JoVE Journal > Neuroscience
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
Please note that all translations are automatically generated. Click here for the English version.
신경과학

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

Published: March 19, 2021
doi:
10.3791/62308
,
1Program for Neuroscience and Mental Health,Hospital for Sick Children, 2Department of Molecular Genetics,University of Toronto

Summary

在这里，我们提出了一种通过延时共聚焦显微镜研究产后小鼠视网膜外植体中神经突形态发生的方法。我们描述了一种使用重组腺相关病毒载体（以 Cre依赖性方式表达膜靶向荧光蛋白）对视网膜细胞类型及其精细过程进行稀疏标记和获取的方法。

Abstract

发现树突状乔木的模式需要方法在发育过程中可视化，成像和分析树突。小鼠视网膜是一个强大的模型系统，用于研究神经元形态发生和连接的细胞类型特异性机制。视网膜亚型的组织和组成是明确的，遗传工具可用于在发育过程中访问特定类型。许多视网膜细胞类型也将其树突和/或轴突限制在狭窄的层，这有助于延时成像。小鼠视网膜外植体培养物非常适合使用共聚焦或多光子显微镜进行活细胞成像，但缺乏针对具有时间和结构分辨率的树突动力学成像优化的方法。这里介绍的是一种稀疏地标记和成像由Cre-Lox系统标记的特定视网膜群体发展的方法。这里使用的市售腺相关病毒（AAV）以Cre依赖性方式表达膜靶向荧光蛋白。新生儿小鼠中AAV的眼内递送在注射后4-5天（dpi）产生靶向细胞类型的荧光标记。膜荧光信号可通过共聚焦成像进行检测，并解析精细的分支结构和动力学。从充满含氧人工脑脊液（aCSF）的视网膜平片成像仪获取跨越2-4小时的高质量视频。还提供了用于反卷积和三维（3D）漂移校正的图像后处理管道。该协议可用于捕获完整视网膜中的几种细胞行为，并确定控制神经突起形态的新因素。在视网膜中学到的许多发育策略将与理解中枢神经系统其他地方的神经回路的形成有关。

Introduction

视网膜神经元的树突形成复杂但特定的模式，影响它们在神经回路中的功能。在脊椎动物视网膜中，不同类型的视网膜神经节细胞（RGC）和柏宁细胞中间神经元具有独特的树突形态，其树突形态在心轴大小、位置、分支长度和密度上有所不同1。在产后发育过程中，RGC和amcrine细胞将旺盛的树突突延伸到称为内丛状层（IPL）的神经瞳孔中，在那里它们接收双极细胞输入，发送光感受器信号2。通过对雏鸡或斑马鱼幼虫中荧光标记的视网膜种群进行延时成像捕获，树突形态发生具有高度动态性3，4，5。在几天内，树突状乔木膨胀，重塑并撞击到IPL的狭窄子层，在那里它们与选定的伙伴突触。乔木在发育过程中表现出不同的结构动力学，分支增加，收缩和稳定的相对速率发生变化。Amacrine和RGC枝晶也表现出不同的生长和重塑行为，这可能反映了特定类型的树木化。然而，这些研究追踪了广泛的amcrine或RGC群体，并专注于层流靶向，这只是形态学的一个方面。

产生在视网膜亚型中观察到的巨大形态多样性的机制知之甚少。该小组的目标是开发一种方法来捕获小鼠中定义的视网膜亚型的树突动力学和乔木重塑。识别树突图案化的细胞类型特异性机制需要可视化和测量目标细胞的树突行为的方法。小鼠视网膜的器官型培养非常适合使用共聚焦或多光子显微镜的活细胞成像研究。将发育中的视网膜解剖并安装到平坦的外植体中，该外植体可以在记录室中成像数小时或在几天内培养，对电路的影响有限6，7。活的视网膜神经元可以通过多种技术进行标记，包括通过电极填充染料，电穿孔，涂有亲脂性染料或编码荧光蛋白的质粒（例如，Gene Gun）的颗粒的生物传递，以及遗传编码的细胞标签7，8，9，10.然而，这些方法对于特定视网膜亚型的树突动力学成像效率低下。例如，染料填充方法的通量较低，需要电生理学设备和额外的遗传标记才能可靠地靶向感兴趣的细胞。此外，体细胞中的强荧光信号会遮挡附近的树突。

