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Bioluminescence Monitoring of Neuronal Activity in Freely Moving Zebrafish Larvae
自由移动的斑马鱼幼鱼中神经元活性的生物发光监测   

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Abstract

The proof of concept for bioluminescence monitoring of neural activity in zebrafish with the genetically encoded calcium indicator GFP-aequorin has been previously described (Naumann et al., 2010) but challenges remain. First, bioluminescence signals originating from a single muscle fiber can constitute a major pitfall. Second, bioluminescence signals emanating from neurons only are very small. To improve signals while verifying specificity, we provide an optimized 4 steps protocol achieving: 1) selective expression of a zebrafish codon-optimized GFP-aequorin, 2) efficient soaking of larvae in GFP-aequorin substrate coelenterazine, 3) bioluminescence monitoring of neural activity from motor neurons in free-tailed moving animals performing acoustic escapes and 4) verification of the absence of muscle expression using immunohistochemistry.

Keywords: GFP-Aequorin-opt(GFP-水母发光蛋白优化基因), Zebrafish(斑马鱼), Bioluminescence(生物发光), Coelenterazine(腔肠素), Escape response(逃避反应)

Background

Unlike fluorescent genetically encoded calcium indicators (GECIs) (Grienberger and Konnerth, 2012), such as the GCaMP family, the bioluminescent indicator GFP-aequorin (Shimomura et al., 1962) does not require light excitation and therefore opens new avenues for monitoring neural activity in moving animals, including flies (Martin et al., 2007), mice (Rogers et al., 2007) and zebrafish larvae (Naumann et al., 2010). However, efficient use of GFP-aequorin remains challenging to achieve in zebrafish larvae, restricting its widespread use as a calcium indicator. The limitation lies in the fact that bioluminescence signals originating from a single muscle fiber are so large they constitute a major pitfall. Once absence of muscle expression is verified for a given transgenic line, bioluminescence signals emanating from neurons only are very small. To overcome these limitations, we developed a codon-optimized variant of GFP-aequorin for zebrafish larvae, achieved selective expression in motor and sensory neurons using existing transgenic lines, modified coelenterazine soaking protocol in order to conduct experiments at 4 days post-fertilization, created a behavioral bioluminescence assay for monitoring neuronal activity during acoustic evoked stereotyped escape responses in zebrafish larvae.

Materials and Reagents

  1. Microscope slide 76 x 26 x 1.1 mm (VINCENT LEERMIDDELEN SCIENTIFIC, catalog number: 29201316 )
  2. Cover glass Knittel Glass 24 x 60 mm
  3. Zebrafish larvae Adult AB and and Tüpfel long fin (TL) strains of Danio rerio aged between 0 and 4 dpf (day post fertilization) were used for this study; transgenic lines used in this protocol (available on request): Tg(mnx1:gal4)icm11;Tg(UAS:GFP-aequorin-opt)icm09
  4. Mammalian GFP-aequorin sequence (provided by Dr. Ludovic Tricoire, Université Pierre et Marie Curie, Paris, France, see text file in Supplement file 1)
  5. PT2 14xUAS plasmid (provided by Pr. Koichi Kawakami, National Institute of Genetics, Mishima, Japan, map can be downloaded at http://kawakami.lab.nig.ac.jp/trans.html)
  6. Instant Ocean® salts
  7. Methylene blue
  8. Fixation solution (4% PFA) paraformaldehyde, powder 95% (Sigma-Aldrich, catalog number: 158127 )
  9. NGS (Abcam, catalog number: ab7481 )
  10. DMSO (Sigma-Aldrich, catalog number: D8418 )
  11. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  12. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 18912014 )
  13. Chicken anti-GFP primary antibody (Abcam, catalog number: ab13970 )
  14. Alexa Fluor 488 goat anti-chicken IgG (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11039 )
  15. Mounting Medium for fluorescence (Vector Laboratories, catalog number: H-1000 )
  16. Coelenterazine-h (Biotium, catalog number: 10111 )
  17. Propylene glycol
  18. Cyclodextrine
  19. Agarose
  20. ‘Blue water’ (see Recipes)
  21. Blocking solution (see Recipes)
  22. Incubation solution (see Recipes)
  23. Washing solution (see Recipes)

