在这里, 我们提出了一个协议 ca2 +成像在神经元和胶质细胞, 使 ca2 +信号的解剖在亚细胞分辨率。此过程适用于允许表达基因编码的 ca2 +指标的所有细胞类型。
钙离子 (Ca2 +) 是一种通用的细胞内信使分子, 它驱动多种信号通路, 从而产生不同的生物输出。两个 Ca2 +信号源的协调–细胞外的 “ca2 +流入” 和细胞内 ca2 + 存储内质网 (Er) 的 “ca2 +释放”–被认为是 ca 的不同时空模式的基础2 +信号, 导致细胞中的多种生物功能。该协议的目的是描述一种新的 Ca2 +成像方法, 该方法能够监视 “ca2 +流入” 和 “ca2 +释放” 的时刻。Oer-gcmp6f 是一种基因编码的 Ca2 +指示器 (geci), 由 GCaMP6f 组成, 针对 er 外膜。Oer-gmp6f 可以以比传统 Gmp6f 更高的时间分辨率监测 Ca2 +释放。结合等离子体膜靶向 GECIs, 时空 Ca2 +信号模式可以描述为亚细胞分辨率。这里描述的亚细胞靶向 ca2 +指标原则上适用于所有细胞类型, 甚至适用于线虫神经元的体内成像. 在这个协议中, 我们介绍 ca2 +成像细胞从细胞系, 神经元, 和胶质细胞在离解的原代培养, 并描述了大鼠皮质神经元冻结存量的制备。
Ca2 +信号表示细胞内 ca2 +浓度的升高。Ca2 +是真核细胞的通用第二信使。使用 Ca2 +, 细胞通过不同的细胞内信号通路发挥作用, 并诱导各种生物输出。例如, 在神经元中, 突触前末端的突触囊泡释放、细胞核中的基因表达以及突触后突触突触的突触可塑性诱导都受到不同的 Ca2 +信号的调节, 这些信号精确地激活了适当的信号。下游酶在正确的地点和精确的时间1。
Ca2 +信号的特定时空模式激活特定的下游酶。Ca2 +信号是由两个不同的 ca2+源之间的协调产生的: ca2 +来自细胞外空间的流入和来自内质网 (er) 的 ca2 +释放, 后者作为细胞内 ca2 +店。在质膜或 ER 膜2上产生的 10–100μmca 2 +纳米值 2 + 也支持了诱导特定细胞功能的有意义的时空 ca 2+信号模式. 重要的是, Ca2 +信号的来源是决定下游生物输出的最关键因素之一。在神经元中, Ca2 +流入和 ca2 +释放对 gabaep 突触下的γ-氨基丁酸 (gaba) a 受体 (gaba a r) 的聚集有相反的影响, 这对其进行抑制.神经元兴奋性3。Ca2 +流入伴随着巨大的神经元兴奋, 导致突触 gabaar 簇的分散, 而来自 er 的持续性 ca2 +释放促进突触 gaba r 的聚类.其他研究小组也报告说, 生长锥的调谐方向在很大程度上取决于 Ca2 +信号的来源: ca2 +流入会引起排斥, 而 ca2+释放则引导神经元生长的吸引力圆锥4。因此, 要充分理解 ca2 +信号通路背后的特定细胞输出, 重要的是通过描述 ca2 +信号的来源在亚细胞分辨率。
在该协议中, 我们描述了一种 Ca2 +成像方法, 用于以亚蜂窝分辨率报告 ca2 +信号, 该方法允许估计 ca2 +信号源 (图 1)。通过在 Src 内连接等离子体膜定位信号 Lck, 基因编码的 Ca2 +指标 (gec) 对质膜下的 ca 2+微域进行了成功的监测。激酶到 GECIs 5的 n-终止。为了以更好的空间和时间分辨率检测 er 附近的 Ca2 +信号模式, 我们最近开发了 Oer-gmmmp6f, 其中 GCaMP6f6使用 er 跨膜蛋白瞄准 er 外膜。Oer-gcmp6 可以通过避免 Ca 2 + 和 GECI 的扩散, 以比传统的非目标 Gmp6f 更好的时空分辨率敏感地报告 er 中的 Ca2 +释放 .我们还证实, Oer-gmpmp6f 报告的培养海马星形胶质细胞的自发钙 2+升高与血浆膜靶向 gcmp6f (Lck-gmp6f) 监测的时空模式不同。 9,表明ca2 +成像与 oer-gmpmp6f 结合 lck-gmpmp6f 有助于解剖 ca 2+信号在亚细胞分辨率, 以确定其来源。