生物基因递送方法可以同时标记数十个细胞，但涉及高压颗粒递送和孤立视网膜过夜孵育的步骤可能会损害细胞生理学和树突生长。本文提出，根据以下实验标准，可以利用最近的遗传工具来捕获具有细胞类型和结构分辨率的早期树突动力学。首先，为了解决主导发育中的乔木的细枝和丝状体，该方法应该用明亮的荧光蛋白标记神经元，这些蛋白填充了整个心轴中的过程。在成像期间，荧光标记不应因光漂白而褪色。已经生成了多种荧光蛋白变体，并根据亮度和光稳定性比较了其对体内/离体成像的适用性11 。其次，荧光蛋白（XFPs）必须在树突形态发生的最早阶段以足够高的水平表达，以便不会错过狭窄的发育窗口。在对小鼠视网膜静态时间点的分析中，树突发育发生在出生后的第一周，包括生长，重塑和稳定阶段10，12，13，14，15。第三，该方法应导致选择性标记或增加感兴趣的神经元亚群的标记概率。第四，目标亚群的标记必须足够稀疏，以便可以识别和追踪整个神经元乔木。虽然RGC和amacrine亚型可以通过其成熟的形态特征和IPL分层模式来区分16，17，18，19，20，但挑战在于基于未成熟结构在发育过程中识别亚型。通过扩增转基因工具在发育过程中标记特定的视网膜细胞类型，可以促进这项任务。

转基因和敲入小鼠品系，其中荧光蛋白或Cre的细胞和时间表达由基因调节元件决定，被广泛用于研究视网膜细胞类型13，21，22，23。对树突发育亚型特异性模式的关键观察来自静态时间点对转基因小鼠视网膜的研究10，14，24，25。特别是Cre-Lox系统，它可以使用各种重组酶依赖性报告基因，传感器和光遗传学激活剂对亚型进行精细的基因操作和监测。这些工具导致了亚型特异性分子程序和功能特性的发现，这些功能特性是视网膜电路组装的基础26，27，28，29，30。然而，它们尚未被用于研究小鼠视网膜中亚型特异性的树突动力学。通过将Cre小鼠品系与通过电穿孔或重组AAV引入的转基因相结合，可以实现低密度标记。如果可行，也可以使用他莫昔芬诱导的Cre系或交叉遗传策略。最后，应以微创方式标记细胞，并使用采集参数进行成像，以免损害组织或干扰树突形态发生所需的细胞功能。

这里介绍的是一种应用转基因工具和共聚焦显微镜来研究活小鼠视网膜外植体中的树突动力学的方法。Cre转基因小鼠品系已与AAV载体相结合，后者在Cre重组时表达荧光蛋白，这允许对感兴趣的视网膜细胞进行稀疏标记。市售的 AAV 通过玻璃体内注射输送到新生儿视网膜。本文表明，AAV以4 dpi产生显着的高和细胞类型特异性荧光表达，允许访问产后时间点。为了说明这种方法，通过在表达胆碱乙酰转移酶（ChAT）-内部核糖体进入位点（IRES）-Cre转基因的新生小鼠中传递Brainbow AAV来标记胆碱能“星爆”amacrine中间神经元，该基因在产后早期视网膜中活跃31，32。Starburst amacrine细胞发展出一种刻板的放射状乔木形态，由簇状的原钙粘蛋白介导的树突自我回避形成33，34。本文表明，通过添加用于Brainbow AAVs31的经过法呢基化的CAAX基序，XFP显着提高了星爆树突和丝状体的分辨率。最后，已经确定了延时成像和后处理方案，可产生适合树突重建和形态量化的高质量图像。该协议可用于识别控制树突形态发生的因素，并捕获完整视网膜中的几种细胞行为。

