前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究
Summary
在这里, 我们提出了一个协议, 以图像钙信号在个别细胞类型的人群在小鼠神经肌肉连接。
Abstract
细胞在组织中的电活动可以通过电生理技术进行监测, 但通常仅限于对单个细胞的分析。由于细胞质中的细胞内钙 (Ca2 +) 的增加往往是由于电活动, 或对无数其他刺激的反应, 这一过程可以监测的影像, 加载与荧光钙敏感染料。 然而, 由于这些染料被组织内的所有细胞类型占去, 很难在整个组织内的单个细胞类型中想象这种反应。相比之下, 基因编码的钙指标 (GECIs) 可以用单个细胞类型和荧光来表达, 以响应胞内 ca2 +的增加, 从而允许在整个人群中的 ca2 +信号的成像单个单元格类型。在这里, 我们应用 GECIs GCaMP3/6 的小鼠神经肌肉连接, 一个三方突触之间的运动神经元, 骨骼肌, 和终端/perisynaptic 雪旺细胞。我们展示了这种技术在经典的活体组织制剂中的效用。使用光学分光器, 我们执行动态 Ca2 +信号的双波长成像和神经肌肉连接的静态标签 (NMJ) 的方法, 可以很容易地适应监测两个细胞特定的 GECI 或基因编码的电压指标 (GEVI) 同时进行。最后, 我们讨论了用于捕捉荧光强度空间图的例程。这些光学、转基因和分析技术可以用来研究 NMJ 中不同细胞亚群在多种环境下的生物活性。
Introduction
NMJ 像所有的突触一样, 由三元素组成: 一个突触前终端, 从神经元, 突触后神经元/效应细胞, perisynaptic 胶质细胞1,2。虽然突触传输的基本方面在这个突触3首先被证明, 这个过程的许多方面仍然不明, 部分由于这个突触的不同的细胞元素表达相同的分子。例如, 由脊椎动物 NMJ 的运动神经元共同释放的嘌呤腺嘌呤核苷酸 ATP 和乙酰胆碱的受体, 由肌肉、雪旺细胞和运动神经元表达, 从而使任何这些物质施加的功能效应 (例如, 发射机释放或反应, 肌肉力量世代)4。此外, 虽然 NMJ 的三方成分比较简单, 例如, 中枢神经系统中的神经元通常呈现多个突触输入, 无论是运动神经元、肌细胞还是雪旺细胞因刺激而变化关于它们的内在异质性 (如胚胎衍生、纤维亚型、形态学) 尚不清楚。为了解决这些问题, 在一个突触元素中同时跟踪许多细胞的反应, 以及在其他任何一个单独的元素中同时跟踪这种反应是有利的。使用化学染料来测量钙信号的常规策略不能达到这两个目标, 因为沐浴应用染料在应用到组织后被多种细胞类型所占, intracellularly 载染料只能用于可视化单个或小的细胞群。在这里, 利用转基因小鼠表达 GECIs 设计来测量细胞特定的钙信号, 连同具体的成像和软件工具5, 我们展示了这两个总体目标的第一个, 并讨论如何添加新的转基因工具将有助于实现第二个目标。这项技术将是有用的任何人感兴趣的跟踪钙动力学或其他细胞信号事件可观测到通过基因编码的光学传感器在多个细胞群体的同时。
Protocol
Representative Results
Discussion
在这里, 我们提供了一些例子, 以测量 GECI 表达小鼠完整的神经肌肉组织中的特定细胞的 Ca2 +反应。为了成功地进行这些实验, 在解剖过程中不应损伤膈神经。要在雪旺细胞中以低功率或大功率 (即20X 或 60X) 图像 Ca2 +响应, 必须使用 BHC 或µ-芋螺毒素来阻止移动。对于在肌肉细胞中的 Ca2 +反应的低功率成像, 有可能在没有这些药物的情况下测量它们, 从而允许同时获得?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了来自国家卫生研究院 (NIH) GM103554 和 GM110767 (东华三院) 和来自国家研究资源5P20RR018751 和国立普通医学科学院 8P20 GM103513 (G.W.H.) 的资金的支持。
Materials
| Myf5-Cre mice | Jax | #007893 | Drives muscle cell expression as early as E136 |
| Wnt1-Cre mice | Jax | #003829 | Drives expression into all Schwann cells at E13 but not P209 |
| Sox10-Cre mice | Jax | #025807 | Drives Schwann cell expression at older ages |
| Conditional GCaMP3 mice | Jax | #029043 | Expresses GCaMP3 in cell-specific fashion |
| Conditional GCaMP6f mice | Jax | #024105 | Expresses GCaMP6f in cell-specific fashion |
| BHC (3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one) | Hit2Lead | #5102862 | Blocks skeletal muscle myosin but not neurotransmission6 |
| CF594-α-BTX | Biotium | #00007 | Labels acetylcholine receptor clusters at NMJ |
| µ-conotoxin GIIIb | Peptides Int'l | #CONO20-01000 | Blocks Nav1.4 voltage-dependent sodium channel8 |
| Silicone Dielectric Gel; aka Sylgard | Ellswoth Adhesives | # Sil Dielec Gel .9KG | Allows for the immobilization of the diaphragm by minutien pins |
| Minutien pins (0.1mm diameter) | Fine Science Tools | 26002-10 | Immobilizes diaphragm onto silicone dielectric gel |