目前, 我们详细介绍了在 HeLa 细胞和神经星形胶质混合培养中的 Ca2 +信号解剖的协议, 并将其涂在玻璃覆盖片上。此处所示的 GECIsca 2 +成像技术, Lck-gmpmp6f、等离子体膜靶向 rmmp210 (LCC-RMPMP2) 和 Oer-gmcmp6f (图 1) 适用于所有可以表达 gecis 的细胞。
不同的生物输出是由 Ca2 +信号启动的。Ca2 +是一种多才多艺的细胞内信号信使。解码 Ca2 +信号以唤起特定的输出一直是一个基本的生物学问题, ca2 +成像技术来描述 ca2 +信号的多样性是必需的。目前详细的协议允许检测不同的 Ca2 +信号在质膜和 Er (图 3和图 4) 和本地 ca 2 + 微域在一个细胞内 (图 4和图 5) 。这有助于描述细胞内 Ca2+信号的多样性。ca2 +信号的时间分辨率也通过针对质膜和 er 中的 geci 得到改善, 因为它可以避免 ca2 +和 geci 本身的三维扩散的影响, 并且有可能检测到Ca2 +流入或ca 2 +释放的时刻, 发生在膜上。
该协议有一些限制。用户应记住, 检测到的信号是 “Ca2 +流入或释放的时刻” 和 “ca2 + 从原始 ca2 +源扩散出来” 的总和, 特别是对于大型 ca2 +信号。例如, 尽管他在 HeLa 细胞中的刺激唤起 Er 中的 ca2 +释放, 但其产生的 ca2 +信号不仅通过 er 靶向的 Oer-gmpmp6f 检测, 而且还通过等离子体膜靶向 Lcc-rcmp2 检测到 (图 3)。另一个限制是 ca2 +信号的时空模式可能不是 ca2 +信号输出的唯一行列式。下游效应蛋白 (如 Ca2 +依赖激酶和磷酸酶) 的分布也可能是一个决定性因素2。要完全解码细胞内 Ca2 +信号, 分析下游酶的行为, 这在本协议中没有涉及, 是绝对必要的。
ca2 +成像成功的最关键的方面之一是成像设置和图像采集条件, 以及其他实时成像研究。我们之前表明, ca2 +反应在细胞中是高度依赖的持续时间和强度的兴奋和图像采集条件, 包括曝光时间和采集频率16。激发照明能力是最关键的因素, 因为它可以引起 Geci 的光毒性和光漂白。应根据实验目的, 优化曝光时间、记录频率、激发光强和记录持续时间的记录条件。我们建议尽可能缩短曝光时间和激发光强度, 以避免光漂白和光氧化对电池的影响。记录频率和记录时间应足以涵盖感兴趣的 Ca2 +高程事件, 但应尽可能保持在较低的水平, 以避免光漂白和光毒性。我们建议首先确定记录频率和持续时间, 并优化光强和曝光时间, 以最大限度地减少 Geci 的光漂白。另一个重要因素是 Geci 的表达水平。Geci 具有 Ca2 +缓冲效应, 因为它们是ca 2 +结合蛋白。因此, Geci 的过度表达导致 Ca2 +的缓冲, 这在生理上是必要的细胞。应尽量减少 GECI 表达的量, 以避免成像细胞表达大量 Geci。
总之, 以亚细胞分辨率分割 ca2 +信号是解码确定输出生物现象的细胞内 ca2 +信号的最重要步骤之一。该协议为 Ca2 +信号的分割提供了一种新的方法来描述这些信号之间的多样性。目前, 该技术仅限于体外实验。然而, Lcc-gmcmp6f 已经被用于小鼠体内ca2 +成像, oer-gmp6 f 被证实在体内监测 ca 2 + 信号在 vd 运动神经元在 c . elegans7.因此, 针对亚细胞舱内的 Geci 有可能在未来扩展到体内成像, 从而使 Ca2 +在体内解剖成为可能。
The authors have nothing to disclose.