Equipment

  1. Wave generator (Agilent Technologies, catalog number: 33210A )
  2. Audio amplifier (Lepai, catalog number: LP-2020A )
  3. Upright confocal microscope (Olympus, model: FV1000 )
  4. 850 nm LED (Effisharp, Effilux, France, EFFI-sharp_CM_850_2)
  5. Long-pass 780 filter (Asahi Spectra USA, catalog number: ZIL0780 )
  6. Long-pass 810 filter (Asahi Spectra USA, catalog number: XIL0810 )
  7. Diffuser (Thorlabs, catalog number: DG10-120-B )
  8. High-speed infrared sensitive camera (Mikrotron, catalog number: Eosens MC1362 )
  9. Objective Nikkor 50 mm f/1.8D (Nikon, Japan)
  10. Photomultiplier tube (PMT) (Hamamatsu Photonics, catalog number: H7360-02 )
  11. Acquisition card (National Instruments, catalog number: PCI 6602 )
  12. Band-pass filter (525 nm/50 nm) (ZEISS, catalog number: 489038-8002-000 )
  13. Short-pass filter (670 nm) (Asahi Spectra USA, catalog number: XVS0670 )
  14. 2-Ohm speaker
  15. TTL chronogram generator (RD Vision, France, EG Chrono)
  16. Black cardboard box protecting the setup from residual photons in the room
  17. Standard equipment for molecular biology
  18. Sonicator (Emerson Electric, Branson, model: B1510-DTH )

Software

  1. Hiris video software (RD Vision, France)
  2. MATLAB (The MathWorks, catalog number: R2012b)

Procedure

  1. Generation of the codon-optimized Tg(UAS:GFP-aequorin-opt) transgenic line
    1. Generate a codon-optimized sequence, GFP-aequorin-opt, for expression in zebrafish from original sequence of mammalian GFP-aequorin (we used to online tool freely available made by Integrated DNA Technology: https://eu.idtdna.com/CodonOpt, original and optimized DNA sequences provided in Supplement file 2).
    2. Subclone GFP-aequorin-opt into a PT2 14xUAS plasmid.
    3. Inject the UAS:GFP-aequorin-opt construct in the Tg(mnx1:gal4)icm11 embryos to generate the Tg(mnx1:gal4;UAS:GFP-aequorin-opt)icm09 double transgenic line. Injection mix is composed as follows: 2 µl ADN (120 ng/µl) + 2 µl ARN transposase (175 ng/µl) + 1 µl KCl 2 M +1 µl 2% phenol red, complete to 10 µl with MilliQ H2O.
    4. Maintain adult AB and Tüpfel long fin (TL) strains of Danio rerio on a 14/10 h light cycle and water is maintained at 28.5 °C, conductivity at 500 μS and pH at 7.4.
    5. Raise embryos in ‘blue water’ (3 g of Instant Ocean® salts and 2 ml of methylene blue at 1% in 10 L of osmosed water, see Recipes) at 28.5 °C during the first 24 h before screening for GFP expression.

  2. Characterization of GFP-aequorin expression with immunohistochemistry
    1. Fix 4 dpf larvae in 4% PFA for 4 h at 4 °C followed by 3 x 5 min washes in PBS.
    2. Block larvae for 1 h in blocking solution (see Recipes) (agitation required).
    3. Incubate larvae with the primary antibody (anti-GFP, dilution 1:500) over night at 4 °C in incubation solution (see Recipes) (agitation required).
    4. Wash three times for 5 min in washing solution (see Recipes), then incubate larvae in the dark with the secondary antibody (Alexa Fluor 488 goat anti-chicken IgG, dilution1:1,000) in PBST (agitation required) for 2 h at RT.
    5. Wash three times for 5 min in PBST, then mount larvae on a slide with mounting medium and image on a standard upright confocal microscope (Olympus FV-1000).
    6. Perform negative IHC controls by omitting the primary antibody.
    7. Image the entire immunostained Tg(mnx1:gal4;UAS:GFP-aequorin-opt)icm09 larvae to confirm selective expression of GFP-aequorin-opt in spinal motor neuron populations and absence from muscle fibers. We noted more prominently primary dorsal motor neurons but also intermediate and ventral secondary motor neurons (Figure 1) without any expression in the muscles and only very limited expression in the brain and hindbrain.


