Name: isolating and culturing vestibular and spiral ganglion somata from neonatal rodents for patch-clamp recordings
Uploaded: 2023-04-21T12:40:00
Description: Watch this Scientific Journal Video about Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings at JoVE.com

我们感谢 Jing Bing Xue 博士和 Ruth Anne Eatock 博士对这些方法的早期贡献。这项工作得到了 NIH NIDCD R03 DC012652 和 NIH NIDCD DC012653S 的支持，R01 DC0155512到 RK 和 T32 DC009975到 DB、NN 和 KR。

此处描述的所有动物用途均已获得南加州大学机构动物护理和使用委员会的批准。该方案中的动物是从查尔斯河实验室获得的P3至P25年龄的两性Long Evans大鼠，但这些方法可以应用于其他啮齿动物品系。在所有程序中必须穿戴实验室外套和手套，并在制备溶液时戴上防溅护目镜。
1. 准备工作
注意：本节中描述的解决方案和工具可以提前制作，以...

<ol>
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L., Crozier, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+firing+properties+of+type+I+spiral+ganglion+neurons.">Dynamic firing properties of type I spiral ganglion neurons.</a> Cell and Tissue Research. 361 (1), 115-127 (2015).</li><li>Reijntjes, D. O. J., Pyott, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+afferent+signaling+complex:+Regulation+of+type+I+spiral+ganglion+neuron+responses+in+the+auditory+periphery.">The afferent signaling complex: Regulation of type I spiral ganglion neuron responses in the auditory periphery.</a> Hearing Research. 336, 1-16 (2016).</li><li>Eatock, R. A., Christov, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ionic Conductances of Vestibular Afferent Neurons: Shaping Head Motion Signals From the Inner Ear. , (2020).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+in+the+biophysical+properties+of+spiral-ganglion+and+vestibular-ganglion+neurons+in+neonatal+rats.">Similarities in the biophysical properties of spiral-ganglion and vestibular-ganglion neurons in neonatal rats.</a> Frontiers in Neuroscience. 15, 710275 (2021).</li><li>Armstrong, C. E., Roberts, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+properties+of+frog+saccular+hair+cells:+distortion+by+enzymatic+dissociation.">Electrical properties of frog saccular hair cells: distortion by enzymatic dissociation.</a> The Journal of Neuroscience. 18 (8), 2962-2973 (1998).</li><li>Rocha-Sanchez, S. M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+expression+of+Kcnq4+in+vestibular+neurons+and+neurosensory+epithelia.">Developmental expression of Kcnq4 in vestibular neurons and neurosensory epithelia.</a> Brain Research. 1139, 117-125 (2007).</li><li>Meredith, F. L., Rennie, K. 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这里介绍的方法特定于来自分离神经元的记录;以前的研究集中在半完整制剂中轴突末端的记录。与现有的终端记录技术相比，隔离记录具有出色的空间钳位和等电位行为。此外，该协议提供了对更广泛的神经元样本的访问，因为在前庭上皮的半完整记录中只能访问带有花萼的亚群。最后，孤立的记录允许使用穿孔贴片技术，该技术可以防止细胞内环境的破坏，而细胞内环境通常会因破裂贴片记录...