这项工作得到以下赠款的支持: 日本科学技术署/胚胎科学技术前体研究 (日本 JJPR15F8);日本科学促进会/资助科学研究赠款 (JP18H05414, JP17H05710, JP16K07316), 武田基金会。作者感谢 Haruhiko Bito (东京大学) 提供 Rcmp2 和 Arthur j. y. huang 和 Thomas McHugh (RIKEN CBS) 提供 AAV 载体和有关 AAV 制备的指示。作者们还要感谢《可视化实验杂志》的编辑们在视频拍摄和编辑方面提供的帮助。
(RS)-3,5-Dihydroxyphenylglycine (DHPG) | Tocris | #0342 | |
0.5% DNase I stock solution | Sigma-Aldrich | #11284932001 | Prepare 0.5% DNase I (w/v) in Hanks' Balanced Salt Solution supplemented with 120 mM MgSO4. Prepare 160 µL aliquots and store at -30°C. |
0.5% Trypsin-EDTA solution | Thermo Fisher Scientific | #25300054 | |
100 mM L-glutamine (×100 stock) | Thermo Fisher Scientific | #25030081 | Preparing small aliquots of 250–750 µL and store at -30°C. |
100 mM Sodium pyruvate (×100 stock) | Thermo Fisher Scientific | #11360070 | Aliquots (10 mL) can be stored at -20°C. After thawing, the solution can be maintained at 4°C for 2 months. |
12-Well multiwell culture plates with low-evaporation lid | Falcon | #353043 | Low-evaporation lid is critical for culturing neuron-glia mixed culture. For cell line cells, alternative culture dishes can be used. |
18-mm diameter circular coverslips | Karl Hecht "Assistent" | #41001118 | Thickness 1, 18-mm diameter circular coverslips; alternative coverslips can be used. |
1M HEPES | Thermo Fisher Scientific | #15630080 | pH 7.2 – 7.6 |
2.5% Trypsin stock solution (×20 stock) | Sigma-Aldrich | #T4674 | Prepare 150 µL aliquot and store at -30°C. |
50% Poly(ethyleneimine) (PEI) solution | Sigma-Aldrich | #P3143 | Prepare 2% (V/V) PEI stock solution (×50) with distilled water sterilized by filtration. Store stock solution at -30°C after preparing small aliquots of 250-750 µL. Prepare 0.04% PEI solution with distilled water on the day of coverslip coating. |
70% Ethanol | Kept in a spray bottle to be used for surface disinfection. | ||
Adeno-associated virus (AAV) for Lck-GCaMP6f, Lck-RCaMP2, and OER-RCaMP2 expression under the direction of the EF1a promoter | AAV can be prepared using AAV Helper Free System (Agilent Technologies) and HEK293 cells, or alternative methods. pAAV.EF1a.Lck-GCaMP6f, pAAV.EF1a.Lck-RCaMP2, and pAAV.EF1a.OER-GCaMP6f are available upon request. | ||
B-27 supplement (×50 stock) | Thermo Fisher Scientific | #17504044 | This can be replaced by B-27 plus supplement (Thermo Fisher Scientific; #A3582801) or MACS NeuroBrew-21 (Miltenyi Biotec, Bergisch Gladbach, Germany; #130-093-566). |
B57BL/6 | Japan SLC, Inc. | ||
Camera for microscopic image recording | The following cameras were available for use: cooled-CCD camera (e.g., Hamamatsu Photonics, OECA-ER), EM-CCD camera (e.g., Hamamatsu Photonics, ImagEM; Andor, iXon) or CMOS camera (e.g., Hamamatsu Photonics ORCA-Flash4.0) | ||
Cell freezing container | Sarstedt K.K. | #95.64.253 | Alternative cell freezing container can be used. |
Cell strainer | Falcon | #352350 | |
CO2 incubator | Maintain at 37°C, 5% CO2. | ||
Cryogenic tube | Corning | #430661 | Alternative cryogenic tubes can be used. |
