凝胶神经元全细胞斑块夹的急性脊髓切片的制备
Summary
在这里, 我们描述了由体外脊髓切片中的明胶 (sg) 神经元制成的全细胞膜片夹记录的基本步骤。该方法可对 sg 神经元的固有膜特性、突触传递和形态表征进行研究。
Abstract
最近的全细胞膜片钳研究从明胶 (sg) 神经元提供了大量的信息, 有关脊柱机制的基础感觉传递, 诱发电位调节, 慢性疼痛或瘙痒的发展。在急性脊髓切片的效用基础上, 实施电生理记录和形态学研究, 进一步提高了我们对 sg 中神经元特性和局部电路组成的认识。在这里, 我们提出了一个详细和实用的指南, 为脊髓切片的制备, 并显示具有代表性的全细胞记录和形态学结果。该协议允许理想的神经元保存, 并能在一定程度上模拟体内条件。总之, 能够获得脊髓切片的体外准备, 可以稳定的电流和电压夹具记录, 从而有助于对固有膜特性、局部电路和神经元结构使用不同的实验方法。
Introduction
胶质 (sg, 脊髓背角的层 ii) 是一个无可争辩的重要中继中心, 用于传输和调节感官信息。它由兴奋和抑制间神经元组成, 接收来自初级传入纤维、局部神经元间和内源性下降抑制系统1的输入。近几十年来, 急性脊髓切片制剂的发展和全细胞膜片夹具记录的出现, 使 sg2的内在电生理和形态特性的各种研究,3 个,4并且地方电路的研究在 sg5,6。此外, 通过体外脊髓切片制剂, 研究人员可以解释神经元兴奋性的变化 7,8, 离子通道9,10, 和突触活动11,12在各种病理条件下。这些研究加深了我们对 sg 神经元在慢性疼痛和神经病理性瘙痒的发展和维持中的作用的理解。
从本质上讲, 实现神经元 soma 的清晰可视化和理想的全细胞修补使用急性脊髓切片的关键先决条件是确保切片的优良质量, 从而获得健康和可修补的神经元。然而, 准备脊髓切片涉及几个步骤, 如进行腹侧椎板切除术和去除皮-蜘蛛核细胞膜, 这可能是获得健康切片的障碍。虽然准备脊髓切片并不容易, 但在体外对脊髓切片进行录音有几个优点。与细胞培养制剂相比, 脊髓切片可以部分保存生理相关条件下的固有突触连接。此外, 使用脊髓切片的全细胞膜片夹具记录可与其他技术相结合, 如双膜片钳13、14、形态学研究15、16和单细胞 rt-pcr17. 因此, 这项技术提供了更多关于特定区域内解剖和遗传多样性特征的信息, 并可以调查当地电路的组成。
在这里, 我们提供了一个基本和详细的描述, 我们的方法准备急性脊髓切片和获取全细胞膜片夹从 sg 神经元。
Protocol
所描述的所有实验方案均获得南昌大学动物伦理委员会 (南昌, 中国, 道德号 2017-010号)。所有的努力都是为了最大限度地减少实验动物的压力和痛苦。在室温下进行的电生理记录 (rt, 22–25°c)。 1. 动物 使用 sprague-dawley 大鼠 (3-5周大) 的任何性别。将这些动物以12小时的光暗循环居住, 让它们获得足够的食物和水。 2. 溶液和材料的制备 …
Representative Results
根据图 1所示的图表制备了急性脊髓切片。切片和恢复后, 脊髓切片被转移到记录室。利用 ir-dic 显微镜, 根据 soma 的外观鉴定了健康的神经元。其次, 当神经元在 rmp 上保持时, 一系列的去极化电流脉冲 (1秒) 激发了 sg 神经元的动作电位。如图 2所示, 在 sg 神经元中观察到的发射模式包括扁桃体发射、延迟发射、杂波发射、初始爆…
Discussion
该协议详细介绍了准备脊髓切片的步骤, 我们在 sg 神经元18、19、20、21上进行全细胞膜片时成功地使用了脊髓切片。通过实施这种方法, 我们最近报告说, 米诺环素, 第二代四环素, 可以显著提高抑制突触传递通过突触前机制在 sg 神经元19。此外, 该制剂可降低 ih 的振幅, 进一?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金的资助 (81560198、31660289号)。
Materials
| NaCl | Sigma | S7653 | Used for the preparation of ACSF and PBS |
| KCl | Sigma | 60130 | Used for the preparation of ACSF, sucrose-ACSF, and K+-based intracellular solution |
| NaH2PO4·2H2O | Sigma | 71500 | Used for the preparation of ACSF, sucrose-ACSF and PBS |
| CaCl2·2H2O | Sigma | C5080 | Used for the preparation of ACSF and sucrose-ACSF |
| MgCl2·6H2O | Sigma | M2670 | Used for the preparation of ACSF and sucrose-ACSF |
| NaHCO3 | Sigma | S5761 | Used for the preparation of ACSF and sucrose-ACSF |
| D-Glucose | Sigma | G7021 | Used for the preparation of ACSF |
| Ascorbic acid | Sigma | P5280 | Used for the preparation of ACSF and sucrose-ACSF |
| Sodium pyruvate | Sigma | A7631 | Used for the preparation of ACSF and sucrose-ACSF |
| Sucrose | Sigma | S7903 | Used for the preparation of sucrose-ACSF |
