应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应
Summary
在这里描述了使用一个适应的 “嗅觉芯片” 的有效钙显像的C. 线虫男性。还显示了男性接触甘油和信息素的研究。
Abstract
使用钙指标大大提高了我们对神经动力学和调节的认识。线虫线虫线虫, 其完全映射的神经系统和透明的解剖, 提出了一个理想的模型, 了解 real-time 神经动力学使用钙指标。结合微流控技术和实验设计, 使用这些指标的钙显像研究在自由和被困动物中都进行。然而, 以前的研究大多使用诱捕器, 如 Chronis et al.中描述的嗅觉芯片, 这些装置的设计用于更常见的雌雄同体, 因为不常见的雄性在形态和结构上都不同.设计和制作了一种适应的嗅觉芯片, 以提高男性神经元成像的效率, 使用年轻的成年动物。一个转弯被并入到蠕虫的装货港旋转的动物和允许分离的单个神经元内的一对双边对2D 成像。正如以前的雌雄同体研究中所描述的, 蠕虫被暴露在微流控装置内受控的气味流中。然后使用开源软件 ImageJ 分析钙瞬变。本文所述的过程应允许增加 male-based 的C. 线虫钙显像研究, 加深我们对性特异性神经元信号机制的理解。
Introduction
微流控设备提供了更高的访问精确控制环境, 其中动物, 如线虫的C. 线虫, 可以在实验上操纵1。这些研究包括行为分析, 钙显像研究, 甚至筛选特定的表型, 导致更精确的实验结果的测量1,2,3,4, 5,6。微提供 small-scale 的液体条件, 通过它可以在使用少量试剂的同时运行详细的实验。有一个不断生产的新的微流控装置设计, 并使用每一个不同的领域, 从允许的自然正弦运动的C. 线虫的行为分析和神经成像研究, 陷阱设备用于神经成像和嗅觉研究, 对设备, 允许高通量表型分析在遗传屏幕4,5,6,7。随着主模的制造, 微流控设备的构建成本低廉, 因为它可以重用主控器, 而且易于使用, 允许通过高通量的研究快速生成数据。使用烷 (聚烯烃) 等聚合物制造设备, 可以在数小时内创造出新的设备。
钙显像研究使用在靶细胞中表达的基因编码的钙指标 (GECIs) 实时测量这些细胞的神经动力学8,9,10,11。C. 线虫的透明性质允许在活体动物中记录这些蛋白质的荧光水平。传统上, GECIs 依赖于绿色荧光蛋白 (GFP) 为基础的传感器 GFP-Calmodulin-M13 肽 (GCaMP), 虽然最近的研究已经适应这些传感器, 以允许更好的信噪比和红色位移的励磁剖面。随着 GCaMP3 的发展, 与这些规格的蛋白质有不同, 包括传感器, 如 GCaMP6s 和 GCaMP6f (慢和快速荧光 off-rates, 分别), 以及 RFP-Calmodulin-M13 肽 (RCaMP), 其中有一个红色转移激活配置文件。这些 GECIs 与C. 线虫细胞特异基因启动子序列的组合可以针对感兴趣的细胞, 特别是感觉神经元12,13,14,15,16。
虽然在微流体研究中使用的线虫的易用性很明显, 但几乎所有的研究都集中在雌雄同体上。尽管男性只占 0.01-0. 02% 的野生类型的人口, 宝贵的发现可能会产生从他们的特征。虽然雌雄同体神经系统的物理连接已经被完全映射了几十年17, 雄性连接仍然不完整, 特别是在动物的头部区域18。使用钙显像在男性将有助于产生一个了解男性神经系统和差异, 出现在两性之间。较小的大小的C. 线虫成年男性防止有效和可靠的诱捕在加载端口的传统嗅觉设备设计为较大的雌雄同体。为了解决这个问题, 修改后的 Chronis 嗅觉芯片19的版本开发了一个较窄的加载端口, 一个较低的通道高度, 并在蠕虫加载端口 (旋转的动物), 允许可视化的双边左/右神经元对。这个设计允许: (1) 年轻成年雄性的有效诱捕, (2) 对双侧配对神经元的可视化的动物更可靠的定位, 以及 (3) 对雄性神经元的神经活动的精确成像。
越来越多的研究表明, 线虫雄性对各种 ascarosides (ascr) 或线虫信息素20、21 、22、23 的响应雌雄同体不同。 ,24。因此, 在雄性连接中培养对神经动力学和表达的理解变得更加贴切。雄性的线虫包含87性特异性神经元不存在于雌雄同体的25,26中, 改变连接的不确定的方式。能够想象这些独特的神经动力学将使我们能够更好地理解性特定的反应和神经表达。
本议定书描述了使用男性适应的嗅觉芯片的神经成像男性线虫chemosensation。伤害的神经元灰反应可靠地对 1 M 甘油在男性, 与早先两性研究一致27。暴露在 ascarosides 可能引起的反应, 从动物到动物的变化, 需要更多的动物进行测试。通过电生理学和钙显像研究表明, 雄性特异性的杰姆神经元的反应, 变对 ascaroside #323。
Protocol
1. 设备制造 注意: 请参阅参考资料 1 。 注意: 硅母模是使用标准的光刻技术制作的, 用于在硅母版上进行图案 SU-8 光刻胶的制造 1 , 7 。硅片图案的光印在 2.5万 dpi。该男性适应装置的特点是 Chronis 嗅觉芯片设计 19 与蠕虫加载端口的变化, 适应一个设计获得了 m. 兹 (个人通?…
Representative Results
在图 1A-b中可以看到整个设备设置的一个示例。图 1A描述了适当的油藏构造和设置。图 1B显示了储层与微流体设备的连接。图 1C描述了一个微流体设备, 其各个端口标记为清晰。 设计的男性适应微流控装置包含一条曲线在装?…
Discussion
男性适应的嗅觉芯片结合了一个转弯到一个狭窄的装货口岸, 允许更多控制方向和为有效地诱捕男性C. 线虫。这使得对神经元双边对的左、右两个成员的可视化, 而无需 z 堆叠。这条曲线导致一个方向远离垂直100% 的时间在蠕虫, 只有一个双边对是目标与荧光标记, 如灰 (图 2D-E)29,30。然而, 在神经元类的四径向对?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢曼努埃尔-兹曼为我们提供的初步设计文件, 适合与男性使用;弗兰克施罗德为 ascr#3 的综合和供应;罗斯 Lagoy 为洞察和协助以成像和分析;劳拉 Aurilio 为大师制作, 并与克里斯托弗滑道, 贡献了对这份手稿的审查。这项工作的经费是在国家卫生研究所赠款 1R01DC016058-01 (国家科学基金会赠款本位 1605679 (D.R.A.), 和在科学接口 (D.R.A.) 的宝来惠康职业奖。