前庭蜗神经的双极神经元将内耳的感觉毛细胞连接到脑干。它们是有关声音和头部运动的信息的主要载体;这些重要细胞的损伤会导致耳聋和平衡障碍。神经的前庭和听觉部分分别由形态和功能多样化的不同细胞类型组成 1,2。在前庭系统中，两个传入亚群以规则或不规则的间隔自发放电2.传入尖峰时间被认为反映了离子通道组成的潜在多样性 3,4。在听觉系统中，螺旋神经节神经元 （SGN） 有两个主要亚群;I 型 SGN 接触单个内毛细胞5，II 型 SGN 接触多个外毛细胞5。来自半完整和器官型培养物的体外记录表明 I 型和 II 型 SGN 的膜特性存在差异 6,7。
在这些神经元末端发现的许多离子通道和神经递质受体也存在于它们的细胞体中。因此，可以在 体外 研究孤立的前庭和螺旋神经节体的培养物，以了解离子通道和神经递质受体如何促进这些神经元的反应。分离细胞体的紧凑形态允许高质量的电记录，适用于电压门控离子通道和神经递质受体的详细表征。易于获得具有代表性的各种神经元亚型，可对细胞多样性进行高通量分析。
本文介绍了一种在出生后第9至P20天从大鼠前庭神经节上部分离和培养解离的神经节细胞体的方法。除了成功提取、解离和接种神经节细胞所需的步骤外，还提供了将这些方法扩展到螺旋神经节的建议。这些方法是各个实验室出版物中设计的方法的演变 8,9,10。本文还包括选择健康细胞进行膜片钳记录的指南。
最后，该协议概述了使用穿孔补丁配置11 进行膜片钳记录的过程。虽然穿孔贴片配置比更常见的破裂贴片配置更耗时，技术上更具挑战性，但它更适合维持细胞质环境，允许长时间稳定的记录会话。这种记录配置的好处在这里通过相对于破裂贴片记录的超极化激活阳离子电流在穿孔贴片中提高的稳定性来说明。
该协议分为五个部分。第 1-3 节描述了可以提前准备和存储的解决方案和工具。第 4 节描述了解剖和接种前庭和 SGN 的步骤，第 5 节描述了在培养一段时间后从神经元记录的步骤。在我们手中，第 4 节和第 5 节是在连续 2 天内执行的。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin</td>
			<td>Sigma-Aldrich</td>
			<td>A4888-100MG</td>
			<td>For perforated patch recordings.</td>
		</tr>
		<tr>
			<td>ATP di-sodium</td>
			<td>Sigma-Aldrich</td>
			<td>A7699</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>B27 Supplement (50x), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td>additive to culture medium, for SGN</td>
		</tr>
		<tr>
			<td>Beakers (1000, 100, 10) milliliter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bench-top centrifuge</td>
			<td>USA Scientific</td>
			<td>2641-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>J.T. Baker</td>
			<td>1311-01</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C5318</td>
			<td>one out of three enzyme to digest tissue</td>
		</tr>
		<tr>
			<td>Coverglass, rectangular, #1 thickness, 22x40&#160;</td>
			<td>Warner Instruments</td>
			<td>64-0707</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Biotium</td>
			<td>90082</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I,from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>11284932001</td>
			<td>one out of three enzyme to digest tissue</td>
		</tr>
		<tr>
			<td>Dumont #3 Forceps (Blunt)</td>
			<td>Fine Science Tools</td>
			<td>11231-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps (Fine)</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps (Fine)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E0396</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>Electrode Puller</td>
			<td>Narashige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Epi-illumination light source&#160;</td>
			<td>Zeiss&#160;</td>
			<td>CL 1500 ECO</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td>for cleaning head and around dissection bench</td>
		</tr>
		<tr>
			<td>Filamented Borosilicate Capillaries for electrodes</td>
			<td>Sutter Instruments</td>
			<td>BF140-117-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-edged dissection blade</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipettes</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td>to pull trituration pipettes</td>
		</tr>
		<tr>
			<td>Heat-inactivated Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16140063</td>
			<td>additive to culture medium</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-100G</td>
			<td>for pH buffering all solutions in protocol</td>
		</tr>
		<tr>
			<td>Hot plate / magnetic stirrers&#160;</td>
			<td>VWR</td>
			<td>76549-914</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulated bucket filled with ice</td>
			<td></td>
			<td></td>
			<td>to keep all samples and solutions cool</td>
		</tr>
		<tr>
			<td>K2SO4, Potassium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>P9458-250G</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P93333</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>KOH (1 M)</td>
			<td>Honeywell</td>
			<td>319376-500ML</td>
			<td>To bring internal solution to desired pH.</td>
		</tr>
		<tr>
			<td>Large Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>14133-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz medium&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L4386</td>
			<td>dissection and bath solutions&#160;</td>
		</tr>
		<tr>
			<td>Low-profile-bath recording chamber for culture dishes</td>
			<td>Warner Instruments</td>
			<td>64-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>luer-lok syringes, 30 ml</td>
			<td>BD</td>
			<td>302832</td>
			<td>for drawing L-15/HEPES/HEPES solution.</td>
		</tr>
		<tr>
			<td>MEM + Glutamax Supplement</td>
