自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动
1Program of Biomedical Science, Graduate School of Integrated Sciences for Life,Hiroshima University, 2Institute for Quantum Life Science,National Institute for Quantum and Radiological Science and Technology, 3National Institute for Radiological Sciences,National Institute for Quantum and Radiological Science and Technology, 4JST, PRESTO
Summary
这里报道的是一个用于自由行为秀 丽隐杆线虫 的钙成像系统，具有良好控制的，非局部化的振动。该系统允许研究人员在纳米级位移下唤起具有良好控制特性的非局部振动，并在 秀丽隐杆线虫 对振动的反应期间量化钙电流。
Abstract
非局部化机械力，如振动和声波，影响从发育到稳态的各种生物过程。动物通过改变它们的行为来应对这些刺激。要了解这种行为修饰背后的机制，需要在感兴趣的行为期间量化神经活动。在这里，我们报告了一种在自由行为秀 丽隐杆线虫 中进行钙成像的方法，该隐杆线虫具有特定频率，位移和持续时间的非局部振动。该方法允许使用声学换能器产生控制良好的非局部振动，并在单细胞分辨率下量化诱发的钙反应。作为原理证明，在 秀丽隐杆线虫 对振动的逃逸反应期间，证明了单个中间神经元AVA的钙反应。该系统将有助于理解对机械刺激的行为反应背后的神经机制。
Introduction
动物经常暴露于非局部化的机械刺激，如振动或声波1，2。由于这些刺激影响体内平衡，发育和繁殖，动物必须改变其行为以应对它们3，4，5。然而，这种行为改变背后的神经回路和机制知之甚少。
线虫中的机械感觉行为，秀丽隐杆线虫，是一个简单的行为范式，其中蠕虫在遇到非局部振动时，通常从向前运动转变为向后逃逸反应6。这种行为背后的神经回路主要由五个感觉神经元，四对中间神经元和几种类型的运动神经元7，8组成。此外，蠕虫在涉及重复刺激的间隔训练9，10，11之后习惯于这种机械刺激。因此，这种简单的行为反应构成了研究非局部振动诱发行为和记忆背后的神经机制的理想系统。图中说明了在非局部振动影响下自由行为蠕虫的钙成像方案。与以前报告的系统相比，该系统很简单，因为它不需要额外的摄像头进行跟踪;但是，它允许我们改变非局部振动的频率，位移和持续时间。由于AVA中间神经元的活化诱导了向后逃逸反应，因此在AVA特异性启动子的控制下，以共表达GCaMP（钙指示剂）和TagRFP（一种对钙不敏感的荧光蛋白）的蠕虫为例（详见材料表）。该协议演示了当蠕虫从向前切换到向后移动时AVA神经元的激活。该协议有助于理解机械感觉行为背后的神经回路机制。
Protocol
1. 蠕虫的培养直至钙成像 在钙成像实验前四天，将两个成年ST12蠕虫转移到新的线虫生长培养基（NGM）板（材料表）上，在该板上，大 肠杆菌 OP50使用细胞扩散器以方形模式（约4 mm x 4 mm）条纹，以便蠕虫在钙成像过程中将大部分时间花在细菌中12。 将此NGM板在培养箱中在20°C下孵育4天（材料表）。 2. 硬件设…
Representative Results
在这里，在AVA中间神经元特异性启动子的控制下表达GCaMP和TagRFP的蠕虫被用作自由行为 秀丽隐杆线虫钙成像的一个例子。GCaMP和TagRFP通道数据以一系列图像的形式获得，其中一些图像如图 6 所示，并显示为电影（补充电影1）。由我们的非局部振动系统引起的培养皿板的位移（图7）也被量化。位移可以通过在振动控制软件（<strong class…
Discussion
通常，神经活动的量化需要引入探针和/或限制动物身体运动。然而，对于机械感觉行为的研究，探针的侵入性引入和约束本身构成机械刺激。 秀丽隐杆线虫 提供了一个系统来规避这些问题，因为它的特征是透明的，并且因为它具有一个简单，紧凑的神经回路，仅包含302个神经元。将这些优点与先前开发的唤起纳米级位移的非局部振动的方法13相结合，这里说明了一种用?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Caenorhabditis 遗传学中心提供本研究中使用的菌株。本出版物由JSPS KAKENHI科学研究补助金（B）（授予号为。JP18H02483），关于创新领域“软机器人科学”项目（授予号：JP18H05474），日本医学研究开发机构（拨款19gm6110022h001）和岛津基金会的PRIME。
Materials
| Data anaylsis software | |||
| DualViewImaging.nb | author | For analysis of acquired data | |
| Mathematica12 | Wolfram | For running data anaysis software DualViewImaging | |
| Escherichia coli and C. elegans strains | |||
| E. coli OP50 | Caenorhabditis Genetics Center | OP50 | Food for C. elegans. Uracil auxotroph. E. coli B. |
| lite-1(ce314) strain | Caenorhabditis Genetics Center | KG1180 | Light-insensitive mutant |
| lite-1(ce314) strain expressing NLS-GCaMP-NLS and TagRFP under the control of the AVA-speciric promoter | author | ST12 | lite-1(ce314) mutant carrying the genes expressing NLS-GCaMP5G-NLS (NLS; nuclear localization signal) and TagRFP under the control of the flp-18 promoter as an extrachoromosomal arrays |
| Laser Doppler vibrometer | |||
| Lase Doppler vibrometer | Polytec Japan | IVS-500 | For quantifying frequency and displacement generated by the accoustic transducer |
| Mouse macro system | |||
| Assay.txt | Author | Script for temporally and specially controlling mouse cursol in Windows | |
| HiMacroEx | Vector | https://www.vector.co.jp/download/file/winnt/util/fh667310.html | Free download software for controling mouse cursor based on a script |
| Nematode growth media plate | |||