Materials
| Silicon Wafer | University Wafer | 452 | |
| SU-8 2035 | MicroChem | Y111070-0500L1GL | |
| Developer | MicroChem | Y020100-4000L1PE | |
| Wafer Mask | Cad/Art Services | – | Custom order. Printed at 25,000 dpi. |
| Sylgard-184 | Ellsworth Adhesives | 184 SIL ELAST KIT 0.5KG | |
| 1.0 mm Dermal Punches | Acuderm Inc. | P150 | |
| Soft Tubing | Cole-Palmer | EW-06419-01 | |
| Hard Tubing | IDEX Health & Science | 1622 | |
| Pins | New England Small Tube | NE-1027-12 | |
| Blocking Pins | New England Small Tube | 0.415/0.425" OD x .500 Long | Batch PB07027 |
| 3 mL syringes | BD | 309657 | |
| 30 mL syringes | Vitality Medical | 302832 | Used as buffer reservoirs. |
| Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer | Component Supply Company | NE-231PL-50 | |
| Stopcocks with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile | Cole-Palmer | EW-30600-07 | |
| Fisherfinest Premium Cover Glass | Fisher Scientific | 12-548-5M | |
| Mercator Control System LF-5 Plasma System | Mercator | LF-5 | |
| Scotch Tape | Scotch | BSN43575 | |
| Series 20 Chamber | Warner Instruments | P-2 | |
| Vacuum Desicator | Bel-Art Scienceware | 420250000 | 24 cm inner diameter. |
| Weigh Boats | Cole-Palmer | EW-01017-27 | |
| Classic Plus Balance | Mettler Toledo | PB1501-S/FACT | |
| Glass Pasteur Pipettes | Cole-Palmer | EW-25554-06 | |
| Transfer pipettes | Genesee Scientific | 30-202 | |
| Oven | Sheldon Manufacturing Inc | 9120993 | Model Number: 1500E. |
| 60 mm, non-vented, sharp edge Petri dishes | TriTech Research | T3308 | |
| Zeiss Axio Observer.A1 | Zeiss | – | |
| Hammamatsu Orca Flash 4.0 Digital CMOS | Hammamatsu | C11440-22CU | |
| Blue Fluorescent Light | Lumencor | SOLA SM6-LCR-SA | 24-30V/7.9A DC. |
| Illumination Adaptor | Zeiss | 423302-0000 | |
| Series 1 and 2 Miniature Inert PTFE Isolation Valve | Parker | 001-0017-900 | 3-way valve for controlling flow. |
| ValveLink8.2® | AutoMate Scientific | 01-18 | Flow Switch Controller |
| Micro Manager | Micro-Manager | – | Free software, can be downloaded at: https://www.micro-manager.org/wiki/Download_Micro-Manager_Latest_Release |
| ImageJ | ImageJ | – | Free software, can be downloaded at: https://imagej.nih.gov/ij/download.html |
| Agar, Bacteriological Grade | Apex | 9012-36-6 | |
| Peptone | Apex | 20-260 | |
| CaCl2 | VWR | BDH0224-1KG | |
| MgSO4 | Sigma-Aldrich | 230391-1kg | |
| Cholesterol | Alfa Aesar | A11470 | |
| Ethanol | Sigma-Aldrich | 270741-4L | |