Protocol

注意：该方案跨越2天，实验日之间的病毒转导最短时间为4-5天（图1A）。动物实验是根据加拿大动物护理委员会根据病童医院动物服务实验室动物使用和护理委员会批准的协议进行的，用于研究和实验动物护理。 1. 新生儿AAV注射和成像实验的准备 选择Cre小鼠线以标记感兴趣的视网膜细胞群。在AAV注射时，通过与Cre转基因报告基因交叉或通过Cre抗…

Representative Results

使用上述协议，采集了星爆细胞枝晶发育的高分辨率3D视频，对其进行了解卷积，并针对3D漂移进行了校正。生成Z平面最大投影以制作用于分析的2D视频（补充视频1， 图5A）。每个时间点的3D反卷积提高了细丝状突起的分辨率（图5B，C）。细丝状突起是视网膜树突发育的特征36 ，在影像学检查过程中应可见。AA…

Discussion

该视频演示了一个实验管道，该管道利用现有的遗传工具，通过共聚焦实时成像对发育中的视网膜神经元的树突动力学进行成像。还证明了将编码膜靶向荧光蛋白的Cre依赖性AAVs的眼内注射到新生小鼠中。遗传靶向群体的单细胞早在4-5 dpi时就被明确标记。为标准成像室准备视网膜平片支架以执行活细胞共聚焦成像。这种方法可以生成单个细胞及其精细投影的高分辨率延时视频。使用开源软件（包?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢麦迪逊·格雷（Madison Gray）在我没有帮助的时候帮了我一把。这项研究得到了NSERC发现补助金（RGPIN-2016-06128），斯隆神经科学奖学金和加拿大研究主席Tier 2（J.L.L）的支持。S. Ing-Esteves得到了视觉科学研究计划和NSERC研究生奖学金博士的支持。

Materials

Addgene viral prep #45185-AAV9
Addgene viral prep #45186-AAV9
Dissection tools
Cellulose filter paper Whatman 1001-070
Dumont #5 fine forceps FST 11252-20 Two Dumont #5 forceps are required for retinal micro-dissection
Dumont forceps VWR 82027-426
Fine Scissors FST 14058-09
Mixed cellulose ester membrane (MCE) filter papers, hydrophilic, 0.45 µm pore size Millipore HABG01 300
Petri Dish, 50 × 15 mm VWR 470313-352
Polyethylene disposable transfer pipette VWR 470225-034
Round tip paint brush, size 3/0 Conventional art supply store Two size 3/0 paint brushes (or smaller) are required for retinal flat-mounting
Surgical Scissors FST 14007-14
Vannas Spring Scissors – 2.5 mm Cutting Edge FST 15000-08
Live-imaging incubation system
Chamber polyethylene tubing, PE-160 10' Warner Instruments 64-0755
Dual channel heater controller, Model TC-344C Warner Instruments 64-2401
HC FLUOTAR L 25x/0.95 W VISIR dipping objective Leica 15506374
Heater controller cable Warner Instruments CC-28
Large bath incubation chamber with slice support Warner Instruments RC-27L
MPII Mini-Peristaltic Pump Harvard Apparatus 70-2027
PM-6D Magnetic Heated Platform (incubation chamber heater) Warner Instruments PM-6D
Pump Head Tubing Pieces For MPII Mini-Peristaltic Pump Harvard Apparatus 55-4148
Sample anchor (Harps) Warner Instruments 64-0260 Sample anchor must be compatible with incubation chamber
Sloflo In-line Solution Heater Warner Instruments SF-28
Neonatal Injections
10 µL Microliter Syringe Series 700, Removable Needle Hamilton Company 80314
30 G Hypodermic Needles (0.5 inch) BD PrecisionGlide 305106
4 inch thinwall glass capillary, no filament (1.0 mm outer diameter/0.75 mm)  WPI World Precision Instruments TW100-4
Ethanol 99.8% (to dilute to 70% with double-distilled water [ddH2O]) Sigma-Aldrich V001229 
AAV9.hEF1a.lox.TagBFP. lox.eYFP.lox.WPRE.hGH-InvBYF Penn Vector Core AV-9-PV2453 Addgene Plasmid #45185 
AAV9.hEF1a.lox.mCherry.lox.mTFP
1.lox.WPRE.hGH-InvCheTF		 Penn Vector Core AV-9-PV2454 Addgene Plasmid #45186
ChAT-IRES-Cre knock-in transgenic mouse line The Jackson Laboratory 6410
Fast Green FCF Dye content ≥85 % Sigma-Aldrich F7252-25G
Flaming/Brown Micropipette Puller, model P-97 Sutter Instrument Co. P-97
Green tattoo paste Ketchum MFG Co 329A
Phosphate-Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell culture Sigma-Aldrich 806552
Pneumatic PicoPump WPI World Precision Instruments PV-820
Oxygenated artifiial cerebrospinal fluid (aCSF) Reagents
Calcium chloride dihydrate (CaCl2·2H2O) Sigma-Aldrich C7902
Carbogen (5% CO2, 95% O2) AirGas X02OX95C2003102 Supplier may vary depending on region
D-(+)-Glucose Sigma-Aldrich G7021
HEPES, Free Acid Bio Basic HB0264
Hydrochloric acid solution, 1 N Sigma-Aldrich H9892
Magnesium chloride hexahydrate (MgCl2·6H2O) Sigma-Aldrich M2670
pH-Test strips (6.0-7.7) VWR BDH35317.604
Potassium chloride (KCl) Sigma-Aldrich P9541
Sodium chloride (NaCl) Bio Basic DB0483
Sodium phosphate monobasic (NaH2PO4) Sigma-Aldrich RDD007
Software
ImageJ National Institutes of Health (NIH) Open source
DOWNLOAD MATERIALS LIST

