Name: preparations and protocols for whole cell patch clamp recording of xenopus laevis tectal neurons
Uploaded: 2018-03-15T15:00:00.000+00:00
Duration: 5 min 25 s
Description: Watch this Scientific Journal Video about Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons at JoVE.com

由 NIH 赠款 SBC COBRE 1P20GM121310-01 支持。

这里描述的所有方法都已被怀俄明州大学的机构动物护理和使用委员会 (IACUC) 批准。所有的程序, 包括电生理记录, 都是在室温下进行的, 大约23摄氏度。这里描述的所有方法都是针对在发育阶段42和 49 (根据 Neiuwkoop 和费伯15) 的蝌蚪记录 tectal 神经元进行优化的。
1.体内准备
<ol><li>麻醉蝌蚪。<ol>
<li>将蝌蚪放在含有斯坦伯格溶液的小培养皿中, 0.01% MS-222 约5分钟。 注: MS-222 名为 "Tricaine" 是一种常见的鱼类和两栖类麻醉剂。在斯坦伯格的溶液中, 蝌蚪在毫米: 0.067 氯化钾, 0.034 Ca (无3)2· 4H2o, 0.083 MgSO4· 7H2o, 5.8 氯化钠, 4.9 HEPES。</li>
		<li>确保蝌蚪深麻醉 (不反应, 不再游泳), 然后再继续步骤2。</li>
	</ol>
</li>
	<li>在解剖/记录盘的地板上, 将麻醉蝌蚪固定在一个浸没的硅胶块上。<ol>
<li>使用一次性转移吸管将麻醉蝌蚪移到含有外部记录溶液的解剖/记录盘 (氯化钠115毫米, 2 毫米氯化钾, 3 毫米 CaCl2, 3 毫米氯化镁2, 5 毫米 HEPES, 1 毫米葡萄糖; 将 pH 值调整为7.25 使用10 N 的氢氧化钠, 渗透 255 mOsm)。<ol>
<li>为减少自发性肌肉抽搐, 将乙酰胆碱受体阻滞剂, 筒箭毒 (100 µM) 添加到外部溶液中。(通常, 这是通过使用200µL 吸管添加100µL 10 毫米筒箭毒库存到10毫升的外部溶液)。</li>
		</ol>
</li>
		<li>使用昆虫引脚, 确保蝌蚪, 背侧向上, 到一个浸没的硅胶弹性体 (如 Sylgard 184, 见材料表的细节), 已粘附在解剖盘地板。 注: 放置针脚是至关重要的: 把他们放在大脑的两边, 足够的尾鳍, 以避免刺穿传入的研资局轴突的项目从眼睛和进入大脑刚刚前的视神经顶盖 (图1 A).</li>
	</ol>
</li>
	<li>沿中线将大脑圆角。<ol>
<li>对于大脑的清晰视图, 通过使用不育的25克针在中线上进行浅切口, 去除大脑上方的皮肤。</li>
		<li>将针头插入神经管并轻轻向上拉 ( 背 ) , 使脑部沿同一中线轴向上切下 , 这样 , 管子的背部就会净地切割 ( 断开 ) , 同时保持底板完好无损 (图 1B) 。 注: 重要的是, 神经管的底板保持完好, 因为传入感觉输入进入顶盖通过地板板。</li>
	</ol>
</li>
	<li>去除覆盖 tectal 神经元的透明心室膜。<ol>
<li>将记录盘移动到电生理平台上, 用碎玻璃吸管尖去除 tectal 神经元细胞体上的心室膜。通过轻轻拖动它穿过一个微妙的任务雨刷打破小费。</li>
		<li>将吸管拧入吸管支架, 并通过机器人将其降低到光学顶盖。</li>
		<li>用破吸管将心室膜从顶盖中剥离出来。 注: 不需要从整个顶盖中取出膜;一个小窗口就足够了, 可以提供大量的神经元。</li>
	</ol>
</li>
	<li>获得整个细胞补丁钳录音。 注意: 从这一点向前的方法类似于执行塞格夫中所述的小鼠脑切片的全细胞贴钳录音, et 。16(图 1C)。</li>
	<li>将 tectal 神经元的反应记录到整个磁场投射到视网膜上的光的闪光。<ol>
<li>在视网膜上投射一整场光, 将一根光纤与蝌蚪的眼睛相邻。在光纤的另一端是一个 LED 线与可变电阻器, 允许光亮度被控制。由放大器的数字输出触发 LED。这样, 就可以记录不同亮度的整个场闪烁 (图 4A)。</li>
	</ol>
</li>
</ol>2. 全脑准备
<ol><li>执行步骤1.1 到1.3。 注意: 没有必要在外部解决方案中筒箭毒此准备。</li>
	<li>隔离大脑。<ol>
<li>使用 25 G 针切断后脑 (图 2a)。</li>
		<li>要将整个大脑从蝌蚪中分离出来, 轻轻地将针头放在脑下, 在尾部向延髓的方向上切断所有的侧向和腹壁结缔组织和神经纤维。</li>
	</ol>
</li>
	<li>把大脑固定在一大块硅胶弹性体上。<ol>
<li>一旦完全释放, 通过在后脑 (图 2B) 中的一个嗅球和另一个 pin 放置一个 pin, 将大脑固定在硅弹性体块上。这是从 tectal 神经元进行记录的最佳配置。</li>
	</ol>
</li>
	<li>将含有固定全脑准备的盘子移动到电生理钻机。采用步骤1.4 所述的碎玻璃吸管去除心室膜。</li>
	<li>将双极刺激电极放在光学交叉上 (从每只眼睛的轴突穿过中脑的地方) 直接激活研资局轴突。<ol>
<li>放置双极刺激电极延髓, 几乎相邻的大中脑室。光学交叉位于延髓的大中脑室 (图 2B) 中。<ol>
<li>轻轻地将刺激电极降低到光学交叉, 这样在组织中形成一个小凹痕。双极电极由脉冲刺激器驱动, 使刺激的强度得到精确控制。</li>
		</ol>
</li>
	</ol>
</li>
	<li>按照塞格夫、等. 的描述, 执行整个单元格膜片钳录制 (图 2C)。16
</li>
</ol>3. 水平脑切片制备
<ol><li>执行步骤2.1 到2.3。</li>
	<li>使用剃刀刀片将最侧向第四 (在体内对应于一个光学顶盖一侧的最背第四)。此切口与延髓尾平面平行, 如图 3A所示。</li>
	<li>将大脑重新钉在硅弹性体的侧面, 切片侧朝上 (这样胞体和 neuropil 可以直接进入录音), 而大脑的心室表面离硅胶弹性体块 (图3B) (使双极电极可以放在光学交叉上)。</li>
	<li>执行整个单元格补丁钳或本地归档的潜在记录 (图 3C), 如塞格夫、等. 所述。16
</li>
</ol>