| Agar purified, powder | Nakarai tesque | 01162-15 | For preparation of NGM plates |
| Bacto pepton | Becton Dickinson | 211677 | For preparation of NGM plates |
| Calcium chloride | Wako | 036-00485 | For preparation of NGM plates |
| Cholesterol | Wako | 034-03002 | For preparation of NGM plates |
| di-Photassium hydrogenphosphate | Nakarai tesque | 28727-95 | For preparation of NGM plates |
| LB broth, Lennox | Nakarai tesque | 20066-95 | For culture of E. coli OP50 |
| Magnesium sulfate anhydrous | TGI | M1890 | For preparation of NGM plates |
| Potassium Dihydrogenphosphate | Nakarai tesque | 28720-65 | For preparation of NGM plates |
| Sodium Chloride | Nakarai tesque | 31320-05 | For preparation of NGM plates |
| Petri dishes (60 mm) | Nunc | 150270 | For preparation of NGM plates |
| Nonlocalized vibration device | |||
| Amplifier | LEPY | LP-A7USB | For stimulation with controllable vibration |
| Acoustic transducer | MinebeaMitsumi | LVC25 | For stimulation with controllable vibration |
| WaveGene Ver. 1.5 | Thrive | http://efu.jp.net/soft/wg/down_wg.html | Free download software for controling vibration property |
| Noninvasive calcium imaging | |||
| 2-Channel benchtop 3-phase brushless DC servo controller | Thorlabs | BBD202 | Compatible controller for MLS203-1 stages |
| 479/585 nm BrightLine dual-band bandpass filter | Semrock | FF01-479/585-25 | For acquisition of two channel images (GCaMP and TagRFP) |
| 505/606 nm BrightLine dual-edge standard epi-fluorescence dichroic beamsplitter | Semrock | FF505/606-Di01-25×36 | For acquisition of two channel images (GCaMP and TagRFP) |
| 512/25 nm BrightLine single-band bandpass filter | Semrock | FF01-512/25-25 | For acquisition of two channel images (GCaMP and TagRFP) |
| 630/92 nm BrightLine single-band bandpass filter | Semrock | FF01-630/92-25 | For acquisition of two channel images (GCaMP and TagRFP) |
| Computer | Dell | Precision T7600 | Windows7 with Intel Xeon CPU ES-2630 and 8 GB of RAM |
| High-speed x-y motorized stage | Thorlabs | MLS203-1 | Fast XY scannning stage |
| Image splitting optics | Hamamatsu photonics | A12801-01 | For acquisition of two channel images (GCaMP and TagRFP) generated by W-VIEW GEMINI Image spliting optics |
| LED light source | CoolLED | pE-4000 | For generating 470 nm and 560 nm excitation light |
| Microscope | Olympus | MVX10 | |
| sCMOS camera | Andor | Zyla | |
| x 2 Objective lens | Olympus | MVPLAPO2XC | Working distance 20 mm and numerical aperture 0.5 |
| Plasmid | |||
| pKDK66 plasmid | author | pKDK66 | Co-injection marker |
| pTAK83 plasmid | author | pTAK83 | Plasmid for expression of TagRFP under the control of the flp-18 promoter |
| pTAK144 plasmid | author | pTAK144 | Plasmid for expression of NLS-GCaMP5G-NLS under the control of the flp-18 promoter |
| Tracking software | |||
| homingback.vi | author | SubVi file for tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage | |
| LabVIEW | National instruments | For running tracking software | |
| Zyla Control ver.2.6CI.vi | author | For tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage |
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Cite This Article
Shigyou, K., Maeoka, H., Igarashi, R., Sugi, T. Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration. J. Vis. Exp. (170), e61626, doi:10.3791/61626 (2021).