References

  1. Lefebvre, J. L., Sanes, J. R., Kay, J. N. Development of dendritic form and function. Annual Review of Cell and Developmental Biology. 31, 741-777 (2015).
  2. Graham, H. K., Duan, X. Molecular mechanisms regulating synaptic specificity and retinal circuit formation. Wiley Interdisciplinary Reviews Developmental biology. 10 (1), 379 (2021).
  3. Godinho, L., et al. Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina. Development. 132 (22), 5069-5079 (2005).
  4. Mumm, J. S., et al. In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells. Neuron. 52 (4), 609-621 (2006).
  5. Wong, W. T., Faulkner-Jones, B. E., Sanes, J. R., Wong, R. O. Rapid dendritic remodeling in the developing retina: dependence on neurotransmission and reciprocal regulation by Rac and Rho. The Journal of Neuroscience. 20 (13), 5024-5036 (2000).
  6. Wei, W., Elstrott, J., Feller, M. B. Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina. Nature Protocols. 5 (7), 1347-1352 (2010).
  7. Morgan, J. L., Wong, R. O. L. Ballistic labeling with fluorescent dyes and indicators. Current Protocols in Neuroscience. 43 (1), 1-10 (2008).
  8. Nickerson, P. E. B., et al. Live imaging and analysis of postnatal mouse retinal development. BMC Developmental Biology. 13, 24 (2013).
  9. Morgan, J. L., Dhingra, A., Vardi, N., Wong, R. O. L. Axons and dendrites originate from neuroepithelial-like processes of retinal bipolar cells. Nature Neuroscience. 9 (1), 85-92 (2006).
  10. Coombs, J. L., Van Der List, D., Chalupa, L. M. Morphological properties of mouse retinal ganglion cells during postnatal development. The Journal of Comparative Neurology. 503 (6), 803-814 (2007).
  11. Cranfill, P. J., et al. Quantitative assessment of fluorescent proteins. Nature Methods. 13 (7), 557-562 (2016).
  12. Stacy, R. C., Wong, R. O. L. Developmental relationship between cholinergic amacrine cell processes and ganglion cell dendrites of the mouse retina. The Journal of Comparative Neurology. 456 (2), 154-166 (2003).
  13. Kay, J. N., et al. Retinal ganglion cells with distinct directional preferences differ in molecular identity, structure, and central projections. The Journal of Neuroscience. 31 (21), 7753-7762 (2011).
  14. Liu, J., Sanes, J. R. Cellular and molecular analysis of dendritic morphogenesis in a retinal cell type that senses color contrast and ventral motion. The Journal of Neuroscience. 37 (50), 12247-12262 (2017).
  15. Diao, L., Sun, W., Deng, Q., He, S. Development of the mouse retina: emerging morphological diversity of the ganglion cells. Journal of Neurobiology. 61 (2), 236-249 (2004).
  16. Coombs, J., vander List, D., Wang, G. Y., Chalupa, L. M. Morphological properties of mouse retinal ganglion cells. 신경과학. 140 (1), 123-136 (2006).
  17. Sanes, J. R., Masland, R. H. The types of retinal ganglion cells: current status and implications for neuronal classification. Annual Review of Neuroscience. 38, 221-246 (2015).
  18. Sümbül, U., et al. A genetic and computational approach to structurally classify neuronal types. Nature Communications. 5, 3512 (2014).