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作者没有什么可透露的。

这项工作中描述的所有方法都是为记录发育阶段42和 49 (根据 Neiuwkoop 和费伯15) 的蝌蚪的 tectal 神经元而优化的。在阶段 42, 蝌蚪是足够大和充分开发的, 以便昆虫别针可以被安置在脑子的任一侧为在体内录音和进行整个脑解剖。在早期阶段, 当蝌蚪本质上是二维 (即, 平), 这里描述的方法是不最佳的。
由于 tectal 神经元可以在整个细胞配置中被访问和记录, 因此这个模型电路一直是关于神经回路在起作用时如何形成的基本发现的温床, 并且在形成19时起作用。这里我们描述了如何从 tectal 神经元获取完整的细胞补丁夹录音, 包括如何执行蝌蚪在体内和整个大脑的准备, 不同的记录配置, 以及不同类型的数据的例子。下面是一个讨论每个准备的长处和局限性, 然后是实现高质量的整个细胞记录的提示。
爪蟾蝌蚪开始显示视觉回避行为的阶段 47/48, 约 7-8 天后受精3,13。这意味着, 即使在这些相对较早的开发阶段, retinotectal 电路也能正常工作并能够处理视觉刺激。关于 retinotectal 电路的发展和可塑性的基本问题已经通过使用体内准备记录 tectal 神经元对投射到视网膜上的视觉刺激的反应来直接解决。与哺乳动物中的等效记录配置相比, 蝌蚪中的体内记录相对简单, 因为神经系统的相对简单和头骨缺乏。由于单个 tectal 神经元 somas 可以可视化在体内, 对给定神经元的密封和实现全细胞补丁钳记录配置实质上等同于在啮齿动物切片中进行全细胞贴片钳记录。准备.蝌蚪通常在钻机上存活几个小时 (通过监测心脏跳动可以很容易地评估)。蝌蚪体内准备的一个局限性是, 只有驻留在光学顶盖的深体细胞层中的 tectal 神经元, 即紧邻大中间脑室的层, 才能进行记录 (图1).这意味着居住在顶盖外体细胞层的 tectal神经元在体内基本上未被探测到。
整个大脑准备的实验优势是, 它从眼睛和皮肤的外围感觉受体分离出大脑, 消除所有虚假感官驱动的活动。虽然不再能够被视觉刺激驱动, 但顶盖的轴突输入仍然存在于整个大脑的准备中, 它们可以通过放置在双极刺激电极 (FHC) 的高度控制的方式直接激活。光学交叉, 两组的研资局轴突束进入大脑。第一个报告使用的整个大脑准备是在 1996年, 在一个重要的研究, 吴et 等。8, 其中, 通过双极刺激电极激活了研资局轴突, 使作者可以将单个研资局轴突的强度量化到突触后 tectal 神经元, 并在这样做的基础上发现了进展突触成熟。与体内准备一样, 整个大脑的准备工作只允许进入深层的神经元, 靠近心室膜。
尽管光学顶盖由大约九个细胞体层组成 17, 所有以前的电生理学研究都集中在深层 tectal 神经元, 因为传统的全脑和体内准备 (描述 以上) 仅允许对深层神经元进行访问。为了访问所有体细胞层的 tectal 神经元, 从最深到最肤浅以及整个 neuropil, 研资局轴突与 tectal 神经元树突形成突触连接, 我们开发了一种改良的全脑准备, 称为 "水平脑切片准备 "(图 314)。与整个大脑的准备工作一样, 水平脑切片制剂在大脑中保持了研资局的轴突, 可以通过将双极刺激电极放在视神经交叉来控制。