      Figure 1. Expression pattern of GFP-aequorin-opt in motor neurons. Fluorescent image (upper panel) and immunohistochemistry for GFP (lower panel) in a 4 dpf Tg(mnx1:gal4;UAS:GFP-aequorin-opt) double transgenic zebrafish larva showing selective expression in spinal motor neurons (arrowhead: dorsal primary, arrow: ventral secondary motor neurons), and strictly no expression in muscle fibers (white arrow in the upper panel).

  3. Soaking of larvae in coelenterazine solution
    1. Prepare 10 mM stock solution from lyophilized coelenterazine-h: e.g.,
      1. For 250 µg of coelenterazine-h, final volume is 60 µl (M = 407.5 g/M).
      2. Add propylene glycol to (25% of final volume, e.g., 15.4 µl).
      3. Sonicate cyclodextrine at 45% (4.5 g in 10 ml).
      4. Add cyclodextrine (75% of final volume, e.g., 46 µl).
    2. Prepare 60 µM coelenterazine-h soaking solution from stock:
      Dilute stock solution in ‘blue water’ (e.g., 6 µl in 1 ml for 10 embryos).
    3. Dechorionate embryos at 1 day post-fertilization under optical magnification.
    4. Soak dechorionated embryos overnight at 26 °C (e.g., 100 µl for each embryo, use 48-well plate sealed with paraffin).
    5. Renew 60 µM soaking solution at 2 days post-fertilization. Embryos are maintained in the dark.
    6. Perform behavioral experiments at 4 days post-fertilization (total soaking time is 72 h).

  4. Monitoring neuronal activity with bioluminescence
    1. Build a lightproof setup for bioluminescence assay (Figure 2)


      Figure 2. Bioluminescence setup for escapes. A. Signals emitted from spinal motor neurons in Tg(mnx1:gal4;UAS:GFP-aequorin-opt) double transgenic zebrafish larvae at 4 dpf were recorded using a photomultiplier tube under infrared illumination during active behaviors elicited by an acoustic stimulus. B. Picture of the setup showing every component: 1: high-speed camera; 2: 50 mm objective; 3: IR 850 nm LED; 4: Diffuser; 5: Long-pass filters; 6: photomultiplier tube.

      1. Using black boards, create a 1 m square lightproof box.
      2. Infrared light illumination is provided by an 850 nm LED mounted with 2 long-pass 780 and 810 filters and a diffuser.
      3. Video acquisition is performed at 1,000 Hz using a high-speed infrared sensitive camera at 320 x 320 pixels resolution controlled by the video software (Hiris®).
      4. Photons are counted with a photomultiplier tube located under the larva arena and sent to an acquisition card. A band-pass filter (525 nm/50 nm) and a short-pass filter (670 nm) are placed between the larva and the PMT.
      5. A custom application-programming interface synchronizes the video acquisition with the photon count and the stimulus delivery using a 30 trials batched TTL chronogram.
    2. Run the bioluminescence assay one larva at a time
      1. Place larva in a circular (2 cm diameter) 3D-printed arena (larva can also be head-embedded in 1.5% low-melting point agarose with the tail free to move).
      2. Place the larva in the arena and attach the arena to a small 2-Ohm speaker.
      3. Deliver sinusoidal stimuli (5 cycles, 500 Hz) produced by the waveform generator and audio amplifier through a 2-Ohm speaker attached to the larva arena.
      4. Adjust intensity to the lowest value reliably eliciting an escape response (between 0.5 and 5 V usually).
      5. Each trial consists in a 500 msec baseline followed by a 10 msec acoustic stimulus and 1,990 msec subsequent recording.
      6. Assays consist of 30 trials with 1-min inter-trial intervals to reduce habituatio.

