Summary

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

Published: January 18, 2019
doi:

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1516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181920、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?…

Disclosures

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

References

  1. Todd, A. J. Neuronal circuitry for pain processing in the dorsal horn. Nature Reviews Neuroscience. 11 (12), 823-836 (2010).
  2. Yoshimura, M., Nishi, S. Blind patch-clamp recordings from substantia gelatinosa neurons in adult rat spinal cord slices: pharmacological properties of synaptic currents. Neuroscience. 53 (2), 519-526 (1993).
  3. Maxwell, D. J., Belle, M. D., Cheunsuang, O., Stewart, A., Morris, R. Morphology of inhibitory and excitatory interneurons in superficial laminae of the rat dorsal horn. The Journal of Physiology. 584 (Pt. 2, 521-533 (2007).
  4. Grudt, T. J., Perl, E. R. Correlations between neuronal morphology and electrophysiological features in the rodent superficial dorsal horn. The Journal of Physiology. 540 (Pt 1), 189-207 (2002).
  5. Lu, Y., et al. A feed-forward spinal cord glycinergic neural circuit gates mechanical allodynia. Journal of Clinical Investigation. 123 (9), 4050-4062 (2013).
  6. Zheng, J., Lu, Y., Perl, E. R. Inhibitory neurones of the spinal substantia gelatinosa mediate interaction of signals from primary afferents. The Journal of Physiology. 588 (Pt 12), 2065-2075 (2010).
  7. Balasubramanyan, S., Stemkowski, P. L., Stebbing, M. J., Smith, P. A. Sciatic chronic constriction injury produces cell-type-specific changes in the electrophysiological properties of rat substantia gelatinosa neurons. Journal of Neurophysiology. 96 (2), 579-590 (2006).
  8. Zhang, L., et al. Extracellular signal-regulated kinase (ERK) activation is required for itch sensation in the spinal cord. Molecular Brain. 7, 25 (2014).
  9. Kopach, O., et al. Inflammation alters trafficking of extrasynaptic AMPA receptors in tonically firing lamina II neurons of the rat spinal dorsal horn. Pain. 152 (4), 912-923 (2011).
  10. Takasu, K., Ono, H., Tanabe, M. Spinal hyperpolarization-activated cyclic nucleotide-gated cation channels at primary afferent terminals contribute to chronic pain. Pain. 151 (1), 87-96 (2010).
  11. Iura, A., Takahashi, A., Hakata, S., Mashimo, T., Fujino, Y. Reductions in tonic GABAergic current in substantia gelatinosa neurons and GABAA receptor delta subunit expression after chronic constriction injury of the sciatic nerve in mice. European Journal of Pain. 20 (10), 1678-1688 (2016).
  12. Alles, S. R., et al. Peripheral nerve injury increases contribution of L-type calcium channels to synaptic transmission in spinal lamina II: Role of alpha2delta-1 subunits. Molecular Pain. 14, 1-12 (2018).
  13. Santos, S. F., Rebelo, S., Derkach, V. A., Safronov, B. V. Excitatory interneurons dominate sensory processing in the spinal substantia gelatinosa of rat. The Journal of Physiology. 581 (Pt 1), 241-254 (2007).
  14. Lu, Y., Perl, E. R. Modular organization of excitatory circuits between neurons of the spinal superficial dorsal horn (laminae I and II). The Journal of Neuroscience. 25 (15), 3900-3907 (2005).
  15. Hantman, A. W., van den Pol, A. N., Perl, E. R. Morphological and physiological features of a set of spinal substantia gelatinosa neurons defined by green fluorescent protein expression. The Journal of Neuroscience. 24 (4), 836-842 (2004).
  16. Yasaka, T., Tiong, S. Y., Hughes, D. I., Riddell, J. S., Todd, A. J. Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach. Pain. 151 (2), 475-488 (2010).