总体而言, 该制剂旨在优化 tectal 神经元胞体的可视化和获取, 以实现全细胞膜片钳的记录。对体细胞的直接访问对于高质量的全细胞膜片钳录音是必不可少的: 进入神经元内部需要形成一个非常强 (> 1 x 109 Ω) 在吸管和神经元的等离子膜之间的密封, 这需要直接将吸管尖端接触到膜上。因此, 充分去除心室膜是成功记录的关键。此外, 成功的研资会诱发录音的关键是将双极刺激电极正确放置在光学交叉上。基本上所有 tectal 神经元从研资局轴突获得直接突触输入。如果没有观察到研资局诱发的反应, 那么这很可能是由于不正确的双极电极的放置, 所以它不是在视神经交叉和不接触视神经。这可以通过简单地重新定位刺激电极来纠正。在双极电极重新定位后未能观察研资局诱发的反应, 可能意味着研资局轴突投射在解剖过程中意外切断。在这种情况下, 有必要重新开始新的准备工作。最后, 未能观察到研资会诱发的反应可能是由于记录从一个细胞不是一个常见的 tectal 神经元。例如, 胶质细胞通常不显示由 tectal 神经元显示的正常的研资局诱发的 monosynaptic 反应, 也没有被发现的大型中脑神经元, 尽管其数量很低, 居住在视神经顶盖20中.由于 tectal 神经元的数量太多, 从这些非 tectal 细胞中记录的可能性非常低。当然, 质量记录也要求 tectal 神经元保持健康。一个健康的 tectal 神经元的特征是一个紧凑的圆形或椭圆形的躯体, 是浅色的, 没有瑕疵。一旦完成了整个细胞的记录配置, 就可以通过测量基本电学特性, 如静止膜电位、输入电阻和电容, 进一步评估神经元的健康状况。.健康 tectal 神经元在发育阶段42和49之间的静止膜电位约为-45 mV, 平均输入电阻约为 1 GΩ, 电容为 10-12 pF3,4,5。由于心室膜的去除, 一小部分神经元不可避免地会受损。受损或不健康的神经元的胞体出现颗粒状、肿胀或萎缩。一个不健康的神经元的例子显示为图 2C。如果大多数神经元看起来不健康, 这可能反映出外部记录解决方案有问题, 因此建议您建立新的外部记录解决方案。除了看起来健康, 记录的最佳神经元通常与大量其他神经元相关, 而不是孤立的。另外, 选择易于访问的单元格。例如, 不要太用力推组织进入细胞, 因为这可能会损害组织或移动组织, 从而将刺激电极移出原处。最后, 录制应限制在顶盖的中间第三个 (图2 B) 中, 以避免由于在 rostro-尾轴4、8中存在的发展渐变而产生的潜在差异,当然, 除非实验的重点是上述的发展梯度。顶盖适当的是增生区的面积延髓, 尾侧为大中脑室的尾缘。
整个细胞膜片钳记录技术不是没有它的局限性。通常, 这种记录方式涉及一次记录一个神经元。这与新世代 GCaMP6 钙指标的成像方法形成对比, 这使得大量神经元的活动在切片准备和体内中同时被成像。
整个电池电压钳记录固有的另一个缺点是空间钳位错误 (无法控制电压远超过 soma), 这限制了电压4的时间控制。总的来说, 这些电压钳问题是最低的, 当记录从 tectal 神经元, 因为它们相对较小的大小, 适度的树突 arborizations, 和相对较小和缓慢的电流相比, 哺乳动物的神经元。这些 tectal 神经元的特性使它们特别适合于电压钳记录, 测量多个参数, 以及构造单个神经元的轮廓5,6。
未来的方向包括优化体内水平脑切片准备, 以表征外层神经元在视觉刺激过程中的功能。