  19. Lin, B., Masland, R. H. Populations of wide-field amacrine cells in the mouse retina. The Journal of Comparative Neurology. 499 (5), 797-809 (2006).
  20. Macneil, M. A., Heussy, J. K., Dacheux, R. F., Raviola, E., Masland, R. H. The shapes and numbers of amacrine cells: Matching of photofilled with Golgi-stained cells in the rabbit retina and comparison with other mammalian species. Journal of Comparative Neurology. 413 (2), 305-326 (1999).
  21. Ivanova, E., Hwang, G. S., Pan, Z. H. Characterization of transgenic mouse lines expressing Cre recombinase in the retina. 신경과학. 165 (1), 233-243 (2010).
  22. Jo, A., Xu, J., Deniz, S., Cherian, S., DeVries, S. H., Zhu, Y. Intersectional strategies for targeting amacrine and ganglion cell types in the mouse retina. Frontiers in Neural Circuits. 12, 66 (2018).
  23. Siegert, S., et al. Genetic address book for retinal cell types. Nature Neuroscience. 12 (9), 1197-1204 (2009).
  24. Kim, I. -. J., Zhang, Y., Meister, M., Sanes, J. R. Laminar restriction of retinal ganglion cell dendrites and axons: subtype-specific developmental patterns revealed with transgenic markers. The Journal of Neuroscience. 30 (4), 1452-1462 (2010).
  25. Peng, Y. -. R., Tran, N. M., Krishnaswamy, A., Kostadinov, D., Martersteck, E. M., Sanes, J. R. Satb1 regulates contactin 5 to pattern dendrites of a mammalian retinal ganglion cell. Neuron. 95 (4), 869-883 (2017).
  26. Duan, X., Krishnaswamy, A., Dela Huerta, I., Sanes, J. R. Type II cadherins guide assembly of a direction-selective retinal circuit. Cell. 158 (4), 793-807 (2014).
  27. Ray, T. A., et al. Formation of retinal direction-selective circuitry initiated by starburst amacrine cell homotypic contact. eLife. 7, 34241 (2018).
  28. Krishnaswamy, A., Yamagata, M., Duan, X., Hong, Y. K., Sanes, J. R. Sidekick 2 directs formation of a retinal circuit that detects differential motion. Nature. 524 (7566), 466-470 (2015).
  29. Caval-Holme, F., Zhang, Y., Feller, M. B. Gap junction coupling shapes the encoding of light in the developing retina. Current Biology. 29 (23), 4024-4035 (2019).
  30. Lucas, J. A., Schmidt, T. M. Cellular properties of intrinsically photosensitive retinal ganglion cells during postnatal development. Neural Development. 14 (1), 8 (2019).
  31. Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W., Sanes, J. R. Improved tools for the Brainbow toolbox. Nature Methods. 10 (6), 540-547 (2013).
  32. Rossi, J., et al. Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis. Cell Metabolism. 13 (2), 195-204 (2011).
  33. Lefebvre, J. L., Kostadinov, D., Chen, W. V., Maniatis, T., Sanes, J. R. Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature. 488 (7412), 517-521 (2012).
  34. Ing-Esteves, S., et al. Combinatorial effects of alpha- and gamma-protocadherins on neuronal survival and dendritic self-avoidance. The Journal of Neuroscience. 38 (11), 2713-2729 (2018).
  35. Williams, P. R., Morgan, J. L., Kerschensteiner, D., Wong, R. O. L. In vitro imaging of retinal whole mounts. Cold Spring Harbor Protocols. 2013 (1), (2013).
