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?...

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 border="0" cellpadding="0" cellspacing="0" style="width:1012px;" width="1011">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Stemi Stereo 508</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:148px;">495009-0006-000&#160;</td>
			<td style="width:299px;">Dissecting microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS-222 &#34;Tricane&#34;</td>
			<td>Finquel</td>
			<td>ARF5G</td>
			<td>Amphibian general anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-1KG</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Calcium nitrate tetrahyrate (Ca(NO3)&#8226;4H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>237124-500G</td>
			<td>Used to prepare Stienberg&#39;s solution&#160;&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Magnesium Sulfate (MgSO4)</td>
			<td>Mallinckrodt Chemicals</td>
			<td>6066-04</td>
			<td>Used to prepare Steinberg&#39;s solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Calcium Chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C5080-500G</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Magnesium Chloride (MgCl2)</td>
			<td>J.T. Baker</td>
			<td>2444-01</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose Anhydrous</td>
			<td>Mallinckrodt Chemicals</td>
			<td>6066-04</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubocurarine hydrochloride pentahydrate</td>
			<td>Sigma</td>
			<td>T2379</td>
			<td>Nicotinic acetylcholine receptor antagonist</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Insect Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>0.1mm diameter stainless steel pins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>761028</td>
			<td>Preweighed monomer and curing agent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Polystyrene Petri Dish - 60x15mm</td>
			<td>Fisher Scientific</td>
			<td>AS4052</td>
			<td>Small petri dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrecisionGlide Needle 25Gx5/8 (.0.5mm X 16mm)</td>
			<td>BD</td>
			<td>305122</td>
			<td>Syringe needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1mL Slip Tip Tuberculin Syringe&#160;</td>
			<td>BD</td>
			<td>309659</td>
			<td>Disposable, sterile syringes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td>Fabricates micropipettes for electrophysiology recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipes Kimtech wipes</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td>Delicate task lint-free wipers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Fiber-Coupled LED (Green)</td>
			<td>Thorlabs</td>
			<td>M530F2</td>
			<td>Fiber optic cable paired with green LED</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cluster Bipolar Electrode (25&#181;m diameter)</td>
			<td>FHC</td>
			<td>30207</td>
			<td>Bipolar stimulating electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISO-Flex Stimulator</td>
			<td>A.M.P.I. (Israel)&#160;</td>
			<td>Contact manufacturer</td>
			<td>Flexible stimulus isolator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axon Instruments 700B Multipatch Amplifier</td>
			<td>Molecular Devices</td>
			<td>2500-0157</td>
			<td>Amplifier for voltage- and current-clamp recording&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1322A digitizer</td>
			<td>Molecular Devices</td>
			<td>2500-135</td>
			<td>Data acquisition system for electrophysiology recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axio Examiner.A1</td>
			<td>Zeiss</td>
			<td>491404-0001-000&#160;</td>
			<td>Microscope for electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-g Lab Table</td>
			<td>TMC</td>
			<td>63-533</td>
			<td>Air table for electrophysiology microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">c2400 CCD camera</td>
			<td>Hamamatsu</td>
			<td>70826-5</td>
			<td>Charge-coupled device camera for electrophysiology imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7 O&#39;Clock Super Platinum Stainless Razorblades</td>
			<td>Gillette</td>
			<td>CMM01049</td>
			<td>Platinum-coated stainless razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 (<strong class="xfig...

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

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

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

科研

神经科学

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

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 ...

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

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

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

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

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 ...

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

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

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的神经植体培养

解剖，文化与分析

全细胞膜片钳红外神经刺激机制的调查

隔离的感觉神经元中

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

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

在脑片全细胞膜片钳记录

通过的嗅球钙监控录像的变化温度引起的神经元电活动

全细胞贴片记录用于电泳生理测定通道视紫质中的离子选择性