| Eclipse FN1 upright microscope | Nikon | MBA74100 | Allows staging and observation of specimen |
| Basic Fixed Microscope Platform with Manual XY Microscope Translator | Autom8 | MXMScr | Allows movement of specimen |
| Manual micromanipulator | Narishige | M-152 | Holds recording and stimulating electrodes |
| Microelectrode amplifier | Molecular Devices | Axoclamp 900A | Allows sharp electrode intracellular electrophysiological recording |
| Microelectrode low-noise data acquisition system | Molecular Devices | Digidata 1550 | Allows electrophysiological data acquisition |
| Microelectrode data analysis system | Molecular Devices | PCLAMP 10 Standard | Performs electrophysiological data analysis |
| Square wave stimulator | Grass | S48 | Stimulates nerve to excite muscle |
| Stimulus Isolation Unit | Grass | PSIU6 | Reduces stimulation artifacts |
| Borosilicate filaments, 1.0 mm outer diameter, 0.5mm internal diameter | Sutter | FG-GBF100-50-15 | Impales and records nerve-evoked muscle potentials |
| Borosilicate filaments, 1.5 mm outer diameter, 1.17mm internal diameter | Sutter | BF150-117-15 | Lengthened and used for suction electrode |
| Micropipette Puller | Sutter | P-97 | Pulls and prepares recording electrodes |
| 1200×1200 pixel, back-illuminated cMOS camera | Photometrics | Prime 95b | Sensitive camera that allows high-resolution, high-speed imaging |
| Light Source | Lumencor | Spectra X | Provides illumination from LEDs for fluorescence obsevation |
| Infinity-corrected fluorescent water immersion objectives, W.D. 2mm | Nikon | CFI60 | Provide long working distances for visualization of specimen |
| Fiber Optic Illuminator with Halogen lamp | Sumita | LS-DWL-N | Provides illumination for brightfield observation |
| W-View Gemini Image Splitter | Hamamatsu | A12801-01 | Projects 1 pair of dual wavelength images separated by a dichroic to single camera |
| Single-band Bandpass Filters (512/25-25 and 630/92-25) | SemRock | FF01-512/25-25; FF01-630/92-25 | Permits dual band imaging |
| 560 nm Single-Edge Dichroic Beamsplitter | Sem Rock | FF560-FDi01-25×36 | Dichroic mirror which separates beams of light to allow dual-wavelength imaging |
| Imaging data acquisition system | Nikon | NIS Elements – MQS31000 | Allows imaging data acquisition |
| Wavelength control module | Nikon | MQS41220 | Module for imaging data acqusiition |
| Emission splitter hardware module | Nikon | MQS41410 | Module for imaging data acqusiition |
| Imaging data analysis system | NA | Volumetry 8D5, Fiji | Allows analysis of fluorescence intensity and other imaging data |
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