  36. Ramoa, A. S., Campbell, G., Shatz, C. J. Transient morphological features of identified ganglion cells in living fetal and neonatal retina. Science. 237 (4814), 522-525 (1987).
  37. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9, 676-682 (2012).
  38. Peng, H., Ruan, Z., Long, F., Simpson, J. H., Myers, E. W. V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets. Nature Biotechnology. 28 (4), 348-353 (2010).
  39. Cuntz, H., Forstner, F., Borst, A., Häusser, M. One rule to grow them all: a general theory of neuronal branching and its practical application. PLoS Computational Biology. 6 (8), 1000877 (2010).
  40. Xiao, H., Peng, H. APP2: automatic tracing of 3D neuron morphology based on hierarchical pruning of a gray-weighted image distance-tree. Bioinformatics. 29 (11), 1448-1454 (2013).
  41. Nanda, S., et al. Design and implementation of multi-signal and time-varying neural reconstructions. Scientific data. 5, 170207 (2018).
  42. Sherry, D. M., Wang, M. M., Bates, J., Frishman, L. J. Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits. The Journal of Comparative Neurology. 465 (4), 480-498 (2003).
  43. Johnson, J., et al. Vesicular neurotransmitter transporter expression in developing postnatal rodent retina: GABA and glycine precede glutamate. The Journal of Neuroscience. 23 (2), 518-529 (2003).
  44. Jüttner, J., et al. Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans. Nature Neuroscience. 22 (8), 1345-1356 (2019).
  45. Zincarelli, C., Soltys, S., Rengo, G., Rabinowitz, J. E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Molecular Therapy. 16 (6), 1073-1080 (2008).
  46. Petros, T. J., Rebsam, A., Mason, C. A. In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells. Journal of Visualized Experiments: JoVE. (31), e1333 (2009).
  47. Lye, M. H., Jakobs, T. C., Masland, R. H., Koizumi, A. Organotypic culture of adult rabbit retina. Journal of Visualized Experiments: JoVE. (3), e190 (2007).
  48. Pignatelli, V., Strettoi, E. Bipolar cells of the mouse retina: a gene gun, morphological study. The Journal of Comparative Neurology. 476 (3), 254-266 (2004).
  49. Huckfeldt, R. M., et al. Transient neurites of retinal horizontal cells exhibit columnar tiling via homotypic interactions. Nature Neuroscience. 12 (1), 35-43 (2009).
  50. Prahst, C., et al. Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy. eLife. 9, 49779 (2020).

Tags

Time-lapse ImagingNeuronal ArborizationSparse Adeno-associated VirusGenetically Targeted Retinal Cell PopulationsSingle-cell LabelingDendritic ArborAxonal ArborNeuronal MorphogenesisConfocal Live ImagingNeonatal Arbor MorphologiesCentral Nervous SystemCre Mouse LineRecombinase-dependent AAVFluorescent ProteinsPlasma MembraneAAV DilutionFast Green FCF DyeNeonatal MiceCorneaIntravitreal SpaceRetinal ACSFCarbogen
check_url/kr/62308?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Ing-Esteves, S., Lefebvre, J. L. Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations. J. Vis. Exp. (169), e62308, doi:10.3791/62308 (2021).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image