Data analysis

  1. Kinematics analysis (blue trace in Figure 3, right panel)
    1. Using a custom MATLAB algorithm (see Supplement file 3), manually locate the base and tip of the tail. The tail is subsequently automatically tracked.
    2. The tail angle is computed for each frame and filtered using median filtering (window size = 10).
    3. The start of the movement is determined as the first frame followed by 3 with a differential tail angle value above 0.08 degree in our conditions.
    4. The end of the movement is determined as the last of 20 frames with a differential tail angle value below 0.1 degree in our conditions.
    5. Local minimal and maximal values of the tail angle occur at least 2 msec apart and 1° above the 5 msec preceding value.
    6. Automated movement categorization is determined as follows: ‘escapes’ for all movements with maximum values of tail angle > 45° and number of cycles > 1; ‘slow swims’ for all movement with maximum values of tail angle < 25° and number of cycles > 1.

  2. Bioluminescence recording and analysis (green trace in Figure 3B)
    1. Photons are counted at 1 kHz (temporal resolution of 1 msec) and then binned every 10 msec.
    2. The signal is filtered using a running average with a window size of 10, giving a typical signal-to-noise ratio (SNR) for active movements of 50 to 1.
    3. Noise is extrapolated from a linear fit of the cumulative photon count before the stimulus and subtracted from the signal.
    4. The start and end of the bioluminescent signal are computed respectively as the first time point followed by 3 points with a value above 0.4 photons/10 msec and below 0.2 photons/10 msec from this first point bioluminescence value.
    5. The time-to-peak is calculated between the start and the peak of the bioluminescent signal while the decay coefficient is derived from the one-term exponential fit between the peak and the end of the signal.


      Figure 3. Example of kinematics and bioluminescence data for an escape response. A. 4 dpf larva in the head-embedded setup with the tail tracked (red dots) in transmitted IR light. B. Superimposed, and color-coded according to delay from stimulus, behaviors elicited by an acoustic stimulus; C. Example traces of typical bioluminescence signals and tail angle observed for an escape response; D. Example of a color-coded slow swim and (E) corresponding bioluminescence signal and tail angle traces for comparison with escapes.

Notes

  1. Soaking in coelenterazine is key to obtaining a good signal-to-noise ratio for bioluminescence: pay attention to soaking conditions (incubation temperature, hour of coelenterazine renewal, keep the wells of the plate air-tight).
  2. Noise due to ambient light must be tested before running the behavioral experiment; make sure the box is completely lightproof.
  3. The Tg(UAS:GFP-aequorin-opt) zebrafish line is freely available to all users that request it.

Recipes

  1. ‘Blue water’
    3 g of Instant Ocean® salts and 2 ml of 1% methylene blue in 10 L of ultrapure water
  2. Blocking solution
    10% NGS
    1% DMSO
    0.5% Triton X-100
    in 0.1 M PBS
  3. Incubation solution
    1% NGS
    1% DMSO
    0.5% Triton X-100
    in 0.1% PBS
  4. Washing solution
    0.1 M PBST with 0.5% Triton X-100

Acknowledgments

We would like to thank Dr. Ludovic Tricoire (University Pierre et Marie Curie, France) for providing the original GFP-aequorin sequence, Dr. Tom Auer and Dr. Filippo Del Bene (Institut Curie, France) for providing the mnx1 construct and Prof. Koichi Kawakami (NIG, Japan) for providing the PT2 14xUAS plasmid. We are indebted to RD Vision for developing the custom API to synchronize photon collection and video acquisition. We thank Bogdan Buzurin and Natalia Maties for fish care. SK received a PhD fellowship from Inserm and Assistance Publique–Hôpitaux de Paris. This work received financial support from the Institut du Cerveau et de la Moelle épinière with the French program ‘Investissements d’avenir’ ANR-10-IAIHU-06, the ENP Chair d’excellence, the Fondation Bettencourt Schueller (FBS), the City of Paris Emergence program, the ATIP/Avenir junior program from INSERM and CNRS, the NIH Brain Initiative grant no. 5U01NS090501 and the European Research Council (ERC) starter grant ‘OptoLoco’ #311673.

