在 Vivo 钙成像中，在 C. elegans身体壁肌肉
Summary
这种方法提供了一种方法，将光遗传学和基因编码的钙传感器相结合，以成像基线细胞钙水平，以及模型生物体C.elegans身体壁肌肉中诱发的钙瞬变的变化。
Abstract
模型有机体C. elegans提供了一个出色的系统，以执行体内钙成像。其透明的身体和遗传的可操作性允许基因编码钙传感器的靶向表达。该协议概述了使用这些传感器在靶细胞中钙动力学的体内成像，特别是蠕虫的身体壁肌肉。通过使用前突触通道性多普辛的共同表达，利用蓝光脉冲诱导兴奋运动神经元的乙酰胆碱释放，导致肌肉去极化和细胞质钙的可重复变化水平。讨论了两种蠕虫固定技术，难度不同。这些技术的比较表明，这两种方法都保留了神经肌肉结的生理，并允许钙瞬变的可重现定量。通过配对光遗传学和功能钙成像，可以在各种突变背景中评估午后钙处理和平衡的变化。提供的数据验证了固定技术，并具体考察了C.elegans sco（内科）质状性视网膜钙ATPase和钙激活的BK钾通道在体壁肌肉钙调节中的作用。
Introduction
本文介绍了利用光遗传学神经元刺激对C.elegans身体壁肌进行体内钙成像的方法。将肌肉表达的遗传编码钙指标（GECI）与蓝光触发神经元去极化，并提供一个系统来清楚地观察诱发的午贴钙瞬变。这避免了使用电刺激，允许对影响午睡钙动力学的突变体进行非侵入性分析。
单荧光基质GECI，如GCaMP，使用单个荧光蛋白分子融合到M13域的肌苷光链激酶在其N端和平静素（CaM）在C端。在钙结合后，对钙具有高亲和力的CaM域会经历一种构象变化，导致荧光蛋白随后的构象变化，导致荧光强度增加1。GCaMP荧光在488nm时激发，因此不适合与通道性荧光结合使用，其激励波长相似，为473nm。因此，为了将钙测量与通道性多普司辛刺激结合使用，GCaMP的绿色荧光蛋白需要替换为红色荧光蛋白mRuby（RCaMP）。使用肌肉表达的RCaMP，结合胆碱能运动神经元表达的渠道多普辛，允许在蠕虫神经肌肉结（NMJ）的研究与同时使用光遗传学和功能成像在同一动物2.
使用通道性多普辛绕过了电刺激的需要，以去极化C.elegans的神经肌肉结，这只能在解剖制剂中实现，从而使这种技术更容易使用和更精确瞄准特定组织时。例如，通道性多普辛以前曾用于C.elegans可逆地激活特定神经元，导致兴奋性神经元或抑制神经元的强健激活3，4。使用光刺激的去极化也绕开神经元损伤的问题，由于直接电刺激。这提供了一个机会来检查许多不同的刺激方案的影响，包括持续和重复的刺激，对后午睡钙动力学4。
C. elegans的透明特性使其成为荧光成像功能分析的理想选择。然而，当刺激NMJ的兴奋性乙酰胆碱神经元时，动物会立即对肌肉收缩4做出反应，使蠕虫的固定性在可视化离散钙的变化时变得至关重要。传统上，药理剂被用来使动物瘫痪。其中一种使用的药物是左旋苯酚，一种胆碱能乙酰胆碱受体激动剂5，6，7。由于左旋基导致兴奋性肌肉受体亚型的持续激活，这种试剂不适合研究肌肉钙动力学。左旋面的作用诱导后脱极化，提升细胞钙，并在登步前刺激后进行闭塞观察。为了避免使用麻痹药物，我们研究了两种替代方法来固定C.elegans。动物要么被粘住，然后解剖打开，露出身体壁的肌肉，类似于现有的C.elegansNMJ电生理学方法8，或纳米珠被用来固定完整的动物9。这两个程序允许可重复的测量休息和唤起肌肉钙瞬变，很容易量化。
本文的方法可用于测量C.elegans中细胞后肝肌肉细胞的基线细胞钙水平和瞬变。给出了采用两种不同固定技术的数据实例。这两种技术都利用光遗传学来消除肌肉细胞的极化，而无需使用电刺激。这些例子证明了这种方法在评估影响蠕虫中贴菌后钙处理的突变的可行性，并指出两种固定方法的优缺点。
Protocol
Representative Results
Discussion
GEC是C.elegans神经生物学的有力工具。以前的工作已经利用钙成像技术检查神经元和肌肉细胞的各种功能，包括感觉和行为反应，具有不同的刺激方法。一些研究已经使用化学刺激触发钙瞬变在感觉ASH神经元22，23或诱导钙波在咽部肌肉24。另一组利用机械刺激，而蠕虫则被关在微流体芯片中，以检查触摸受体神经元?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢亚历山大·戈特沙尔克博士的ZX1659，含有蠕虫菌株的RCaMP和通道多普辛，金红金博士的slo-1（eg142）蠕虫菌株，以及sca-1（tm5339）蠕虫菌株的国家生物资源项目。
Materials
| all-trans retinal | Sigma-Aldrich | R2500 | Necessary for excitation of channel rhodopsin |
| Amber LED | RCaMP illumination | ||
| Arduino UNO | Mouser | 782-A000066 | Controls fluorescence illumination |
| Blue LED | channelrhodopsin illumination | ||
| BX51WI microscope | Olympus | Fixed state compound microscope | |
| Current controlled low noise linear power supply | Ametek | Sorenson | Controls LED intensity |
| Igor Pro | Wavemetrics | Wavemetrics.com | Graphing software |
| ImageJ | NIH | imagej.nih.gov | Image processing software |
| LUMFLN 60x water NA 1.4 | Olympus | Water immersion objective for dissected preparation | |
| Master-8 Stimulator | A.M.P.I | Master timer for image acquisition and LED illumination | |
| Micro-Manager | micro-manager.org | Controls camera acquisition and LED excitiation | |
| Microsoft Excel | Microsoft | Spreadsheet software | |
| pco.edge 4.2 CMOS camera | pco. | 4.2 | High-speed camera |
| PlanApo N 60x oil NA 1.4 | Olympus | Oil immersion objective for nanobead preparation | |
| Polybead microspheres | Polysciences, Inc. | 00876-15 | For worm immobilization |
| solid state switches | Sensata Technologies | Crydom CMX100D6 | Controls timing of LED illumination |
| Transgenic strain, sca-1(tm5339); [zxIs6{Punc17::chop-2 (h134R)::yfp,lin-15(+)}; Pmyo3::RCaMP35] |
Richmond Lab | SY1627 | Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with SERCA mutant allele |
| Transgenic strain, slo-1 (eg142); [zxIs6{Punc17::chop-2 (h134R)::yfp,lin-15(+)}; Pmyo3::RCaMP35] |
Richmond Lab | Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with calcium-activated BK potassium channel mutant allele | |
| Transgeneic strain, [zxIs6{Punc17::chop-2 (h134R)::yfp,lin-15(+)}; Pmyo3::RCaMP35] |
Gottschalk Lab | ZX1659 | Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line |
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