retinotectal 电路是两栖视觉系统的主要组成部分。它由眼睛中的 RGCs 组成, 它将它们的轴突投射到视神经顶盖, 与突触后 tectal 神经元形成突触连接。爪蟾蝌蚪 retinotectal 电路是研究神经回路形成和功能的一种流行的发展模式。这个蝌蚪的 retinotectal 电路有许多属性, 使它成为一个强大的实验模型1,2,3。这篇文章的主要特点之一, 是能够从 tectal 神经元、体内或使用整个大脑准备进行完整的细胞贴片钳录音。有一个电生理学钻机配备了一个放大器, 支持电压和电流钳记录模式, 整个细胞补丁钳录音允许神经元的电生理学的特点是高分辨率。因此, 整个细胞补丁钳录音从 tectal 神经元跨越关键阶段的 retinotectal 电路形成提供了详细和全面的理解的发展和可塑性的内在4,5,6,7和突触8,9,10,11属性。结合全细胞贴片钳 tectal 神经元记录, 表达基因或 morpholinos 在这些神经元中感兴趣的能力12, 以及通过已建立的视觉避免测试 (13 ) 来评估视觉引导行为的方法, 可促进识别分子、电路功能和行为之间的链接。
重要的是要注意的是, 从整个细胞膜片钳录音获得的高分辨率数据的类型是不可能使用新的成像方法, 如基因钙指示器 GCaMP6, 因为虽然使用钙指标允许成像在大量神经元的钙动力学同时, 没有直接或明显的方法可以通过测量胞体中的三角洲荧光来获得特定的电学参数, 也没有办法对神经元进行电压钳位测量。电流-电压关系。显然, 这两种截然不同的方法, 电生理记录和钙成像, 具有不重叠的优势和产生不同类型的数据。因此, 最好的方法取决于正在处理的具体实验问题。
在这里, 我们描述了我们的方法, 从蝌蚪光学顶盖神经元, 使用体内准备, 全脑准备, 和一个新的修改全脑准备, 在我们的实验室中开发的全细胞补丁钳录音 14 .在 "代表性结果" 部分中, 我们演示了每个准备的实验优势和可以获得的不同类型的数据。讨论部分包括了不同准备工作的极限和强度, 以及排除故障的提示。