			<td>Fisher Scientific</td>
			<td>41-090-101</td>
			<td>base of the culture medium</td>
		</tr>
		<tr>
			<td>MgCl2-Hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>microFil needle for filling micropipettes - 34 gauge&#160;</td>
			<td>World Precision Instruments</td>
			<td>MF34G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narashige</td>
			<td>MF-90</td>
			<td>For electrode polishing.</td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td>additiive to culture medium, for SGN</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>NaOH (1 M)</td>
			<td>Thomas Scientific</td>
			<td>319511-500ML</td>
			<td>for titration pH</td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>Advanced Instruments Inc.</td>
			<td>3320</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen, Medical grade, with adequate regulator and tubing</td>
			<td>USC Material Management</td>
			<td>MEDOX200 (Identifier: 00015)</td>
			<td>for dissolving into dissection and bath solutions</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette bulb (3 ml)</td>
			<td>Fisher Scientific</td>
			<td>03-448-25</td>
			<td>bulb for trituration pipettes</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>additive to prevent contamination of culture medium</td>
		</tr>
		<tr>
			<td>Pentobarbital based euthanasia solution (e.g., Fatal Plus. 50 &#8211; 60 mg/kg dosing)&#160;</td>
			<td>MWI Animal Health</td>
			<td>15199</td>
			<td>for euthanasia</td>
		</tr>
		<tr>
			<td>PES membrane filters ,&#160; 0.2 micrometer&#160;</td>
			<td>Nalgene</td>
			<td>566-0020</td>
			<td>for filtering solutions</td>
		</tr>
		<tr>
			<td>PES membrane sterile syringe filters, 0.22 um, 30 mm&#160;</td>
			<td>CELLTREAT</td>
			<td>229747</td>
			<td>for filtering solutions drawn into syringes</td>
		</tr>
		<tr>
			<td>Petri dishes, 35 x 10 mm</td>
			<td>Genessee Scientific</td>
			<td>32-103</td>
			<td>for micro dissection and to hold Tip dip solution in perforated-patch configuration</td>
		</tr>
		<tr>
			<td>Petri Dishes, 60 x 15 mm</td>
			<td>Midland Scientific</td>
			<td>P7455</td>
			<td>for gross dissection</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>Model S20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors (1000, 200, 10) microliter</td>
			<td>USA Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-d-lysine coated glass bottomed culture dish</td>
			<td>Mattek</td>
			<td>P35GC-0-10-C</td>
			<td>to plate neurons for culture</td>
		</tr>
		<tr>
			<td>Quick change platform, heated base, for 35 mm culture dishes</td>
			<td>Warner Instruments</td>
			<td>64-0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Reference Cell</td>
			<td>World Precision Instruments</td>
			<td>RC1T</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Miltex</td>
			<td>4-315</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Scale</td>
			<td>Mettler Toledo</td>
			<td>XS64</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes (10, 25) milliliter</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Grease Kit (for sealing coverglass and chamber)</td>
			<td>Warner Instruments</td>
			<td>64-0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Guillotine</td>
			<td>Kent Scientific</td>
			<td>DCAP</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal guillotine</td>
			<td>Kent Scientific</td>
			<td>DCAP</td>
			<td>for decapitation if dissecting rats older than P15.</td>
		</tr>
		<tr>
			<td>Stereo Dissection Microscope&#160;</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (3, 10, 30) milliliter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma Aldrich</td>
			<td>T1426</td>
			<td>one out of three enzyme to digest tissue</td>
		</tr>
		<tr>
			<td>Tuberculin syringe&#160;</td>
			<td>Covidien</td>
			<td>8881500105</td>
			<td>for delivering euthanasia solution by intraperitoneal injection</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissor, 2.5 mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric flask, 1000 milliliter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td>945300</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, sterile u ltrapure, R&#62;18.18 megaOhms cm (e.g., filtered by a Millipore-Sigma water purification system)</td>
			<td>Millipore-Sigma</td>
			<td>CDUFBI001</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating and culturing vestibular and spiral ganglion somata from neonatal rodents for patch-clamp recordings