| Tetramisole | Sigma-Aldrich | L9756-10(G) | Store at 4 °C. |
| Fluorescein | Sigma-Aldrich | FD2000S-250mg | Light Sensitive. Store in photoprotective vials. |
| Glycerol | Sigma-Aldrich | G6279-1L | |
| Ascaroside #3 | – | – | Synthesized in the Schroeder Lab (Cornell University). |
| NaCl | Genesee Scientific | 18-215 | |
| KH2PO4 | BDH | BDH9268.25 | |
| K2HPO4 | J.T. Baker | 3252-025 | |
| ASH GCaMP3 line | – | – | CX10979 (KyEx2865 [psra-6::GCAMP3 @ 100 ng/uL]). Developed in Bargmann lab. Provided from Albrecht Lab library. |
| CEM GCaMP6 line | – | – | JSR49 (FkEx98[ppkd-2::GCaMP::SL2::dsRED + pBX-1]; pha-1(e2123ts); him-5(e1490); lite-1(ce314)). Developed by Robyn Lints. Provided from Srinivasan Lab library. |
| E. coli (OP50) | Caenorhabditis Genetics Center | OP50 | |
| "Reservoir" | – | – | To create a Reservoir: A "30 mL syringe", is connected to a "Stopcock with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile", which is connected to a "3 mL syringe" and a "Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer". The "Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer" is then inserted into "Soft Tubing" approximately 1/3 of the way down the needle. |
References
- Lagoy, R. C., Albrecht, D. R. Microfluidic Devices for Behavioral Analysis, Microscopy, and Neuronal Imaging in Caenorhabditis elegans. Methods Mol Biol. 1327, 159-179 (2015).
- Ben-Yakar, A., Chronis, N., Lu, H. Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans. Curr Opin Neurobiol. 19 (5), 561-567 (2009).
- Chronis, N. Worm chips: Microtools for C. elegans biology. Lab on a Chip. 10 (4), 432-437 (2010).
- Lee, H., Crane, M. M., Zhang, Y., Lu, H. Quantitative screening of genes regulating tryptophan hydroxylase transcription in Caenorhabditis elegans using microfluidics and an adaptive algorithm. Integr Biol (Camb). 5 (2), 372-380 (2013).
- Lockery, S. R., et al. A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans. Lab Chip. 12 (12), 2211-2220 (2012).
- Mondal, S., et al. Large-scale microfluidics providing high-resolution and high-throughput screening of Caenorhabditis elegans poly-glutamine aggregation model. Nat Commun. 7, 13023 (2016).
- Larsch, J., Ventimiglia, D., Bargmann, C. I., Albrecht, D. R. High-throughput imaging of neuronal activity in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 110 (45), E4266-E4273 (2013).
- Akerboom, J., et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neuro. 6, 2 (2013).
- Badura, A., Sun, X. R., Giovannucci, A., Lynch, L. A., Wang, S. S. H. Fast calcium sensor proteins for monitoring neural activity. Neurophotonics. 1 (2), 025008 (2014).