<table><tbody><tr><td>Stemi Stereo 508</td><td>Zeiss</td><td>495009-0006-000&amp;nbsp;</td><td>Dissecting microscope</td></tr><tr><td>MS-222 &quot;Tricane&quot;</td><td>Finquel</td><td>ARF5G</td><td>Amphibian general anesthetic</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-3</td><td>Used to prepare Stienberg&#039;s solution and external solution</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>P217-500</td><td>Used to prepare Stienberg&#039;s solution and external solution</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375-1KG</td><td>Used to prepare Stienberg&#039;s solution and external solution</td></tr><tr><td>Calcium nitrate tetrahyrate (Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>237124-500G</td><td>Used to prepare Stienberg&#039;s solution&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Mallinckrodt Chemicals</td><td>6066-04</td><td>Used to prepare Steinberg&#039;s solution</td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C5080-500G</td><td>Used to prepare external recording solution</td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>J.T. Baker</td><td>2444-01</td><td>Used to prepare external recording solution</td></tr><tr><td>D-glucose Anhydrous</td><td>Mallinckrodt Chemicals</td><td>6066-04</td><td>Used to prepare external recording solution</td></tr><tr><td>Tubocurarine hydrochloride pentahydrate</td><td>Sigma</td><td>T2379</td><td>Nicotinic acetylcholine receptor antagonist</td></tr><tr><td>Insect Pins</td><td>Fine Science Tools</td><td>26002-10</td><td>0.1mm diameter stainless steel pins</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>761028</td><td>Preweighed monomer and curing agent kit</td></tr><tr><td>Sterile Polystyrene Petri Dish - 60x15mm</td><td>Fisher Scientific</td><td>AS4052</td><td>Small petri dishes</td></tr><tr><td>PrecisionGlide Needle 25Gx5/8 (.0.5mm X 16mm)</td><td>BD</td><td>305122</td><td>Syringe needles</td></tr><tr><td>1mL Slip Tip Tuberculin Syringe&amp;nbsp;</td><td>BD</td><td>309659</td><td>Disposable, sterile syringes</td></tr><tr><td>Borosilicate pipette glass</td><td>Sutter Instrument</td><td>BF150-86-10HP</td><td>Pulled to desired specifications using pipette pulling machine</td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter Instruments</td><td>P-97</td><td>Fabricates micropipettes for electrophysiology recording</td></tr><tr><td>Kimwipes Kimtech wipes</td><td>Kimberly-Clark</td><td>34120</td><td>Delicate task lint-free wipers</td></tr><tr><td>Axon Instruments MultiClamp 700B Headstage CV-7B</td><td>Molecular Devices</td><td>1-CV-7B</td><td>Current clamp and voltage clamp headstage</td></tr><tr><td>MP-285 Motorized Manipulator with Tabletop Controller</td><td>Sutter Instrument</td><td>MP-285/T</td><td>Control for headstage on electrophysiology rig</td></tr><tr><td>Fiber-Coupled LED (Green)</td><td>Thorlabs</td><td>M530F2</td><td>Fiber optic cable paired with green LED</td></tr><tr><td>Cluster Bipolar Electrode (25&amp;micro;m diameter)</td><td>FHC</td><td>30207</td><td>Bipolar stimulating electrode</td></tr><tr><td>ISO-Flex Stimulator</td><td>A.M.P.I. (Israel)&amp;nbsp;</td><td>Contact manufacturer</td><td>Flexible stimulus isolator</td></tr><tr><td>Axon Instruments 700B Multipatch Amplifier</td><td>Molecular Devices</td><td>2500-0157</td><td>Amplifier for voltage- and current-clamp recording&amp;nbsp;</td></tr><tr><td>Digidata 1322A digitizer</td><td>Molecular Devices</td><td>2500-135</td><td>Data acquisition system for electrophysiology recording</td></tr><tr><td>Axio Examiner.A1</td><td>Zeiss</td><td>491404-0001-000&amp;nbsp;</td><td>Microscope for electrophysiology</td></tr><tr><td>Micro-g Lab Table</td><td>TMC</td><td>63-533</td><td>Air table for electrophysiology microscope</td></tr><tr><td>Inspiron 620 Personal Desktop Computer with Windows 7 64-bit</td><td>Dell</td><td>D06D001</td><td>Computer running electrophysiology software</td></tr><tr><td>c2400 CCD camera</td><td>Hamamatsu</td><td>70826-5</td><td>Charge-coupled device camera for electrophysiology imaging</td></tr><tr><td>7 O&#039;Clock Super Platinum Stainless Razorblades</td><td>Gillette</td><td>CMM01049</td><td>Platinum-coated stainless razor blades</td></tr><tr><td>Transfer Pipets</td><td>Fisher Scientific</td><td>13-711-7M</td><td>Disposable Polyethylene transfer pipets</td></tr></tbody></table>

preparations and protocols for whole cell patch clamp recording of xenopus laevis tectal neurons