分离和培养的内耳神经节神经元的紧凑形态允许详细表征离子通道和神经递质受体，这些离子通道和神经递质受体有助于该群体的细胞多样性。该协议概述了成功解剖，解离和短期培养内耳双极神经元的体细胞以进行膜片钳记录所需的步骤。提供了制备前庭神经节神经元的详细说明，以及铺设螺旋神经节神经元所需的必要修改。该协议包括在穿孔贴片配置中执行全细胞膜片钳记录的说明。表征超极化激活的环核苷酸门控 （HCN） 介导电流的电压钳位记录的示例结果突出了与更标准的破裂贴片配置相比，穿孔贴片记录配置的稳定性。这些方法的组合，分离体体和穿孔膜片钳记录，可用于研究需要长期、稳定记录和细胞内环境保存的细胞过程，例如通过 G 蛋白偶联受体的信号传导。

通过应用一系列电压阶跃来运行电压钳位协议，揭示了各种不同电流系列的电压依赖性激活。图1A，B显示了从VGN唤起并改编自已发表的记录13的全细胞电流的代表性示例。施加去极化电压（图1B）会激活一个内向电流（按照惯例为负），该电流会非常迅速地激活和失活（图1A）。这是钠通道 14,15 的电?...

Watch this Scientific Journal Video about Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings at JoVE.com