References

  1. Grienberger, C. and Konnerth, A. (2012). Imaging calcium in neurons. Neuron 73(5): 862-885.
  2. Martin, J. R., Rogers, K. L., Chagneau, C. and Brulet, P. (2007). In vivo bioluminescence imaging of Ca2+ signalling in the brain of Drosophila. PLoS One 2(3): e275.
  3. Naumann, E. A., Kampff, A. R., Prober, D. A., Schier, A. F. and Engert, F. (2010). Monitoring neural activity with bioluminescence during natural behavior. Nat Neurosci 13(4): 513-520.
  4. Rogers, K. L., Picaud, S., Roncali, E., Boisgard, R., Colasante, C., Stinnakre, J., Tavitian, B. and Brulet, P. (2007). Non-invasive in vivo imaging of calcium signaling in mice. PLoS One 2(10): e974.
  5. Shimomura, O., Johnson, F. H. and Saiga, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59: 223-239.

简介

以前已经描述了基因编码的钙指示剂GFP-aequorin的斑马鱼中神经活性的生物发光监测的概念验证(Naumann等人,2010),但是仍然存在挑战。 首先,源自单一肌纤维的生物发光信号可能构成主要缺陷。 第二,仅从神经元发出的生物发光信号非常小。 为了改善信号,同时验证特异性,我们提供了一个优化的4步骤协议,实现:1)选择性表达斑马鱼密码子优化的GFP水母发光蛋白,2)在GFP-水母发光蛋白底物coelenterazine中有效浸泡幼虫3)生物发光监测神经活动 来自运动神经元的自由运动的动物执行声学逃逸和4)使用免疫组织化学验证肌肉的表达缺乏。
【背景】不同于荧光基因编码的钙指示剂(GECIs)(Grienberger和Konnerth,2012),如GCaMP家族,生物发光指示剂GFP-水母发光蛋白(Shimomura等,1962)不需要光激发,因此开启了监测神经元的新途径包括苍蝇在内的动物(Martin等,2007),小鼠(Rogers et al。,2007)和斑马鱼幼虫(Naumann et al。,2010)的活动。然而,GFP-水母发光蛋白的有效使用在斑马鱼幼虫中仍然具有挑战性,限制了其广泛用作钙指示剂。限制在于,源自单个肌纤维的生物发光信号如此之大,构成主要缺陷。一旦给定的转基因品系证实肌肉表达缺失,则仅从神经元发出的生物发光信号非常小。为了克服这些局限,我们开发了斑马鱼幼虫GFP-aequorin的密码子优化变体,使用现有的转基因品系在运动和感觉神经元中进行选择性表达,修饰的肠道浸润方法在受精后4天进行实验用于监测斑马鱼幼虫声音诱发定型逃避反应过程中神经元活动的行为生物发光测定。

关键字:GFP-水母发光蛋白优化基因, 斑马鱼, 生物发光, 腔肠素, 逃避反应

材料和试剂

  1. 显微镜幻灯片76 x 26 x 1.1 mm(VINCENT LEERMIDDELEN SCIENTIFIC,目录号:29201316)
  2. 盖玻璃Knittel玻璃24 x 60毫米
  3. 本研究使用斑马鱼幼虫成年AB和Tüpfel长鳍(TL)菌株,年龄在0〜4 dpf(受精后1天);在本方案中使用的转基因品系(可根据要求提供):Tg(mnx1:gal4) ; Tg(UAS:GFP-aequorin-opt)
  4. 哺乳动物GFP-aequorin序列(由Dr.Ludovic Tricoire,UniversitéPierre et Marie Curie,Paris,France提供,见 http://kawakami.lab.nig.ac.jp/trans.html
  5. 即时海洋®盐
  6. 亚甲蓝
  7. 固定溶液(4%PFA)多聚甲醛,粉末95%(Sigma-Aldrich,目录号:158127)
  8. NGS(Abcam,目录号:ab7481)
  9. DMSO(Sigma-Aldrich,目录号:D8418)
  10. Triton X-100(Sigma-Aldrich,目录号:T9284)
  11. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:18912014)
  12. 鸡抗GFP一抗(Abcam,目录号:ab13970)
  13. Alexa Fluor 488山羊抗鸡IgG(Thermo Fisher Scientific,Invitrogen TM,目录号:A-11039)
  14. 荧光固定介质(Vector Laboratories,目录号:H-1000)
  15. Coelenterazine-h(Biotium,目录号:10111)
  16. 丙二醇
  17. 环糊精
  18. 琼脂糖
  19. “蓝水"(见食谱)
  20. 阻塞解决方案(见配方)
  21. 孵化解决方案(参见食谱)
  22. 洗涤液(参见食谱)