爪蟾蝌蚪 retinotectal 电路, 由眼睛中的视网膜神经节细胞 (RGCs) 组成, 直接在视神经顶盖神经元上形成突触, 是研究神经回路自组装的流行模型。能够从 tectal 神经元进行完整的细胞修补钳录音, 并记录研资局诱发的反应, 无论是在体内还是使用全脑准备, 都产生了大量高分辨率的数据, 关于正常的机制、异常、电路的形成和功能。在这里, 我们描述如何执行在体内准备, 原来的整个大脑准备, 和最近开发的水平脑切片准备, 以获得整个细胞补丁钳录音从 tectal 神经元。每个制剂都具有独特的实验优势。通过体内准备, 可以记录 tectal 神经元对投射到眼睛上的视觉刺激的直接反应。整个大脑的准备, 使研资局的轴突被激活的高度控制的方式, 和水平脑切片准备允许记录从所有层面的顶盖。

为了记录光诱发的反应, 整个领域的闪光投射到视网膜上, 而由此产生的响应是从单个的 tectal 神经元 (图 4a) 记录下来的。这个特殊的协议旨在测量神经元对光的响应 ("on" 响应), 然后在十五年代关闭以测量 "关闭响应"。Tectal 神经元通常表现出强健的和关闭的响应 (这里显示的电压钳模式, 与神经元钳位 60mV, 以测量突触电流 (图 4B))。任何一个神经元接收到的突触驱动量可以通过记录自发突触事件 (图 4C) 来量化。电流-电压关系 (I-V 曲线) 可以通过步进神经元越来越偏振光电位和记录产生的电压激活 Na+和 K+电流产生, 称为 "混合电流录制 "(图 4D, E)。已经确定的是, 对于这些两栖神经元, 峰值 Na+和 K+电流可以通过混合的电流记录进行量化, 因为峰值不会与另一个47之间的世俗重叠。
在光学交叉直接活化研资局轴突, 绕过所有在眼中发生的视觉刺激的上游计算和处理, 并允许研究突触前和突触后 tectal 神经元之间的突触传递。最纯净和减少的形式。Tectal 神经元对研资局激活的反应是由光刺激的 RGCs 或直接使用由两个成分组成的双极刺激电极产生的: 一个 monosynaptic 成分 (从活化 RGCs 直接突触输入) 和 polysynaptic组件 (本地经常性网络活动反馈到被记录的神经元)。在光诱发反应的情况下, monosynaptic 和 polysynaptic 组分相互重叠由一些 undeterminable 量。这种时间重叠限制了在视网膜和视神经顶盖之间的突触传递上可以得出的结论。然而, 使用一个完整的大脑准备并通过放置在光学交叉上的电极直接激活研资局轴突道 (图 5a), 就会产生一个单一的和 polysynaptic 的组件, 本质上是不重叠的。双极性电极的刺激强度可以调整。随着刺激强度的增加, 有更多的研资局轴突激活 (图 5B)。极小的刺激协议确定一个单独的研资局轴突的突触强度到单个 tectal 神经元;最大刺激协议反映了所有研资局轴突组合的总或最大的突触输入 (图 5C)。由单个 tectal 神经元接收的兴奋性和抑制性突触输入 (E: I 比值) 的适当比率对于正常的视觉功能18是至关重要的。图 5D显示了从单个 tectal 神经元记录的研资局诱发的兴奋和抑制电流跟踪的例子。从研资局突触前轴突终端发射器释放的概率由配对脉冲记录测量。为此, 突触后的 tectal 神经元被记录在电压钳模式中以测量突触电流, 而研资局轴突则连续激活, 用50毫秒时间间隔 (图 5E) 隔开。
整个细胞补丁钳录音可以从 tectal 神经元居住在任何躯体层。与整个大脑的准备工作类似, 研资局轴突可以通过在光学交叉上放置一个双极记录电极 (图 6a) 来激活。所有的录音在整个大脑准备也可以执行使用这种水平脑切片准备。图 6B显示了从表面层的神经元记录的研资局诱发反应的示例跟踪。这项准备还允许研资会突触输入到 tectal 树突的空间模式进行测量。为此, 在 neuropil (图 6C) 的内侧侧轴 (即, 远端近轴) 上每10µm 记录了研资局诱发的场电位。这就产生了一个高分辨率的功能研资会输入模式的轮廓。通过计算第二空间导数14 , 可以将场电位转换为电流源密度, 并将当前源密度转换为图像图。图 6C中的图像图形显示, 最大的研资局输入目标是 neuropil 的远端区域。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57465/57465fig1.jpg"> 图 1。体内准备过程.(A) 蝌蚪固定在一块硅胶弹性体上。针脚由白色箭头指示。注意更多的尾鳍定位, 以避免刺穿的光束。白色虚线指示中线。(B) 在沿背中线切割并去除覆盖皮肤后, 放大整个大脑。(C) 全单元格补丁-钳位记录体内。(L: 侧向。M: 内侧。R: 延髓。尾鳍。D: 背部。腹部。嗅球。光学顶盖。