ,BL 36342)
  6. 琼脂神户我(卡尔罗斯,目录编号:5210.4)
  7. 琼脂平板(见食谱)
  8. 飞食物(见食谱)
    1. &nbsp;琼脂(字符串)(GewürzmühleBrecht,Eggenstein,目录号:00262)
    2. 玉米粉(Davert,Newstartcenter,目录号:17080)
    3. 大豆粉(Davert,Newstartcenter,目录号:46985)
    4. 啤酒酵母(地面)(GewürzmühleBrecht,Eggenstein,目录号:03462)
    5. 麦芽糖浆(MeisterMarken-Ulmer Spatz,莱茵河畔宾根,目录号:728985)
    6. 糖蜜(糖蜜)(Grafschafter Krautfabrik,Meckenheim,目录编号:01939)
    7. 尼泊金(4-羟基苯甲酸甲酯)(Sigma-Aldrich,目录号:54752-1KG-F)
    8. 丙酸(Carl Roth,目录号:6026.3)

设备

  1. 刷子(规格1,Boesner,型号:Da Vinci Nova Serie 1570,产品目录号:D15701)
  2. 镊子(Dumont,#3)(精细科学工具,目录号:11231-30)
  3. 光源(白光)LED Schott KL 1500 LCD(Pulch und Lorenz,产品目录编号:150.200)
    制造商:SCHOTT,型号:KL 1500 LCD。
  4. SZX7立体显微镜(奥林巴斯,型号:SZX7)

软件

  1. Origin Pro 9.0(OriginLab,Northampton,USA)或类似的统计分析

程序

  1. 所有的飞行物保持在25℃和70%湿度下,在标准飞行食物上进行12小时黑暗/光照周期。在产蛋后(AEL)96小时(小时),使用3日龄幼虫进行所有实验。为了确保所有幼虫的年龄大致相同,产蛋时间限制在4-6小时。在25℃,70%湿度和12小时光照/ 12小时黑暗周期的标准飞行食物中提高实验杂交。
  2. 准备你的基因十字架前两天准备约。 20-30个处女和每个10-15个男性(如果你使用弱基因型更多)。 2天后,将苍蝇转移至新鲜食物瓶中,在25℃定时产卵4-6小时(图1A)。将成年果蝇转移到新鲜的小瓶中(在同一天或第二天进行另一轮分娩)。原始小瓶维持在25°C直到96±3小时AEL。所有幼虫应该在第三龄期(L3)觅食阶段,并且还没有离开食物(图1B)。
    注意:准确的分期很重要,因为120 h AEL后幼虫的伤害性反应会减少,这可能是由于向徘徊阶段过渡和蛹化准备所致。食物中幼虫的密度也会影响分期:太少的动物不能有效地处理食物,而幼虫过多会导致食物竞争,这两者都会影响发育进程,并会扩大幼虫种群的发育阶段。在使用具有延迟发育的突变动物的情况下,分期必须相应地调整(通过动物大小或蜕皮计数嘴钩牙进行分期)。


    图1.正确的分期和幼虫密度。 :一种。显示使用的小瓶尺寸的合适胚胎数量的照片(4-6小时内放置大约100-150个胚胎)。 B.在96小时AEL,幼虫应该已经很好地处理了食物,仍然处于觅食阶段。

  3. 工具准备
    1. 切割全长单丝钓鱼线(莎士比亚,6磅测试,直径0.009英寸[0.23毫米])长度为18毫米。将10mm的牙签连接到牙签上,使得8mm的牙签从牙签的末端突出(图2A)。
    2. 通过使用它来平衡纤维的力量,直到看到钓鱼线弯曲。记录力(单位为克),并将测得的克乘以9.81倍,转换为毫牛顿(mN)。通常,这段长丝产生45-50mN的力(图2B-2D)。改变灯丝长度会改变机械力(时间越长,力越小),如果要测试不同的力,这可能是可取的。幼虫的伤害性反应的概率随着施加的力从30至120mN而增加(Kim等人,2012; Almeida-Carvalho等人,2017)。
    3. 检查立体镜下的灯丝,确保没有锋利的边缘,这可能会伤害动物。穿刺体壁会导致行为反应改变。