设备

  1. 波发生器(Agilent Technologies,目录号:33210A)
  2. 音频放大器(Lepai,目录号:LP-2020A)
  3. 直立共焦显微镜(Olympus,型号:FV1000)
  4. 850 nm LED(Effisharp,Effilux,France, EFFI-sharp_CM_850_2
  5. 长通780过滤器(Asahi Spectra USA,目录号:ZIL0780)
  6. 长通810过滤器(Asahi Spectra USA,目录号:XIL0810)
  7. 扩散器(Thorlabs,目录号:DG10-120-B)
  8. 高速红外敏感相机(Mikrotron,目录号:Eosens MC1362)
  9. 目标Nikkor 50 mm f / 1.8D(尼康,日本)
  10. 光电倍增管(PMT)(滨松光子学,目录号:H7360-02)
  11. 采集卡(National Instruments,目录号:PCI 6602)
  12. 带通滤波器(525nm / 50nm)(ZEISS,目录号:489038-8002-000)
  13. 短路滤波器(670nm)(Asahi Spectra USA,目录号:XVS0670)
  14. 2欧姆扬声器
  15. TTL计时码表发生器(RD Vision,France, EG Chrono
  16. 黑色纸板箱保护设备免受房间残留光子的影响
  17. 分子生物学标准设备
  18. 超声波发生器(艾默生电气,布兰森,型号:B1510-DTH)

软件

  1. Hiris视频软件(RD Vision,法国)
  2. MATLAB(MathWorks,目录号:R2012b)

程序

  1. 产生密码子优化的Tg(UAS:GFP-aequorin-opt)转基因株系
    1. 产生密码子优化的序列,GFP-aequorin-opt,用于在哺乳动物GFP-aequorin的原始序列中在斑马鱼中表达(我们曾经用于由Integrated DNA技术: https://eu.idtdna.com/CodonOpt ,原件和优化的DNA序列,提供在补充文件2 )。
    2. 将亚克隆GFP-aequorin-opt
    3. 在TM(gnx1:gal4) icm11 2 O。
    4. 在14/10小时的光循环下维持成年的AB和Tüpfel长鳍(TL)菌株,水保持在28.5℃,电导率为500μS,pH为7.4。
    5. 在第一个24小时内,在28.5℃下,在10升渗透水中,在“蓝色水"(3g的Instant Ocean&lt;&amp;&gt;盐和2ml亚甲基蓝中加入1%的胚胎,见Recipes)筛选GFP表达之前。

  2. 用免疫组化法表征GFP-aequorin表达
    1. 在4℃下将4 dpf幼虫在4℃下固定4小时,然后在PBS中洗涤3×5分钟。
    2. 在封闭溶液中封闭幼虫1小时(见食谱)(需要搅拌)
    3. 在孵育溶液(4℃)(参见食谱)(需要搅拌)下,用初级抗体(抗GFP,稀释1:500)孵育幼虫过夜。
    4. 在洗涤溶液中洗涤3次5分钟(参见食谱),然后在PBST(需要搅拌)中培养2小时的二抗(Alexa Fluor 488山羊抗鸡IgG,稀释1:1000)在黑暗中孵化幼虫。
    5. 在PBST中洗涤3次5分钟,然后将载体上的载体载入载玻片上,并在标准直立共聚焦显微镜(Olympus FV-1000)上进行成像。
    6. 通过省略一级抗体来进行阴性IHC对照
    7. 将整个免疫染色的Tg(mnx1:gal4; UAS:GFP-aequorin-opt)icm09 幼虫成像以确认GFP-aequorin-opt 在脊髓运动神经元群体和缺乏肌肉纤维。我们注意到更突出的主要背侧运动神经元,而且中间和腹侧次要运动神经元(图1),肌肉中没有任何表达,并且在脑和后脑中的表达非常有限。