- Tatro, E. T. Brain-wide imaging of neurons in action. Front Neural Circuits. 8, 31 (2014).
- Tian, L., et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods. 6 (12), 875-881 (2009).
- Greene, J. S., et al. Balancing selection shapes density-dependent foraging behaviour. Nature. 539 (7628), 254-258 (2016).
- Greene, J. S., Dobosiewicz, M., Butcher, R. A., McGrath, P. T., Bargmann, C. I. Regulatory changes in two chemoreceptor genes contribute to a Caenorhabditis elegans QTL for foraging behavior. Elife. 5, (2016).
- Kim, K., et al. Two Chemoreceptors Mediate Developmental Effects of Dauer Pheromone in C. elegans. Science. 326 (5955), 994-998 (2009).
- McGrath, P. T., et al. Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes. Nature. 477 (7364), 321-325 (2011).
- Schmitt, C., Schultheis, C., Husson, S. J., Liewald, J. F., Gottschalk, A. Specific Expression of Channelrhodopsin-2 in Single Neurons of Caenorhabditis elegans. PLoS ONE. 7 (8), e43164 (2012).
- White, J. G., Southgate, E., Thomson, J. N., Brenner, S. The Structure of the Nervous System of the Nematode Caenorhabditis elegans. Phil Trans of the Royal Soc of Lon. 314 (1165), 1 (1986).
- White, J. Q., et al. The sensory circuitry for sexual attraction in C. elegans males. Curr Biol. 17 (21), 1847-1857 (2007).
- Chronis, N., Zimmer, M., Bargmann, C. I. Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans. Nat Meth. 4 (9), 727-731 (2007).
- Chute, C. D., Srinivasan, J. Chemical mating cues in C. elegans. Semin Cell Dev Biol. 33, 18-24 (2014).
- Izrayelit, Y., et al. Targeted metabolomics reveals a male pheromone and sex-specific ascaroside biosynthesis in Caenorhabditis elegans. ACS Chem Biol. 7 (8), 1321-1325 (2012).
- Ludewig, A. H., Schroeder, F. C. Ascaroside signaling in C. elegans. WormBook. , 1-22 (2013).
- Narayan, A., et al. Contrasting responses within a single neuron class enable sex-specific attraction in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 113 (10), E1392-E1401 (2016).
- Srinivasan, J., et al. A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature. 454 (7208), 1115-1118 (2008).
- Sammut, M., et al. Glia-derived neurons are required for sex-specific learning in C. elegans. Nature. 526 (7573), 385-390 (2015).
- Sulston, J. E., Albertson, D. G., Thomson, J. N. The Caenorhabditis elegans male: postembryonic development of nongonadal structures. Dev Biol. 78 (2), 542-576 (1980).
- Hilliard, M. A., et al. In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. The EMBO Journal. 24 (1), 63-72 (2005).
- Evans, T. C. Transformation and microinjection. WormBook. , (2006).
- Cáceres, I. d. C., Valmas, N., Hilliard, M. A., Lu, H. Laterally Orienting C. elegans Using Geometry at Microscale for High-Throughput Visual Screens in Neurodegeneration and Neuronal Development Studies. PLoS ONE. 7 (4), e35037 (2012).
- Schrodel, T., Prevedel, R., Aumayr, K., Zimmer, M., Vaziri, A. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat Methods. 10 (10), 1013-1020 (2013).
- García, L. R., Portman, D. S. Neural circuits for sexually dimorphic and sexually divergent behaviors in Caenorhabditis elegans. Curr Opin Neurobiol. 38, 46-52 (2016).
- Clokey, G. V., Jacobson, L. A. The autofluorescent "lipofuscin granules" in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech Ageing Dev. 35 (1), 79-94 (1986).
- Coburn, C., et al. Anthranilate Fluorescence Marks a Calcium-Propagated Necrotic Wave That Promotes Organismal Death in C. elegans. PLoS Biology. 11 (7), e1001613 (2013).
- Macosko, E. Z., et al. A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature. 458 (7242), 1171-1175 (2009).
- Park, D., et al. Interaction of structure-specific and promiscuous G-protein-coupled receptors mediates small-molecule signaling in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 109 (25), 9917-9922 (2012).
Cite This Article
Reilly, D. K., Lawler, D. E., Albrecht, D. R., Srinivasan, J. Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons. J. Vis. Exp. (127), e56026, doi:10.3791/56026 (2017).