Cryopreservation medium | Zenoaq | "CELLBANKER1" | |
Culture medium (for HeLa cells) | Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, and penicillin-streptomycin solution (final concentration: Penicillin 100 units/mL and Streptomycin 100 µg/mL) | ||
Dissection medium | One milliliter of 1 M HEPES (final concentration 20 mM) to 49 mL DMEM | ||
DMEM | Nacalai | #08456-65 | Alternative DMEM can be used. |
DMEM | Nacalai | #08456-65 | Low glucose |
DNA transfection reagent | Sigma-Aldrich | #6366244001 | "X-tremegene HP DNA transfection reagent" Alternative transfection reagents can be used. |
Glass jar with a lid | 500 mL jar (for mouse) or 1500 mL jar (for rat) to anesthetize the animal | ||
HBSS | Thermo Fisher Scientific | #14170161 | HBSS free of calcium and magnesium |
Heat inactivated bovine serum | Thermo Fisher Scientific | #10100147 | |
HeLa cells | RIKEN BioResource Center | #RCB0007 | |
Histamine | Sigma-Aldrich | #H7125 | |
Image analysis software | Such as Metamorph (Molecular Devices), Image J (NIH), and TI Workbench14 (custom made) | ||
Image splitting optics | Hamamatsu Photonics | #A12801-01 | W-view GEMINI |
Image splitting optics dichroic mirror | Semrock | #FF560-FDi01-25×36 | For separation of green fluorescent protein/red fluorescent protein (GFP/RFP) signal |
Image splitting optics emission filters | Semrock | #FF01-512/25-25, #FF01-630/92-25 | For emission of GFP/RFP signal, respectively |
Imaging medium and buffer | Use optimal medium or buffer for the experiment. When medium is used, medium without phenol red is desirable to reduce background fluorescence. Add 20 mM HEPES to maintain pH outside of CO2 incubator. | ||
Incubation saline | Add 1 mL of 1 M HEPES (20 mM) to 49 mL HBSS | ||
Inverted fluorescence microscope | Such as IX73 (Olympus) or Eclipse TI (Nikon Instech) | ||
Isoflurane | Pfizer | Used for anesthesia | |
Maintenance medium (for 4 × 12 well dishes) | 48.5 mL Neurobasal-A medium supplemented with 1 mL B-27, 500 µL of L-glutamine stock and 25 µL Penicillin-Streptomycin solution. | ||
Maintenance medium for frozen cortical cells (for 1 × 12 well dishes) | 12.2 mL Neurobasal plus medium supplemented with 250 µL B-27, 125 µL of L-glutamine stock and 6.2 µL Penicillin-Streptomycin solution. | ||
MEM (Minimum Essential Medium) | Thermo Fisher Scientific | #11090-081 | |
Microscope filter set for GCaMP6f imaging | Appropriate filter for GFP (excitation, 480 ± 10 nm; emission, 530 ± 20 nm) | ||
Microscope filter set for RCaMP2 imaging | Appropriate filter for RFP (excitation, 535 ± 50 nm; emission, 590 nm long pass) | ||
Microscope filter sets for double imaging of RCaMP2 and GCaMP6f | Semrock | #FF01-468/553-25, #FF493/574-FDi01-25×36, #FF01-512/630-25 | Dual excitation filter, Dual dichroic mirror, and emission filter for GFP/RFP imaging. |
Microscope heating system | A heating system to maintain cells at 37°C during the imaging. To avoid drift caused by thermal expansion, heating systems covering the entire microscope itself (e.g., Tokai Hit, Thermobox) are recommended. | ||