      图1.运动神经元中GFP-aequorin-opt 荧光图像(上图)和GFP(下图)的免疫组织化学显示选择性表达的4dpf Tg(mnx1:gal4; UAS:GFP-水平曲霉素-opt)双转基因斑马鱼幼虫在脊髓运动神经元(箭头:背部原发性,箭头:腹侧继发运动神经元),并且在肌肉纤维中严格没有表达(上图中的白色箭头)。

  3. 在coelenterazine溶液中浸泡幼虫
    1. 从冻干的coelenterazine-h制备10mM储备溶液:例如,
      1. 对于250μg肠腔素-H,终体积为60μl(M = 407.5g / M)
      2. 加入丙二醇至(最终体积的25%,例如15.4μl)
      3. 超声波环糊精45%(4.5克,10毫升)
      4. 加入环糊精(最终体积的75%,例如
    2. 从库存中准备60μMcoelenterazine-h浸泡溶液:
      在“蓝色水"(例如
    3. 光学放大后受精1天后的脱毛胚胎
    4. 在26℃(例如,每个胚胎100μl,使用用石蜡密封的48孔板)中浸泡脱钙胚胎过夜。
    5. 在受精后2天更新60μM浸泡溶液。胚胎保持在黑暗中。
    6. 在受精后4天进行行为实验(总浸泡时间为72 h)。

  4. 用生物发光监测神经元活动
    1. 建立生物发光测定的防光设置(图2)


      图2.逃逸的生物发光设置。 :一种。在4dpf下,使用光电倍增管在红外照射下记录在Tg(mnx1:gal4; UAS:GFP-aequorin-opt))的脊髓运动神经元发出的信号,声刺激。 B.设置显示每个组件的图片:1:高速摄像头; 2:50mm物镜; 3:红外850nm LED; 4:扩散器5:长通滤波器; 6:光电倍增管
      1. 使用黑板,创建一个1平方米的防光盒。
      2. 红外光照明由安装有2个长通780和810滤光片的850nm LED和漫射器提供。
      3. 使用由视频软件(Hiris ®)控制的320 x 320像素分辨率的高速红外敏感摄像机以1,000 Hz进行视频采集。
      4. 光子计数在位于幼虫竞技场下面的光电倍增管,并发送到采集卡。在幼虫和PMT之间放置带通滤波器(525nm / 50nm)和短路滤波器(670nm)。
      5. 定制的应用编程接口使用30个试验批次的TTL计时码表,将视频采集与光子计数和刺激传送同步。
    2. 一次运行生物发光测定一只幼虫
      1. 将幼虫置于圆形(2厘米直径)3D印刷的竞技场(幼虫也可以头部嵌入1.5%低熔点琼脂糖,尾部可自由移动)。
      2. 将幼虫放在舞台上,并将竞技场连接到一个小的2欧姆扬声器。
      3. 由波形发生器和音频放大器通过连接到幼体场的2欧姆扬声器提供正弦波刺激(5个周期,500 Hz)。
      4. 将强度调整到最低值可靠地引发逃生响应(通常在0.5和5 V之间)。
      5. 每个试验包括500毫秒的基线,接着是10毫秒的声学刺激和1,990毫秒的后续记录
      6. 测试包括30次试验间隔时间为1分钟,以减少习惯。

数据分析

  1. 运动学分析(图3中的蓝色轨迹,右图)
    1. 使用自定义MATLAB算法(参见补充文件3 ),手动定位尾部的底部和末端。尾巴随后自动跟踪。
    2. 对每个帧计算尾角,并使用中值滤波(窗口大小= 10)进行滤波
    3. 移动的开始被确定为第一帧,后跟3,在我们的条件下,差分尾角值高于0.08度。
    4. 运动的结束被确定为在我们的条件下差分尾角值低于0.1度的20帧中的最后一帧。
    5. 尾部角度的局部最小值和最大值发生在距离5毫秒前值至少2毫秒和1度之间。
    6. 自动运动分类确定如下:对于具有最大尾角值的所有运动的“逃逸"&gt; 45°,循环次数&gt; 1;所有运动的“慢游泳",尾角的最大值< 25°,循环次数&gt;