HB: 后脑)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57465/57465fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57465/57465fig2.jpg"> 图 2.全脑准备程序.(A) 在大脑沿中线鱼片后, 后脑被切断。(B) 整个大脑被解剖并固定在硅胶弹性体上。白色星号表示左嗅球中的针脚。红色框表示顶盖的中间第三个, 即是要录制的目标区域. (C) 整个细胞贴片钳记录使用全脑准备。黑色星号表示未被擦伤的心室膜区域, 无法在其中记录细胞。如果膜已成功移除, 胞体显得更加清晰和明确。箭头表示一个不健康的神经元。(PZ: 增生区。LMV: 大内侧脑室。嗅球。光学顶盖。HB: 后脑。L: 侧面。M: 内侧。R: 延髓。尾鳍。D: 背部。V: 腹侧)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57465/57465fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57465/57465fig3.jpg"> 图 3.水平脑切片准备程序.(A) 在整个大脑准备后, 光学顶盖一侧的最侧面部分将被切片 (由虚线表示)。(B) 被固定在硅胶弹性体侧面后, 水平脑切片准备工作。双白线代表一个刺激电极放置在光学交叉。(C) 通过水平脑切片准备, 放大了 neuropil 层的视图。虚线曲线指示了 neuropil 层和图元之间的边界。此数字已从 Hamodi ( et .) 中进行了修改。14 (L: 侧向。M: 内侧。R: 延髓。尾鳍。D: 背部。腹部。嗅球。光学交叉。光学顶盖。HB: 后脑)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57465/57465fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57465/57465fig4.jpg"> 图 4.体内准备中的录制.(A) 示意图, 显示体内录制的典型配置。(B) 光诱发反应, 包括光照和光响应。嵌入的痕迹会被放大--针对的是-和不响应, 分别。(C) 记录自发性兴奋后突触电流。(D) Na+和 K+电流, 其响应电压钳位步骤从-60 mv 到 30 mv 从单个神经元。(E) IV 从 (D) 中的跟踪生成的图。所有的录音都是与神经元保持在-60 mV, 除了 (D) 生成 IV 地块;神经元被加强到越来越多的偏振光电位, 在 10 mV 增量, 激活电压依赖电流。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57465/57465fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57465/57465fig5.jpg"> 图 5.全脑准备的录音(A) 示意图, 显示整个脑记录的典型配置, 包括放置在光学交叉上的刺激电极 (绿色和红色线代表研资局轴突)。(B) 覆盖了研资局对研资局刺激力度变化的反应的痕迹。(C) 最大刺激响应 (左) 和最小刺激响应 (右)。(D) 由研资局诱发的兴奋和抑制对单个神经元的反应。(E) 配对脉冲促进的研究人员的诱发反应的单个神经元。(OB: 嗅球。光学交叉。OT: 光学顶盖)。所有录音都是用-60 mV 的神经元进行的, 除了 (D) 测量兴奋性和抑制性输入: 兴奋性输入是通过持有神经元在伽玛丁酸 (GABA) 介导的氯化物的逆转电位记录的。电流 (-45 mv);在α-氨基-3-羟基-5 甲基-4-isoxazolepropionic 酸 (AMPA) 介导的电流 (+ 5 mv) 的反转电位下, 抑制输入。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57465/57465fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57465/57465fig6.jpg"> 图 6.水平脑切片准备的录音.(A) 示意图, 显示水平脑切片准备的配置。注意, 虽然刺激电极保持在光学交叉, 记录吸管现在定位, 以访问所有 tectal 层暴露的水平脑切片准备的细胞。(B) 由居住在顶盖表层的神经元所作出的反应。(C) 在 neuropil 和转换后的电流源密度 (CSDs) 中记录的场电位, 由图像绘制热图显示。(OC: 光学交叉。光学顶盖。HB: 后脑)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57465/57465fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons at JoVE.com