      图2.工具准备和校准。 :一种。根据上述说明制备的8毫米长丝连接到牙签上。 B-d。校准灯丝:灯丝应该施加45-50mN(4.59-5.10g)的力。 B.重复按压灯丝应产生可比较的力(这里为46±5 mN)。 B”。如图所示(图45-60),通过正确的压力和灯丝弯曲成直角可获得可重复的力。 C'。太小的压力和/或错误的偏转角度会导致较小的力。 C'。

      太大的压力和/或错误的角度会导致太大的力量
  4. 在2%琼脂平板上分配2毫升dH 2 O 2以形成薄水膜,使动物能够自由爬行并执行其滚动行为。没有水,幼虫不能充分移动,不会显示一致的行为反应。
  5. 通过在dH 2 O中洗涤来准备10-20个分级的幼虫以去除任何残留的食物,并使用刷子将它们轻轻地放在琼脂平板上(图3A-3D)。


    图3.机械伤害性测定的制备。 A和B.通过使用刷子将10-20个阶段的3龄幼虫轻轻放置在10cm 2的琼脂平板上来制备测定。 C.为了一致的行为反应,需要2毫升薄水膜(用于说明的彩色洋红色)。 D.将具有测试动物的琼脂平板置于具有光源的立体镜下。 E.一只3龄幼虫的插图。盒装区域表示腹部片段A3-A6的背外侧区域,其应该以针对所选择的力(30-120mN)校准的丝线为目标。 F-小时。显示了在幼虫上正确和不正确放置丝线的例子。 F.正确放置在背外侧的A3区域。 G.错误的放置与动物远离灯丝导致刺激角度不佳。 H.通常不会引起滚动行为的前段细丝放置。

  6. 通过在背侧(腹节4,5或6)快速压下幼虫约1秒钟来提供机械刺激。快速释放允许幼虫执行逃生行为。
    停顿2-3秒后,重新对相同的幼虫施加刺激。
  7. 根据Hwang等人的评分表,立即对反应进行评分。 (2007):没有反应,停止,停止和转向(非伤害性反应)或滚动(伤害性反应);此外,我们引入了弯曲评分以获得不会导致滚动的不完全伤害性反应(C形同时抽动性头部和尾部运动)(反应分类:1 =无反应,2 =停止,3 =停止和转动,4 =弯曲,5 =滚动)。如果响应机械刺激(图4A-4D;视频1和2)发生沿着身体轴线的至少一个360°旋转,则对正面滚动响应进行评分。
    注意:为了发展精确的力量并向动物施加正确的力量,实验者应该在天平上练习正确的动作。用牙线拿住牙签,并以45-60°的角度压下牙线直至弯曲,每次都会产生一致的力。与对照动物一起练习将进一步确保一致的结果。
    50-70%的动物在第二次刺激后应该会出现滚动逃跑行为

    图4.对机械伤害性刺激的行为反应。 :一种。机械刺激后伤害性滚动反应的蒙太奇。请注意,幼虫在主气管后方沿着身体轴线执行完整的360°滚动。 B.部分伤害性反应(“弯曲”)的蒙太奇导致C形身体弯曲,但没有完整的360°滚动。 C.停止和转动响应的蒙太奇(停止并且至少45°改变方向)。 D.停止响应的蒙太奇(移动方向没有重大变化)。

    视频1

    视频2

数据分析

  1. 机械伤害性反应的统计学差异可以使用χ2检验来计算,这允许比较2种基因型之间的分类数据(例如 2 / ,控制与 TrpA1 1 )。仅区分伤害性和非伤害性行为允许使用2×2列联表提供使用 2 检验的最高统计功效拥有1个自由度。可根据以下公式确定: 2 :

    O:观测值,E:预期值。
    χ 2 - 测试需要动物的实际数量,因为它无法计算比率,百分比或频率。大多数统计程序(Origin Pro,SigmaPlot,Prism,SAS,Statistica,R)可以用来计算 2 2 并计算统计显着性。
    注意:在应该比较两种以上基因型的情况下,应该进行适当的多重比较统计显着性的校正,例如Bonferroni校正因子(如果p
  2. 每种基因型至少应有60只动物进行测试,以确保20%或更高的效应以足够的力量产生统计学显着性( <0.05) 2 -test(> 0.9)。
    注意:所有的行为实验应该是盲法的,随机的,以避免不必要的偏见。机械伤害感受测定的基因型应该被编码(例如数字或字母),实验者不知道被测试的基因型。