  2. 生物发光记录和分析(图3B中的绿色迹线)
    1. 光子计数为1 kHz(时间分辨率为1毫秒),然后每10毫秒进行合并。
    2. 信号使用窗口大小为10的运行平均值进行滤波,给出了主动移动为50比1的典型信噪比(SNR)。
    3. 从刺激前的累积光子计数的线性拟合推断出噪声,并从信号中减去噪声
    4. 分别计算生物发光信号的开始和结束为第一时间点,随后是具有高于0.4光子/ 10毫秒的值的3个点,并且从该第一点生物发光值开始低于0.2个光子/ 10毫秒。
    5. 在生物发光信号的起始和峰值之间计算时间峰值,而衰减系数是从信号的峰值和结束之间的一项指数拟合得出的。


      图3.逃逸反应的运动学和生物发光数据的实例。 :一种。 4 dpf幼虫在头部嵌入式设置中,尾部跟踪(红色点)在传输的红外线。 B.叠加,根据刺激的延迟进行颜色编码,由声学刺激引起的行为;典型生物发光信号的痕迹和逃逸响应观察到的尾角; D.颜色缓慢游泳的例子和(E)相应的生物发光信号和尾角痕迹,以便与逃逸进行比较。

笔记

  1. 浸泡在肠培养液中是获得生物发光的良好信噪比的关键:注意浸泡条件(培养温度,肠肠菌丝更新时间,保持板的气密性)。
  2. 在运行行为实验之前必须对环境光的噪声进行测试;确保盒子完全防光。
  3. 斑马鱼线(UAS:GFP-aequorin-opt)

食谱

  1. '蓝水'
    3克Instant Ocean&lt;&amp;&gt;盐和2ml 1%亚甲基蓝10升超纯水
  2. 阻塞解决方案
    10%NGS
    1%DMSO
    0.5%Triton X-100
    在0.1M PBS中
  3. 孵化解决方案
    1%NGS
    1%DMSO
    0.5%Triton X-100
    在0.1%PBS中
  4. 洗涤液
    含0.5%Triton X-100的0.1M PBST

致谢

我们要感谢Ludovic Tricoire博士(法国Pierre et Marie Curie大学)提供原始的GFP-aequorin序列,Tom Auer博士和Filippo Del Bene博士(法国居里)为了提供 mnx1

参考

  1. Grienberger,C.和Konnerth,A.(2012)。&lt; a class =“ke-insertfile"href =“http://www.ncbi.nlm.nih.gov/pubmed/22405199"target =“_ blank" >成像神经元中的钙。神经元73(5):862-885。
  2. Martin,JR,Rogers,KL,Chagneau,C.和Brulet,P。(2007)。在自然行为过程中用生物发光监测神经活动。 Nat Neurosci 13(4):513-520。
  3. Rogers,KL,Picaud,S.,Roncali,E.,Boisgard,R.,Colasante,C.,Stinnakre,J.,Tavitian,B.and Brulet,P。(2007)。&lt; a class = -insertfile“href ="http://www.ncbi.nlm.nih.gov/pubmed/17912353“target ="_ blank“>小鼠中钙信号传导的体内非侵入性成像/ a> PLoS One 2(10):e974。
  4. Shimomura,O.,Johnson,FH和Saiga,Y.(1962)。水母发光蛋白的提取,纯化和性质,来自发光的氢溴酸盐的生物发光蛋白质,Amequorea。J Cell Comp Physiol 59:223-239 。
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Copyright Knafo et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Knafo, S., Prendergast, A., Thouvenin, O., Figueiredo, S. N. and Wyart, C. (2017). Bioluminescence Monitoring of Neuronal Activity in Freely Moving Zebrafish Larvae. Bio-protocol 7(18): e2550. DOI: 10.21769/BioProtoc.2550.
  2. Knafo, S., Fidelin, K., Prendergast, A., Tseng, P. B., Parrin, A., Dickey, C., Bohm, U. L., Figueiredo, S. N., Thouvenin, O., Pascal-Moussellard, H. and Wyart, C. (2017). Mechanosensory neurons control the timing of spinal microcircuit selection during locomotion. Elife 6.
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