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

本文讨论了三种用于全细胞膜片钳记录的脑准备, 研究了爪蟾蝌蚪的 retinotectal 电路。每个准备, 以它自己的具体优势, 贡献的实验驯良的爪蟾蝌蚪作为模型, 研究神经回路功能。

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

preparations-protocols-for-whole-cell-patch-clamp-recording-xenopus

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

9.4K 次观看.  University of Wyoming. 本文讨论了三种用于全细胞膜片钳记录的脑准备, 研究了爪蟾蝌蚪的 retinotectal 电路。每个准备, 以它自己的具体优势, 贡献的实验驯良的爪蟾蝌蚪作为模型, 研究神经回路功能。

视频: Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

科研

生物学

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

分离的原发性上皮细胞从附睾的全细胞贴片钳记录

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

发育生物学

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

全细胞膜片钳红外神经刺激机制的调查

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

神经科学

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

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 ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Establishing a Whole-Cell Configuration through the Patch Clamp Method

Source: Segev, A. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54024">Whole-cell Patch-clamp Recordings in Brain Slices.</a>&#160;J. Vis. Exp.&#160;(2016)
This video demonstrates the whole-cell patch clamp technique in a mouse brain slice. This technique establishes a stable connection between micropipette and neuron membrane, which is further disrupted to establish a whole-cell configuration to study the neuron&#39;s health and physiological responses.

通过膜片钳方法建立全细胞配置

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Slice ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Whole-Cell Patch Clamp Electrophysiology: A Method to Study Electrical Properties of Neurons

Source: Roy, B., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/50551/" target="_blank">Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells</a>. J. Vis. Exp. (2013).
This protocol describes a technique called whole-cell patch clamp electrophysiology, which is a method to study electrical properties of Mauthner neurons and other reticulospinal cells in zebrafish embryos.

全细胞膜片钳电生理学:一种研究神经元电特性的方法

Source: Roy, B., et al. Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells. J. Vis. Exp. (2013).
This protocol describes a technique called ...

蓝斑马（斑马鱼）

胚胎

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from a Flat-Mount Mouse Retina

Source: Tu, H., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54608">Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina.</a> J. Vis. Exp. (2016).
This video demonstrates a whole-cell patch-clamp technique for electrophysiological recording from a flat-mount mouse retina. A thin glass pipette with an electrode is used to approach starburst amacrine cells (SACs) within the retina. After forming a seal, suction is applied to rupture the membrane and form the whole-cell patch-clamp configuration. Finally, the flow of ions through the SAC synaptic receptors is measured using the configuration.

建立用于平装小鼠视网膜电生理记录的全细胞膜片钳

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. External and Internal Solutions

	...

Whole-Cell Patch Clamp Recording in a Substantia Gelatinosa Neuron of a Spinal Cord Slice

Source: Zhu, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58479">Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for performing electrophysiological recordings in substantia gelatinosa (SG) neurons of spinal cord slices, using whole-cell patch-clamp techniques to analyze neuron firing patterns. This method allows the study of neural responses to stimuli in SG neurons of the spinal cord.