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

这里介绍的方法提供了从内耳前庭神经节和螺旋神经节神经元解剖、解离、培养和膜片钳记录的详细说明。

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

在听觉和前庭传入神经元末端发现的许多离子通道和神经递质受体也存在于它们的细胞体中。可以在体外研究分离细胞体的培养物，以了解这些离子通道和受体如何影响神经元的反应。细胞体的紧凑形态允许高质量的电记录，以表征离子通道和神经递质受体的电压依赖性。此外，易于获得多种细胞类型，可通过对细胞多样性进行物理分析实现高通量。这些程序将由研究生凯瑟琳·雷加拉多（Katherine Regalado）以及我实验室的博士后研究员丹尼尔·布朗森（Daniel Bronson）博士和纳撒尼尔·诺瓦克（Nathaniel Nowak）进行演示。首先，将玻璃牧场移液管放在本生燃烧器的火焰上，以制备用于细胞解离的氚移液器。玻璃尖端开始熔化后，将玻璃拉伸至所需的尖端直径。将移液器的一端从火焰上拉开，形成一个小弯曲。将弯曲的移液器放在显微镜下。使用刻痕瓷砖，以所需的直径刻划并打破玻璃。将移液器吸头越过燃烧器火焰的顶部。这将快速抛光尖端附近的粗糙边缘。使用封闭的手术剪刀舀出安乐死小鼠的大脑。切断并切除颅神经，使大脑与颅骨完全分离。从头部后部开始，用镊子将透明的膜状材料拉出，然后用手术剪刀剪掉颅骨的顶部。去除头部和颈部后部的多余组织，使解剖区域和耳囊更清洁，更易于取用。将组织转移到装有新鲜L-15溶液的第二个培养皿中。然后定位耳囊和听觉、上、前庭和下前庭神经。用小弹簧剪刀将上神经节和下神经节分开，切掉听觉神经。使用手术刀轻轻剃掉骨脊，以削弱神经潜入骨囊的骨区域。小心地用手术刀清除碎屑，露出整个肿胀的神经节部分。使用细弹簧剪刀将神经节与向椭圆囊俯冲的周围神经分支切割并分开。使用细镊子去除上神经节，并将其转移到装有新鲜L-15溶液的35毫米培养皿中。预热酶溶液后，将酶混合物倒入 35 毫米培养皿中，并将其放入 37 摄氏度的培养箱中 10 至 15 分钟。使用细镊子和小弹簧剪刀清洁神经节，去除骨头、多余的组织、神经纤维和任何其他多余的结构。注意尽量减少任何神经节组织的切除。将清洁的神经节转移到预热的酶溶液中，并将其放回培养箱中 10 至 40 分钟。然后用新鲜的L-15溶液将神经节转移到35毫米培养皿中。两到三分钟后，将神经节转移到另一个装有过滤培养基的35毫米培养皿中。将约150微升过滤的培养基转移到镀膜的玻璃底培养皿上。然后，将所需数量的神经节转移到盖玻片上。从培养皿中抽出少量培养基，并用培养基冲洗涕流移液器，以防止组织粘在玻璃移液器的侧面。通过轻轻和反复地将组织通过移液管直到神经节充分解离来研磨。五分钟后，在光学显微镜下检查细胞是否沉淀在盖玻片上。小心地将培养皿放入 37 摄氏度的培养箱中 12 至 24 小时。接下来，在将头部一分为二后找到耳囊，并使用镊子切掉边缘将其从颅骨中取出。耳囊的螺旋部分容纳耳蜗和Corti器官。切掉骨头，用平行于骨骼曲线的细镊子覆盖耳蜗。使用小弹簧剪刀切断耳蜗底部的 modiolus，以将其从耳囊的其余部分释放出来。用小弹簧剪刀将Corti的器官切成两到三圈，使其平放，然后从每一圈切除modiolus。用细镊子捏住底部的纹，去除血管纹。在螺旋周围展开，一直到顶点，将其从Corti器官中剥离出来。通过切割螺旋神经节神经元纤维束向毛细胞投射的边缘来去除螺旋神经节，然后如前所述清洁神经节。酶促处理螺旋神经节后，在补充有N 2和B27的培养基中枘化螺旋神经节。将含有解离神经节的培养皿置于 37 摄氏度、5% 二氧化碳培养箱中 12 至 24 小时。将移液管的尖端浸入培养皿的清洁溶液中，以填充不含两性霉素的清洁溶液。用含有两性霉素的内部溶液填充移液器的背面。将移液管的尖端浸入培养皿的干净溶液中，同时两性霉素从移液器背面缓慢进入吸头。接下来，用两性霉素溶液填充移液器，使其达到移液器的三分之二。将移液器放入记录室的浴槽中，并将移液器吸头定位在视野中间。确保吸头没有气泡或其他碎屑。将移液器靠近膜并牢固地落在细胞中心。使用注射器或口腔移液器施加负压或抽吸。打开负 60 毫伏的保持电位。密封电阻应增加，直到它超过千兆欧姆。一旦形成千兆欧姆密封，释放负压。在酪氨酸开始起作用之前，输入电阻缓慢降低，响应于五毫伏电压步进的电流随着两性霉素进入膜而逐渐增加。下图显示了从前庭神经节神经元诱发的全细胞电流的代表性示例。净内向钠电流在这里通过其瞬时活化和失活来识别。净向外电流主要由钾离子携带，其活化和失活动力学比钠电流慢得多。超极化激活的环状核苷酸门控电流或HCN电流由一系列长时间的超极化电压激活。在记录过程中，穿孔贴片中离子通道表征的稳定性如图所示。HCN电流的电压相关激活曲线是在穿孔贴片配置中测量的。该曲线呈 S 形，在长时间的记录中保持稳定。破裂贴片模式的相对不稳定性由不同时间点的非重叠S 形曲线来说明。此处显示了补丁配置破裂期间 HCN 电流的激活曲线。这里显示了五个前庭神经节体、五个螺旋神经节神经元的放电模式，以及相应的当前步骤。放电模式的这种异质性反映了前庭神经节神经元和螺旋神经节神经元中潜在钠和钾通道组成的基本多样性。为了在此过程中最大限度地提高细胞存活率，请避免形成气泡，不要让组织过度劳累，并在将样品放入培养箱之前让细胞沉淀。穿孔膜片钳可以研究由胞质第二信使调节的离子通道，这对于研究传出神经递质对神经节神经元的影响至关重要。这些双极神经元的膜片钳记录有助于揭示生物物理多样性如何促进感觉神经元的功能多样性。

Research

JoVE Journal

Neuroscience

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

NRCC的膜片钳芯片上细胞的培养和电

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

<sub&gt; 1</sub&gt; F<sub&gt; O</subATP酶微囊的制备和表演膜片钳记录的Submitochondrial泡膜技术

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...