预期结果

  1. 机械伤害感受测定的功能可以通过使用破伤风毒素轻链(UAS-TnT),内向整流钾通道Kir2.1的遗传沉默或遗传突变体的C4a神经元的失活来评估机械敏感通道(例如, TrpA1 1 )(Hwang et ,2007; Zhong等人,2012; Hu等人,2017)。
  2. 使用TrpA1突变体幼虫进行机械撞击测定与对照相比,显示出伤害性反应的减少。 1 1118 幼虫(视频3)。第一次刺激后,76%的对照幼虫表现出伤害性反应(弯曲+滚动行为)(图5A)。相比之下, TrpA1 <1> 幼虫没有表现出对机械力的伤害性反应。第二次处理幼虫导致85%的伤害性反应(50%滚动),而只有10%的TrpA1 动物与伤害感受反应弯曲但不滚动(图5B)。

    视频3


    图5:TrpA1 突变幼虫在机械感受行为中表现出缺陷 3龄幼虫对机械伤害性反应的反应刺激(50 mN)。行为反应分为伤害性(滚动,弯曲)和非伤害性反应(停止和转动,停止,无反应)。 3龄幼虫和2龄幼虫之后的伤害性和非伤害性行为之间的行为差异在<2> <1> <1> <2> <2>使用χ 2 - 测试来比较刺激。 P &lt; 0.0001( 2 2 - 测试,1个自由度,n = 60 /基因型)。

  3. 或者,可以使用细胞类型特异性基因操作或沉默来通过使用破伤风毒素轻链(UAS-TnT)的表达来探索电路功能。与对照动物相比,TnT( ppk-Gal4 / UAS-TnT )的C4da特异性表达导致机械痛觉缺陷,显示出伤害性行为减少和非伤害性反应增加(图6)。
  4. 这些结果证实了实验设计和C4da神经元机械感觉功能。这种方法应该能够鉴定C4da或伤害感受网络下游神经元机械伤害感受所需的基因。


    图6. C4da神经元沉默与TnT削弱机械伤害行为。 显示在机械痛觉刺激(50mN)后分类的反应的3岁幼虫的百分比。比较具有C4da神经元特异性过表达TnT( ppk-Gal4 / UAS-tnt )的动物的行为反应。反应分为伤害性(滚动,弯曲)和非伤害性反应(停止和转动,停止,无反应)。通过TnT表达阻断C4da神经元中的突触传递导致在第二次刺激后机械伤害性反应中的强烈缺陷。 0.0001(***)( 2 2 - 测试,1自由度,n = 63,62,64) >

笔记

在最佳条件下(正确的分期,相同的长丝,等),结果在不同日期和不同训练的实验者进行的实验之间具有高度的可重现性。根据我们的经验,严格的分期,密度控制的小瓶和对动物的轻柔处理对于重现性结果至关重要。行为反应率高度依赖于始终适当地应用刺激。由于手动操作,需要不断刺激幼虫才能练习。我们建议先用天平练习,以确保施加的力量不变。接下来,用野生型和突变幼虫显示受损的机械伤害感受(例如,TrpA1突变幼虫)来实践将确保一致的和可重复的行为反应。

食谱

  1. 琼脂平板
    注意:对于机械感染试验,使用2%琼脂平板。
    将神户琼脂I溶解在dH 2 O中,并填充定容量为12ml的培养皿(Ø10cm)。
  2. 飞食物

    1L标准飞行食品使用以下成分:
    1升标准飞行食品配料:

    溶于1 L dH 2 O 0

致谢

这项工作得到LandesforschungsförderungLFF-FV27(至P.S.)和Deutsche Forschungsgemeinschaft优先计划SPP1926(项目SO1337 / 2-1至P.S.)的支持。作者想要感谢在果蝇中开发和应用机械感受性测定的几个小组以前的工作和努力,特别是D. Tracey,A. Patapoutian,Y.N.的实验室。 Jan和Z. Wang。作者声明不存在利益冲突或利益冲突。

