利用膜片钳技术标记脑片神经元的方法

doi:10.12047/j.cjap.6035.2021.031

中国应用生理学杂志 ›› 2021, Vol. 37 ›› Issue (4): 445-448.doi: 10.12047/j.cjap.6035.2021.031

• 技术方法 • 上一篇

利用膜片钳技术标记脑片神经元的方法

高洁^1,2, 黄燕云^1,2, 张雨彤^1,2, 杜相欣^1,2, 张利娜^1,2, 郝娜^1,2, 郭霞^1,2, 李建国^1,2, 张宇^1,2△

1. 山西医科大学细胞生理学教育部重点实验室,
2. 山西医科大学生理学系, 太原 030001

出版日期:2021-07-28 发布日期:2021-08-09
通讯作者: ^△Tel: (0351)4135560或3985163; E-mail: zhyucnm@163.com
基金资助:
^*国家自然科学基金项目(81371254);山西省‘1331工程'重点学科建设计划经费(XK201708);山西省自然科学基金(201801D121318)

A method of marking neurons on brain slices using patch clamp technique

GAO Jie^1,2, HUANG Yan-yun^1,2, ZHANG Yu-tong^1,2, DU Xiang-xin^1,2, ZHANG Li-na^1,2, HAO Na^1,2, GUO Xia^1,2, LI Jian-guo^1,2, ZHANG Yu^1,2△

1. Key Laboratory of Cellular Physiology, Ministry of Education, Shanxi Medical University;
2. The Department of Physiology, Shanxi Medical University, Taiyuan 030001, China

Online:2021-07-28 Published:2021-08-09

摘要/Abstract

摘要： 目的: 介绍一种利用膜片钳技术标记脑片神经元形态的方法。方法: 利用振动切片机切好实验目标部位的脑片,用含有Neurobiotin^TM Tracer的电极内液灌注玻璃微电极,并进行全细胞膜片钳记录;实验结束后将脑片先用4％多聚甲醛固定、漂洗,再用含有Streptavidin-Texas Red和Triton X-100的PB染色,2 h后即可用荧光显微镜观察着色的神经元。结果: 将细胞膜电压钳制在-70 mV,阶跃刺激后神经元表现为逐渐增大的膜电流。电流钳模式记录时,阶跃刺激使神经元去极化,达到阈电位后爆发动作电位。荧光显微镜下可看到胞体和主要突起清晰完整的神经元形态。结论: 本方法适用于在膜片钳实验后观察所记录的神经元的形态特征,操作方便,图像直观清晰。

关键词: 全细胞膜片钳, 脑片, 神经元形态, 荧光标记

Abstract: Objective: To introduce a method of marking neurons using patch clamp technique. Methods: The brain slices of the target area was cut with a vibrating microtome. The glass microelectrode was perfused with the electrode liquid containing Neurobiotin^TM Tracer, and the whole-cell patch-clamp recording was performed. After recording, the brain slices were fixed and rinsed with 4% paraformaldehyde. After stained in phosphate buffer with Streptavidin-Texas Red and Triton X-100 for at least 2 hours, the neurons can be observed under a fluorescence microscope. Results: The cell membrane voltage was clamped at -70 mV, and the neuron showed a gradually increasing membrane current after step stimulation. When recording in the current clamp mode, the step stimulus caused the neuron to depolarize to the threshold potential and then burst into action potentials. The morphology of intact neurons with clear cell body and protrusions of a neuron could be observed under a fluorescence microscope. Conclusion: This method is suitable for observing the morphological features of the recorded neuron after patch clamp experiments, which is easy to operate, and the image is intuitive and clear.

Key words: whole-cell patch clamp, brain slices, neuronal morphology, fluorescence tags

中图分类号:

R338.8

高洁, 黄燕云, 张雨彤, 杜相欣, 张利娜, 郝娜, 郭霞, 李建国, 张宇. 利用膜片钳技术标记脑片神经元的方法[J]. 中国应用生理学杂志, 2021, 37(4): 445-448.

GAO Jie, HUANG Yan-yun, ZHANG Yu-tong, DU Xiang-xin, ZHANG Li-na, HAO Na, GUO Xia, LI Jian-guo, ZHANG Yu. A method of marking neurons on brain slices using patch clamp technique[J]. CJAP, 2021, 37(4): 445-448.

参考文献 11

[1]	Huang Q, Zhou D, DiFiglia M.Neurobiotin, a useful neuroanatomical tracer for in vivo anterograde, retrograde and transneuronal tract-tracing and for in vitro labeling of neurons[J]. J Neurosci Methods, 1992, 41(1): 31-43.
[2]	Tuchina OP, Zhukov VV, Meyer-Rochow VB. Central and peripheral neuronal pathways revealed by backfilling with neurobiotin in the optic, tentacular and small labial nerves of Lymnaea stagnalis[J]. Acta Zoologica, 2012, 93(1): 28-47.
[3]	Kanjhan R, Bellingham MC. Neurobiotin electroporation for combined structural and functional analysis of neurons in developing mouse brain slices[J]. Neuromethods, 2013, 78(2): 151-165.
[4]	Lim KH, Huang H, Pralle A, et al. Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection[J]. Biotechnol Bioeg, 2013, 110(1): 57-67.
[5]	Sun XJ, Tolbert LP, Hildebrand JG, et al. A rapid method for combined laser scanning confocal microscopic and electron microscopic visualization of biocytin or neurobiotin-labeled neurons[J]. J Histochem Cytochem, 1998, 46(2): 263-273.
[6]	Fogarty MJ, Kanjhan R, Bellingham MC, et al. Glycinergic neurotransmission: A potent regulator of embryonic motor neuron dendritic morphology and synaptic plasticity[J]. J Neurosci, 2016, 36(1): 80-87.
[7]	Imperadore P, Lepore MG, Ponte G, et al. Neural pathways in the pallial nerve and arm nerve cord revealed by neurobiotin backfilling in the cephalopod mollusk Octopus vulgaris[J]. Invertebr Neurosci, 2019, 19(2): 5.
[8]	Xi XZ, Xu ZC. The effect of neurobiotin on membrane properties and morphology of intracellularly labeled neurons[J]. J Neurosci Methods, 1996, 65(1): 27-32.
[9]	Schlsser B, Bruggencate GT, Sutor B. The intracellular tracer Neurobiotin alters electrophysiological properties of rat neostriatal neurons[J]. Neurosci Let, 1998, 249(1): 13-16.
[10]	Fan RJ, Marin-BurginA , French KA, et al. A dye mixture (Neurobiotin and Alexa 488) reveals extensive dye-coupling among neurons in leeches; physiology confirms the connections[J]. J Comp Physiol A, 2005, 191(12): 1157-1171.
[11]	Kanjhan R, Vaney DI. Semi-loose seal Neurobiotin electroporation for combined structural and functional analysis of neurons[J]. Pflugers Arch, 2008, 457(2): 561-568.

利用膜片钳技术标记脑片神经元的方法

A method of marking neurons on brain slices using patch clamp technique

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