  17. Yin, H., Park, S. A., Han, S. K., Park, S. J. Effects of 5-hydroxytryptamine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in immature mice. Brain Research. 1368, 91-101 (2011).
  18. Hu, T., et al. Lidocaine Inhibits HCN Currents in Rat Spinal Substantia Gelatinosa Neurons. Anesthesia and Analgesia. 122 (4), 1048-1059 (2016).
  19. Peng, H. Z., Ma, L. X., Lv, M. H., Hu, T., Liu, T. Minocycline enhances inhibitory transmission to substantia gelatinosa neurons of the rat spinal dorsal horn. Neuroscience. 319, 183-193 (2016).
  20. Peng, S. C., et al. Contribution of presynaptic HCN channels to excitatory inputs of spinal substantia gelatinosa neurons. Neuroscience. 358, 146-157 (2017).
  21. Liu, N., Zhang, D., Zhu, M., Luo, S., Liu, T. Minocycline inhibits hyperpolarization-activated currents in rat substantia gelatinosa neurons. Neuropharmacology. 95, 110-120 (2015).
  22. Brown, T. H. Methods for whole-cell recording from visually preselected neurons of perirhinal cortex in brain slices from young and aging rats. Journal of Neuroscience Methods. 86 (1), 35-54 (1998).
  23. Rothman, S. M. The neurotoxicity of excitatory amino acids is produced by passive chloride influx. The Journal of Neuroscience. 5 (6), 1483-1489 (1985).
  24. Rice, M. E. Use of ascorbate in the preparation and maintenance of brain slices. Methods. 18 (2), 144-149 (1999).
  25. Takasu, K., Ogawa, K., Minami, K., Shinohara, S., Kato, A. Injury-specific functional alteration of N-type voltage-gated calcium channels in synaptic transmission of primary afferent C-fibers in the rat spinal superficial dorsal horn. European Journal of Pharmacology. 772, 11-21 (2016).
  26. Tian, L., et al. Excitatory synaptic transmission in the spinal substantia gelatinosa is under an inhibitory tone of endogenous adenosine. Neuroscience Letters. 477 (1), 28-32 (2010).
  27. Funai, Y., et al. Systemic dexmedetomidine augments inhibitory synaptic transmission in the superficial dorsal horn through activation of descending noradrenergic control: an in vivo patch-clamp analysis of analgesic mechanisms. Pain. 155 (3), 617-628 (2014).
  28. Yamasaki, H., Funai, Y., Funao, T., Mori, T., Nishikawa, K. Effects of tramadol on substantia gelatinosa neurons in the rat spinal cord: an in vivo patch-clamp analysis. PLoS One. 10 (5), e0125147 (2015).
  29. Furue, H., Narikawa, K., Kumamoto, E., Yoshimura, M. Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patch-clamp recording. The Journal of Physiology. 521 (Pt 2), 529-535 (1999).
  30. Ting, J. T., Daigle, T. L., Chen, Q., Feng, G. Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods in Molecular Biology. 1183, 221-242 (2014).
  31. Ting, J. T., et al. Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method. Journal of Visualized Experiments. (132), e53825 (2018).
  32. Li, J., Baccei, M. L. Neonatal Tissue Damage Promotes Spike Timing-Dependent Synaptic Long-Term Potentiation in Adult Spinal Projection Neurons. The Journal of Neuroscience. 36 (19), 5405-5416 (2016).
  33. Ford, N. C., Ren, D., Baccei, M. L. NALCN channels enhance the intrinsic excitability of spinal projection neurons. Pain. , (2018).
  34. Cui, L., et al. Modulation of synaptic transmission from primary afferents to spinal substantia gelatinosa neurons by group III mGluRs in GAD65-EGFP transgenic mice. Journal of Neurophysiology. 105 (3), 1102-1111 (2011).
  35. Yang, K., Ma, R., Wang, Q., Jiang, P., Li, Y. Q. Optoactivation of parvalbumin neurons in the spinal dorsal horn evokes GABA release that is regulated by presynaptic GABAB receptors. Neuroscience Letters. , 55-59 (2015).

Play Video

Cite This Article
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).

View Video