参考

  1. Almeida-Carvalho,MJ,Berh,D.,Braun,A.,Chen,YC,Eichler,K.,Eschbach,C.,Fritsch,PMJ,Gerber,B.,Hoyer,N.,Jiang,X.,Kleber ,J.,Klambt,C.,Konig,C.,Louis,M.,Michels,B.,Miroschnikow,A.,Mirth,C.,Miura,D.,Niewalda,T.,Otto,N.,Paisios E.,Pankratz,MJ,Petersen,M.,Ramsperger,N.,Randel,N.,Risse,B.,Saumweber,T.,Schlegel,P.,Schleyer,M.,Soba,P.,Sprecher, SG,Tanimura,T.,Thum,AS,Toshima,N.,Truman,JW,Yarali,A。和Zlatic,M.(2017)。 Ol1mpiad:第一阶段和第三阶段行为能力的一致性果蝇 幼虫。 J Exp Biol 220(Pt 13):2452-2475。
  2. Caldwell,J.C。和Tracey,W.D。(2010)。 替代哺乳动物疼痛模型2:使用果蝇识别涉及新基因in nociception。 Methods Mol Biol 617:19-29。
  3. Gorczyca,D.A.,Younger,S.,Meltzer,S.,Kim,S.E.,Cheng,L.,Song,W.,Lee,H.Y.,Jan,L.Y。和Jan,Y.N。(2014)。 ppk26的识别,DEG / ENaC通道以相互依赖的方式与Ppk1一起起作用以指导运动行为在果蝇。 Cell Rep 9(4):1446-1458。
  4. Guo,Y.,Wang,Y.,Wang,Q.和Wang,Z。(2014)。 PPK26在果蝇幼虫机械伤害感受中的作用。 Cell Rep 9(4):1183-1190。
  5. Hu,C.,Petersen,M.,Hoyer,N.,Spitzweck,B.,Tenedini,F.,Wang,D.,Gruschka,A.,Burchardt,LS,Szpotowicz,E.,Schweizer,M.,Guntur ,AR,Yang,CH和Soba,P。(2017)。 感觉整合和神经调节反馈促进果蝇机械感受行为。 Nat Neurosci 20(8):1085-1095。
  6. Hwang,R.Y.,Zhong,L.,Xu,Y.,Johnson,T.,Zhang,F.,Deisseroth,K。和Tracey,W.D。(2007)。 伤害感受神经元保护果蝇幼虫免受寄生蜂的伤害 Curr Biol 17(24):2105-2116。
  7. Kim,S.E.,Coste,B.,Chadha,A.,Cook,B。和Patapoutian,A。(2012)。 果蝇压电在机械伤害中的作用 <自然 483(7388):209-212。
  8. Mauthner,SE,Hwang,RY,Lewis,AH,Xiao,Q.,Tsubouchi,A.,Wang,Y.,Honjo,K.,Skene,JH,Grandl,J.和Tracey,WD,Jr.(2014) 。 Balboa在体内与扒手绑定,并且在果蝇幼虫。 Curr Biol 24(24):2920-2925。
  9. Tracey,W.D.,Jr.,Wilson,R.I.,Laurent,G。和Benzer,S。(2003)。 无痛,果蝇基因对伤害感受至关重要。 Cell 113(2):261-273。
  10. Xiang,Y.,Yuan,Q.,Vogt,N.,Looger,L.L.,Jan,L.Y.and Jan,Y.N。(2010)。 避光调解光感受器对果蝇幼虫体壁进行平铺。< / a> Nature 468(7326):921-926。
  11. Zhong,L.,Bellemer,A.,Yan,H.,Ken,H.,Jessica,R.,Hwang,R.Y.,Pitt,G.S。和Tracey,W.D。(2012)。 果蝇黑热病菌TRPA1的热感受和非热感觉亚型揭示了热感应域一个thermoTRP频道。 Cell Rep 1(1):43-55。
  12. Zhong,L.,Hwang,R.Y。和Tracey,W.D。(2010)。 扒手是果蝇幼虫机械伤害感受所需的DEG / ENaC蛋白。 Curr Biol 20(5):429-434。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hoyer, N., Petersen, M., Tenedini, F. M. and Soba, P. (2018). Assaying Mechanonociceptive Behavior in Drosophila Larvae. Bio-protocol 8(4): e2736. DOI: 10.21769/BioProtoc.2736.
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