Microscope light source for excitation | Mercury lamp (100 W), xenon lamp (75 W), Light-emitting diode (LED) illumination system (e.g., CoolLED Ltd., precisExcite; Thorlabs Inc., 4-Wavelength LED Source; Lumencor, SPECTRA X light engine). In case of mercury lump and xenon lamp, use ND filter to reduce the excitation intensity. | ||
Microscope objective lens | Plan-Apochromat oil immersion objective with numerical aperture higher than 1.3 is highly recommended for the recording of spontaneous Ca2+ activity in neurons and astrocytes. | ||
Neurobasal plus medium | Thermo Fisher Scientific | #A3582901 | |
Neurobasal-A Medium | Thermo Fisher Scientific | #10888022 | Neurobasal plus medium (Thermo Fisher, A3582901) can be used instead of Neurobasal-A medium. |
PBS(-): Phosphate-buffered saline free of Ca2+ and Mg2+ | Fujifilm Wako Pure Chemical Cooperation | #164-23551 | The absence of Ca2+ and Mg2+ is critical not to inhibit the trypsin activity. An alternative to PBS(-) can be used. |
PC and image acquisition software | Such as Metamorph (Molecular Devices); Micromanager; TI Workbench14. | ||
Penicillin-Streptomycin solution | Thermo Fisher Scientific | #15140122 | Penicillin 10,000 units/mL and Streptomycin 10,000 µg/mL |
Plasmid for Lck-GCaMP6f, Lck-RCaMP2, and OER-RCaMP2 expression under cytomegalovirus promoter 7-9 | Available upon request | ||
Plating medium (for 4 × 12 well dishes) | 48 mL MEM supplemented with 1 mL B-27 supplement, 500 µL L-glutamine stock (final concentration: 2 mM), 500 µL of sodium pyruvate stock (1 mM) and 25 µL Penicillin-Streptomycin solution (penicillin 5 u/mL, streptomycin 5 µg/mL). This concentration of Penicillin-Streptomycin, which is 1/20 of the concentration recommended by the manufacturer, is critical for neuronal survival. | ||
Recording chamber | Elveflow | Ludin Chamber | This recording chamber is for 18 mm diameter round coverslips. |
Reduced serum media | Thermo Fisher | #11058021 | Opti-MEM |
Stereomicroscope | Used to dissect hippocampi. Olympus SZ60 or equivalent stereomicroscopes are available. | ||
Surgical instruments | Standard dissecting scissors to cut the abdomen of a mouse or a rat, tweezers to pinch the uterus, delicate dissecting scissors to cut the uterus and the head of embryo, ring forceps to pinch the embryos, 13-cm curved Semken forceps (Fine Science Tools #11009-13) to extract brains, 3 forceps with fine tips (Dumont Inox #5) | ||
Transfection reagent for neuron | Thermo Fisher Scientific | #L3000008 | "Lipofectamine 3000" reagent. It is composed of the the "supplement (P3000)" that should be mixed with plasmid DNA in the step 2.2.3, and the "transfection reagent (lipofectamine 3000)" used in the step 2.2.4. |
Trypan blue (0.4%) | Thermo Fisher Scientific | #15250061 | |
Wash medium for frozen cortical cells | 25 mL DMEM, supplemented with 250 µL heat-inactivated fetal bovine serum + 12.5 µL Penicillin Streptomycin. | ||
Wistar rats | Japan SLC, Inc | Pregnant rats (E18) |