| K-gluconate | Wako | 169-11835 | Used for the preparation of K+-based intracellular solution |
| Na2-Phosphocreatine | Sigma | P1937 | Used for the preparation of intracellular solution |
| EGTA | Sigma | E3889 | Used for the preparation of intracellular solution |
| HEPES | Sigma | H4034 | Used for the preparation of intracellular solution |
| Mg-ATP | Sigma | A9187 | Used for the preparation of intracellular solution |
| Li-GTP | Sigma | G5884 | Used for the preparation of intracellular solution |
| CsMeSO4 | Sigma | C1426 | Used for the preparation of Cs+-based intracellular solution |
| CsCl | Sigma | C3011 | Used for the preparation of Cs+-based intracellular solution |
| TEA-Cl | Sigma | T2265 | Used for the preparation of Cs+-based intracellular solution |
| Neurobiotin 488 | Vector | SP-1145 | 0.05% neurobiotin 488 could be used for morphological studies |
| Agar | Sigma | A7002 | 3% agar block was used in our protocol |
| Paraformaldehyde | Sigma | P6148 | 4% paraformaldehyde was used for immunohistochemical processing |
| Na2HPO4 | Hengxing Chemical Reagents | Used for the preparation of PBS | |
| Mount Coverslipping Medium | Polyscience | 18606 | |
| Urethan | National Institute for Food and Drug Control | 30191228 | 1.5 g/kg, i.p. |
| Borosilicate glass capillaries | World Precision Instruments | TW150F-4 | 1.5 mm OD, 1.12 mm ID |
| Micropipette puller | Sutter Instrument | P-97 | Used for the preparation of micropipettes |
| Vibratome | Leica | VT1000S | |
| Vibration isolation table | Technical Manufacturing Corporation | 63544 | |
| Infrared CCD camera | Dage-MIT | IR-1000 | |
| Patch-clamp amplifier | HEKA | EPC-10 | |
| Micromanipulator | Sutter Instrument | MP-285 | |
| X-Y stage | Burleigh | GIBRALTAR X-Y | |
| Upright microscope | Olympus | BX51WI | |
| Osmometer | Advanced | FISKE 210 | |
| PH meter | Mettler Toledo | FE20 | |
| Confocol microscope | Zeiss | LSM 700 |
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Tags
Acute Spinal Cord SlicesWhole-cell Patch-clamp RecordingSubstantia Gelatinosa NeuronsSensory TransmissionNociceptive RegulationChronic PainAgar BlockSucrose-based ACSFTranscardial PerfusionSpinal Cord ExtractionLumbosacral EnlargementMeninges RemovalPia Arachnoid MembraneVentral/dorsal RootsTransverse SlicesParasagittal SlicesVibratome
Citar este artigo
Zhu, M., Zhang, D., Peng, S., Liu, N., Wu, J., Kuang, H., Liu, T. Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons. J. Vis. Exp. (143), e58479, doi:10.3791/58479 (2019).